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United States Patent Application 20110123544
Kind Code A1
Salfeld; Jochen G. ;   et al. May 26, 2011

HUMAN ANTIBODIES THAT BIND HUMAN IL-12 AND METHODS FOR PRODUCING

Abstract

Human antibodies, preferably recombinant human antibodies, that specifically bind to human interleukin-12 (hIL-12) are disclosed. Preferred antibodies have high affinity for hIL-12 and neutralize hIL-12 activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. The antibodies, or antibody portions, of the invention are useful for detecting hIL-12 and for inhibiting hIL-12 activity, e.g., in a human subject suffering from a disorder in which hIL-12 activity is detrimental. Nucleic acids, vectors and host cells for expressing the recombinant human antibodies of the invention, and methods of synthesizing the recombinant human antibodies, are also encompassed by the invention.


Inventors: Salfeld; Jochen G.; (North Grafton, MA) ; Roguska; Michael; (Ashland, MA) ; Paskind; Michael; (Sterling, MA) ; Banerjee; Subhashis; (Hamden, CT) ; Tracey; Daniel Edward; (Harvard, MA) ; White; Michael; (Framingham, MA) ; Kaymakcalan; Zehra Edward; (Westborough, MA) ; Labkovsky; Boris; (Marlborough, MA) ; Sakorafas; Paul; (Newton Highlands, MA) ; Veldman; Geertuida M.; (Sudbury, MA) ; Venturini; Amy; (Lexington, MA) ; Widom; Angela; (Acton, MA) ; Friedrich; Stuart; (Morpeth, CA) ; Warne; Nicholas W.; (Andover, MA) ; Kantor; Angela; (Pepperell, MA) ; Elvin; John Gawain; (Royston, GB) ; Duncan; Alexander Robert; (Cambridge, GB) ; Derbyshire; Elaine Joy; (Berks, GB) ; Carmen; Sara; (Cambridge, GB) ; Smith; Stephen; (Ely, GB) ; Holtet; Thor Las; (Ronde, DK) ; Du Fou; Sarah Leila; (Hitchen, GB)
Assignee: Abbott GmbH & Co. KG
Wiesbaden
DE

Serial No.: 910461
Series Code: 12
Filed: October 22, 2010

Current U.S. Class: 424/158.1; 436/501; 530/351; 530/387.1
Class at Publication: 424/158.1; 530/387.1; 436/501; 530/351
International Class: C07K 16/24 20060101 C07K016/24; A61K 39/395 20060101 A61K039/395; G01N 33/566 20060101 G01N033/566; C07K 14/54 20060101 C07K014/54; A61P 19/02 20060101 A61P019/02; A61P 37/00 20060101 A61P037/00; A61P 9/10 20060101 A61P009/10; A61P 13/12 20060101 A61P013/12; A61P 25/28 20060101 A61P025/28; A61P 25/16 20060101 A61P025/16; A61P 31/00 20060101 A61P031/00; A61P 7/06 20060101 A61P007/06; A61P 3/10 20060101 A61P003/10; A61P 9/00 20060101 A61P009/00; A61P 35/04 20060101 A61P035/04; A61P 17/06 20060101 A61P017/06


Claims



1. An isolated antibody, or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of said p40 subunit of IL-12.

2. The isolated antibody of claim 1, or antigen binding portion thereof, which dissociates from the p40 subunit of human IL-12 with a K.sub.d of 1.times.10.sup.-10 M or less or a k.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by surface plasmon resonance.

3. The isolated antibody of claim 1, or antigen binding portion thereof, which is a neutralizing antibody

4. The isolated antibody of claim 3, or antigen binding portion thereof, which inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less, or which inhibits human IFN.gamma. production with an IC.sub.50 of 1.times.10.sup.-10 M or less.

5. The isolated antibody of claim 1, or antigen binding portion thereof, which is a human antibody.

6. A pharmaceutical composition comprising the antibody or an antigen binding portion thereof of claim 1, and a pharmaceutically acceptable carrier.

7. A method for detecting the p40 subunit of an interleukin comprising contacting the p40 subunit of IL-12 with the antibody, or antigen-binding portion thereof, of claim 1 such that said p40 subunit is detected.

8. A method for detecting the p40 subunit of an interleukin comprising contacting the p40 subunit of the interleukin with the antibody, or antigen-binding portion thereof, of claim 1 such that the p40 subunit of the interleukin is detected

9. The method of claim 7, wherein the p40 subunit of the interleukin is detected in vitro.

10. The method of claim 7, wherein the p40 subunit of the interleukin is detected in a biological sample for diagnostic purposes.

11. A method for inhibiting an activity of an interleukin comprising a p40 subunit, comprising contacting the interleukin with the antibody, or antigen-binding portion thereof, of claim 1 such that the activity is inhibited.

12. A method for inhibiting an activity of an interleukin comprising a p40 subunit in a human subject suffering from a disorder in which the activity is detrimental, comprising administering to the human subject the antibody, or antigen-binding portion thereof, of claim 1 such that the activity in the human subject is inhibited.

13. The method of claim 11, wherein said interleukin is IL-12.

14. The method of claim 11, wherein said interleukin comprises a p40 subunit and a p19 subunit.

15. The method of claim 11, wherein said interleukin is IL-23.

16. The method of claim 12, wherein the disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyoarthropathy, ankylosing spondylitis, systemic lupus erythematosis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, multiple sclerosis, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, thyroiditis, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, polyarteritis nodosa, Wegener's granulomatosis, Henoch-Schonlein purpura, microscopic vasculitis of the kidneys, chronic active hepatitis, Sjogren's syndrome, uveitis, sepsis, septic shock, sepsis syndrome, adult respiratory distress syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, myasthenia gravis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, fibrotic lung diseases, hemolytic anemia, malignancies, heart failure and myocardial infarction.

17. The method of claim 12, wherein the disorder is psoriasis.

18. The method of claim 12, wherein the disorder is rheumatoid arthritis.

19. A method for altering the conformational structure of the p40 subunit of IL-12, the method comprising contacting said subunit with an antibody, or antigen binding portion thereof, that is capable of binding to said subunit and is capable of altering the conformational structure of said subunit in an amount effective to alter the conformational structure of said subunit, thereby altering the conformational structure of said subunit.

20. A method for inhibiting the activity of an interleukin comprising a p40 subunit, the method comprising contacting the interleukin with an antibody, or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of the interleukin in an amount effective to alter the conformational structure of the interleukin, thereby inhibiting the activity of the interleukin.
Description



RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/534,717, filed Mar. 24, 2000, which issued on Jul. 5, 2005 as U.S. Pat. No. 6,914,128. This application also claims priority to U.S. provisional application Ser. No. 60/126,603, filed Mar. 25, 1999. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Human interleukin 12 (IL-12) has recently been characterized as a cytokine with a unique structure and pleiotropic effects (Kobayashi, et al. (1989) J. Exp Med. 170:827-845; Seder, et al. (1993) Proc. Natl. Acad. Sci. 90:10188-10192; Ling, et al. (1995) J. Exp Med. 154:116-127; Podlaski, et al. (1992) Arch. Biochem. Biophys. 294:230-237). IL-12 plays a critical role in the pathology associated with several diseases involving immune and inflammatory responses. A review of IL-12, its biological activities, and its role in disease can be found in Gately et al. (1998) Ann. Rev. Immunol. 16: 495-521.

[0003] Structurally, IL-12 is a heterodimeric protein comprising a 35 kDa subunit (p35) and a 40 kDa subunit (p40) which are both linked together by a disulfide bridge (referred to as the "p70 subunit"). The heterodimeric protein is produced primarily by antigen-presenting cells such as monocytes, macrophages and dendritic cells. These cell types also secrete an excess of the p40 subunit relative to p70 subunit. The p40 and p35 subunits are genetically unrelated and neither has been reported to possess biological activity, although the p40 homodimer may function as an IL-12 antagonist.

[0004] Functionally, IL-12 plays a central role in regulating the balance between antigen specific T helper type (Th1) and type 2 (Th2) lymphocytes. The Th1 and Th2 cells govern the initiation and progression of autoimmune disorders, and IL-12 is critical in the regulation of Th1-lymphocyte differentiation and maturation. Cytokines released by the Th1 cells are inflammatory and include interferon .gamma. (IFN.gamma.), IL-2 and lymphotoxin (LT). Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13 to facilitate humoral immunity, allergic reactions, and immunosuppression.

[0005] Consistent with the preponderance of Th1 responses in autoimmune diseases and the proinflammatory activities of IFN.gamma., IL-12 may play a major role in the pathology associated with many autoimmune and inflammatory diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), and Crohn's disease.

[0006] Human patients with MS have demonstrated an increase in IL-12 expression as documented by p40 mRNA levels in acute MS plaques. (Windhagen et al., (1995) J. Exp. Med. 182: 1985-1996). In addition, ex vivo stimulation of antigen-presenting cells with CD40L-expressing T cells from MS patients resulted in increased IL-12 production compared with control T cells, consistent with the observation that CD40/CD40L interactions are potent inducers of IL-12.

[0007] Elevated levels of IL-12 p70 have been detected in the synovia of RA patients compared with healthy controls (Morita et al (1998) Arthritis and Rheumatism. 41: 306-314). Cytokine messenger ribonucleic acid (mRNA) expression profile in the RA synovia identified predominantly Th1 cytokines. (Bucht et al., (1996) Clin. Exp. Immunol. 103: 347-367). IL-12 also appears to play a critical role in the pathology associated with Crohn's disease (CD). Increased expression of INF.gamma. and IL-12 has been observed in the intestinal mucosa of patients with this disease (Fais et al. (1994) J. Interferon Res. 14:235-238; Parronchi et al., (1997) Am. J. Path. 150:823-832; Monteleone et al., (1997) Gastroenterology. 112:1169-1178, and Berrebi et al., (1998) Am. J. Path 152:667-672). The cytokine secretion profile of T cells from the lamina propria of CD patients is characteristic of a predominantly Th1 response, including greatly elevated IFN.gamma. levels (Fuss, et al., (1996) J. Immunol. 157:1261-1270). Moreover, colon tissue sections from CD patients show an abundance of IL-12 expressing macrophages and IFN.gamma. expressing T cells (Parronchi et al (1997) Am. J. Path. 150:823-832).

[0008] Due to the role of human IL-12 in a variety of human disorders, therapeutic strategies have been designed to inhibit or counteract IL-12 activity. In particular, antibodies that bind to, and neutralize, IL-12 have been sought as a means to inhibit IL-12 activity. Some of the earliest antibodies were murine monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with IL-12 (see e.g., World Patent Application Publication No. WO 97/15327 by Strober et al.; Neurath et al. (1995) J. Exp. Med. 182:1281-1290; Duchmann et al. (1996) J. Immunol. 26:934-938). These murine IL-12 antibodies are limited for their use in vivo due to problems associated with administration of mouse antibodies to humans, such as short serum half life, an inability to trigger certain human effector functions and elicitation of an unwanted immune response against the mouse antibody in a human (the "human anti-mouse antibody" (HAMA) reaction).

[0009] In general, attempts to overcome the problems associated with use of fully-murine antibodies in humans, have involved genetically engineering the antibodies to be more "human-like." For example, chimeric antibodies, in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived, have been prepared (Junghans, et al. (1990) Cancer Res. 50:1495-1502; Brown et al. (1991) Proc. Natl. Acad. Sci. 88:2663-2667; Kettleborough et al. (1991) Protein Engineering. 4:773-783). However, because these chimeric and humanized antibodies still retain some murine sequences, they still may elicit an unwanted immune reaction, the human anti-chimeric antibody (HACA) reaction, especially when administered for prolonged periods.

[0010] A preferred IL-12 inhibitory agent to murine antibodies or derivatives thereof (e.g., chimeric or humanized antibodies) would be an entirely human anti-IL-12 antibody, since such an agent should not elicit the HAMA reaction, even if used for prolonged periods. However, such antibodies have not been described in the art and, therefore are still needed.

SUMMARY OF THE INVENTION

[0011] The present invention provides human antibodies that bind human IL-12. The invention also relates to the treatment or prevention of acute or chronic diseases or conditions whose pathology involves IL-12, using the human anti-IL-12 antibodies of the invention.

[0012] In one aspect, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that binds to human IL-12.

[0013] In one embodiment, the invention provides a selectively mutated human IL-12 antibody, comprising:

[0014] a human antibody or antigen-binding portion thereof, selectively mutated at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue such that it binds to human IL-12.

[0015] In a preferred embodiment, the invention provides a selectively mutated human IL-12 antibody, comprising:

[0016] a human antibody or antigen-binding portion thereof, selectively mutated at a preferred selective mutagenesis position with an activity enhancing amino acid residue such that it binds to human IL-12.

[0017] In another preferred embodiment, the selectively mutated human IL-12 antibody or antigen-binding portion thereof is selectively mutated at more than one preferred selective mutagenesis position, contact or hypermutation positions with an activity enhancing amino acid residue. In another preferred embodiment, the selectively mutated human IL-12 antibody or antigen-binding portion thereof is selectively mutated at no more than three preferred selective mutagenesis positions, contact or hypermutation positions. In another preferred embodiment, the selectively mutated human IL-12 antibody or antigen-binding portion thereof is selectively mutated at no more than two preferred selective mutagenesis position, contact or hypermutation positions. In yet another preferred embodiment, the selectively mutated human IL-12 antibody or antigen-binding portion thereof, is selectively mutated such that a target specificity affinity level is attained, the target level being improved over that attainable when selecting for an antibody against the same antigen using phage display technology. In another preferred embodiment, the selectively mutated human IL-12 antibody further retains at least one desirable property or characteristic, e.g., preservation of non-cross reactivity with other proteins or human tissues, preservation of epitope recognition, production of an antibody with a close to a germline immunoglobulin sequence.

[0018] In another embodiment, the invention provides an isolated human antibody, or antigen-binding portion thereof, that binds to human IL-12 and dissociates from human IL-12 with a K.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6 M or less. More preferably, the isolated human antibody or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-2 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-7 M or less. More preferably, the isolated human antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-8 M or less. More preferably, the isolated human antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-4 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less. More preferably, the isolated human antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-5 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-10 M or less. Even more preferably, the isolated human antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-5 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-11 M or less.

[0019] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics:

[0020] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or less;

[0021] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 1; and

[0022] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 2.

[0023] In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and has a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5; and has a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, the isolated human antibody, or antigen binding portion thereof, has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and has a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0024] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics:

[0025] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0026] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and

[0027] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0028] In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11; and has a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13; and has a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14. In a preferred embodiment, the isolated human antibody has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15; and has a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

[0029] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which

[0030] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0031] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and

[0032] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

[0033] In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In a preferred embodiment, the isolated human antibody comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions or any allelic variation thereof as discussed in Kabat et al. (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), included herein by reference. In a more preferred embodiment, the antibody heavy chain constant region is IgG1. In another preferred embodiment, the isolated human antibody is a Fab fragment, or a F(ab').sub.2 fragment or a single chain Fv fragment.

[0034] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which

[0035] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0036] b) has a heavy chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 404-SEQ ID NO: 469; and

[0037] c) has a light chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 534-SEQ ID NO: 579.

[0038] In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:335-SEQ ID NO: 403; and a light chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 506-SEQ ID NO: 533. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 288-SEQ ID NO: 334; and a light chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 470-SEQ ID NO: 505. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, comprising a the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In a preferred embodiment, the isolated human antibody comprises a heavy chain constant region, or an Fab fragment or a F(ab').sub.2 fragment or a single chain Fv fragment as described above.

[0039] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which

[0040] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0041] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and

[0042] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0043] In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27; and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30. In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, which has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32. In a preferred embodiment, the isolated human antibody comprises a heavy chain constant region, or an Fab fragment, or a F(ab').sub.2 fragment or a single chain Fv fragment as described above.

[0044] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which

[0045] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or less;

[0046] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, or a mutant thereof having one or more amino acid substitutions at a contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5; and

[0047] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a mutant thereof having one or more amino acid substitutions at a contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6.

[0048] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which

[0049] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0050] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, or a mutant thereof having one or more amino acid substitutions at a contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13; and

[0051] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position, contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

[0052] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which

[0053] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0054] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position, contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21; and

[0055] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, or a mutant thereof having one or more amino acid substitutions at preferred selective mutagenesis position, contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22.

[0056] The invention also provides nucleic acid molecules encoding antibodies, or antigen binding portions thereof, of the invention. A preferred isolated nucleic acid encodes the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17. The isolated nucleic acid encoding an antibody heavy chain variable region. In another embodiment, the isolated nucleic acid encodes the CDR2 of the antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19. In another embodiment, the isolated nucleic acid encodes the CDR1 of the antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21. In another embodiment, the isolated nucleic acid encodes an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23. In another embodiment, the isolated nucleic acid encodes the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18. The isolated nucleic acid encoding an antibody light chain variable region. In another embodiment, the isolated nucleic acid encodes the CDR2 of the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In another embodiment, the isolated nucleic acid encodes the CDR1 of the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In another embodiment, the isolated nucleic acid encodes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 24.

[0057] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which

[0058] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0059] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position, contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29; and

[0060] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position, contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30.

[0061] A preferred isolated nucleic acid encodes the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25. The isolated nucleic acid encoding an antibody heavy chain variable region. In another embodiment, the isolated nucleic acid encodes the CDR2 of the antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27. In another embodiment, the isolated nucleic acid encodes the CDR1 of the antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29. In another embodiment, the isolated nucleic acid encodes an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31. In another embodiment, the isolated nucleic acid encodes the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26. The isolated nucleic acid encoding an antibody light chain variable region. In another embodiment, the isolated nucleic acid encodes the CDR2 of the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 28. In another embodiment, the isolated nucleic acid encodes the CDR1 of the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 30. In another embodiment, the isolated nucleic acid encodes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 32.

[0062] In another aspect, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0063] a) that binds to human IL-12 and dissociates from human IL-12 with a k.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0064] b) has a heavy chain variable region comprising an amino acid sequence selected from a member of the V.sub.H3 germline family, wherein the heavy chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue. [0065] c) has a light chain variable region comprising an amino acid sequence selected from a member of the V.sub..lamda.1 germline family, wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact position or hypermutation position with an activity enhancing amino acid residue.

[0066] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0067] a) that binds to human IL-12 and dissociates from human IL-12 with a k.sub.off rate constant of 0.1s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0068] b) has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 595-667, wherein the heavy chain variable region has a mutation at a preferred selective mutagenesis position, contact position or hypermutation position with an activity enhancing amino acid residue. [0069] c) has a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 669-675, wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue.

[0070] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0071] a) that binds to human IL-12 and dissociates from human IL-12 with a k.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0072] b) has a heavy chain variable region comprising the COS-3 germline amino acid sequence, wherein the heavy chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue. [0073] c) has a light chain variable region comprising the DPL8 germline amino acid sequence, wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue.

[0074] In another embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0075] a) that binds to human IL-12 and dissociates from human IL-12 with a k.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0076] b) has a heavy chain variable region comprising an amino acid sequence selected from a member of the V.sub.H3 germline family, wherein the heavy chain variable region comprises a CDR2 that is structurally similar to CDR2s from other V.sub.H3 germline family members, and a CDR1 that is structurally similar to CDR1s from other V.sub.H3 germline family members, and wherein the heavy chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue; [0077] c) has a light chain variable region comprising an amino acid sequence selected from a member of the V.sub..lamda.1 germline family, wherein the light chain variable region comprises a CDR2 that is structurally similar to CDR2s from other V.sub..lamda.1 germline family members, and a CDR1 that is structurally similar to CDR1s from other V.sub..lamda.1 germline family members, and wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue.

[0078] In a preferred embodiment, the isolated human antibody, or antigen binding portion thereof, has a mutation in the heavy chain CDR3. In another preferred embodiment, the isolated human antibody, or antigen binding portion thereof, has a mutation in the light chain CDR3. In another embodiment, the isolated human antibody, or antigen binding portion thereof, has a mutation in the heavy chain CDR2. In another preferred embodiment, the isolated human antibody, or antigen binding portion thereof, has a mutation in the light chain CDR2. In another preferred embodiment, the isolated human antibody, or antigen binding portion thereof, has a mutation in the heavy chain CDR1. In another preferred embodiment, the isolated human antibody, or antigen binding portion thereof, has a mutation in the light chain CDR1.

[0079] In another aspect, the invention provides recombinant expression vectors carrying the antibody-encoding nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing the host cells of the invention.

[0080] In another aspect, the invention provides an isolated human antibody, or antigen-binding portion thereof, that neutralizes the activity of human IL-12, and at least one additional primate IL-12 selected from the group consisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12, cynomolgus IL-12 and rhesus IL-12, but which does not neutralize the activity of the mouse IL-12.

[0081] In another aspect, the invention provides a pharmaceutical composition comprising the antibody or an antigen binding portion thereof, of the invention and a pharmaceutically acceptable carrier.

[0082] In another aspect, the invention provides a composition comprising the antibody or an antigen binding portion thereof, and an additional agent, for example, a therapeutic agent.

[0083] In another aspect, the invention provides a method for inhibiting human IL-12 activity comprising contacting human IL-12 with the antibody of the invention, e.g., J695, such that human IL-12 activity is inhibited.

[0084] In another aspect, the invention provides a method for inhibiting human IL-12 activity in a human subject suffering from a disorder in which IL-12 activity is detrimental, comprising administering to the human subject the antibody of the invention, e.g., J695, such that human IL-12 activity in the human subject is inhibited. The disorder can be, for example, Crohn's disease, multiple sclerosis or rheumatoid arthritis.

[0085] In another aspect, the invention features a method for improving the activity of an antibody, or an antigen binding portion thereof, to attain a predetermined target activity, comprising:

[0086] a) providing a parent antibody a antigen-binding portion thereof;

[0087] b) selecting a preferred selective mutagenesis position selected from group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94.

[0088] c) individually mutating the selected preferred selective mutagenesis position to at least two other amino acid residues to hereby create a first panel of mutated antibodies, or antigen binding portions thereof;

[0089] d) evaluating the activity of the first panel of mutated antibodies, or antigen binding portions thereof to determined if mutation of a single selective mutagenesis position produces an antibody or antigen binding portion thereof with the predetermined target activity or a partial target activity;

[0090] e) combining in a stepwise fashion, in the parent antibody, or antigen binding portion thereof, individual mutations shown to have an improved activity, to form combination antibodies, or antigen binding portions thereof.

[0091] f) evaluating the activity of the combination antibodies, or antigen binding portions thereof to determined if the combination antibodies, or antigen binding portions thereof have the predetermined target activity or a partial target activity.

[0092] g) if steps d) or f) do not result in an antibody or antigen binding portion thereof having the predetermined target activity, or result an antibody with only a partial activity, additional amino acid residues selected from the group consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 are mutated to at least two other amino acid residues to thereby create a second panel of mutated antibodies or antigen-binding portions thereof;

[0093] h) evaluating the activity of the second panel of mutated antibodies or antigen binding portions thereof, to determined if mutation of a single amino acid residue selected from the group consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 results an antibody or antigen binding portion thereof, having the predetermined target activity or a partial activity;

[0094] i) combining in stepwise fashion in the parent antibody, or antigen-binding portion thereof, individual mutations of step g) shown to have an improved activity, to form combination antibodies, or antigen binding portions thereof;

[0095] j) evaluating the activity of the combination antibodies or antigen binding portions thereof, to determined if the combination antibodies, or antigen binding portions thereof have the predetermined target activity or a partial target activity;

[0096] k) if steps h) or j) do not result in an antibody or antigen binding portion thereof having the predetermined target activity, or result in an antibody with only a partial activity, additional amino acid residues selected from the group consisting of H33B, H52B and L31A are mutated to at least two other amino acid residues to thereby create a third panel of mutated antibodies or antigen binding portions thereof;

[0097] l) evaluating the activity of the third panel of mutated antibodies or antigen binding portions thereof, to determine if a mutation of a single amino acid residue selected from the group consisting of H33B, H52B and L31A resulted in an antibody or antigen binding portion thereof, having the predetermined target activity or a partial activity;

[0098] m) combining in a stepwise fashion in the parent antibody, or antigen binding portion thereof, individual mutation of step k) shown to have an improved activity, to form combination antibodies, or antigen binding portions, thereof;

[0099] n) evaluating the activity of the combination antibodies or antigen-binding portions thereof, to determine if the combination antibodies, or antigen binding portions thereof have the predetermined target activity to thereby produce an antibody or antigen binding portion thereof with a predetermined target activity.

[0100] In another aspect, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0101] a) providing a parent antibody or antigen-binding portion thereof;

[0102] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected preferred selective mutagenesis position, contact or hypermutation position;

[0103] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0104] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

[0105] e) repeating steps b) through d) for at least one other contact or hypermutation position;

[0106] f) combining, in the parent antibody, or antigen-binding portion thereof, individual mutations shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0107] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0108] In one embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0109] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity is not further improved by mutagenesis in said phage-display system;

[0110] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected contact or hypermutation position;

[0111] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0112] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

[0113] e) repeating steps b) through d) for at least one other contact or hypermutation position;

[0114] f) combining, in the parent antibody, or antigen-binding portion thereof, individual mutations shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0115] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof; until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0116] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96. In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93. In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94. In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94.

[0117] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0118] a) providing a recombinant parent antibody or antigen-binding portion thereof;

[0119] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected contact or hypermutation position;

[0120] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expressing said panel in an appropriate expression system;

[0121] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0122] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristics, wherein the property or characteristic is one that needs to be retained in the antibody;

until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0123] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0124] In another embodiment of the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0125] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0126] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected preferred selective mutagenesis position, contact or hypermutation position;

[0127] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0128] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0129] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristic, wherein the property or characteristic is one that needs to be retained, until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0130] f) repeating steps a) through e) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0131] g) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and at least on retained property or characteristic, to form combination antibodies, or antigen-binding portions thereof; and

[0132] h) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0133] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0134] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0135] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0136] b) selecting a contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected contact or hypermutation position;

[0137] c) individually mutating said selected contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0138] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0139] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristics, wherein the property or characteristic is one that needs to be retained;

until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0140] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0141] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0142] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0143] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected preferred selective mutagenesis position contact or hypermutation position;

[0144] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0145] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0146] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristic, wherein the property or characteristic is one that needs to be retained, until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0147] f) repeating steps a) through e) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0148] g) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and at least on retained other characteristic, to form combination antibodies, or antigen-binding portions thereof; and

[0149] h) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0150] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence. In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0151] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0152] a) providing a parent antibody or antigen-binding portion thereof;

[0153] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0154] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0155] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0156] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other property or characteristic;

until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0157] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence

[0158] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0159] a) providing a parent antibody or antigen-binding portion thereof;

[0160] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0161] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0162] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0163] e) repeating steps b) through d) for at least one other CDR position which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0164] f) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0165] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0166] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0167] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0168] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and;

[0169] c) individually mutating said selected contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0170] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0171] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other property or characteristic until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0172] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0173] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0174] a) providing a parent antibody or antigen-binding portion thereof that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0175] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0176] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expression in a non-phage display system;

[0177] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0178] e) repeating steps b) through d) for at least one other position within the CDR which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94;

[0179] f) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0180] g) evaluating the activity and other property or characteristic of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof; until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0181] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0182] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0183] a) providing a parent antibody or antigen-binding portion thereof;

[0184] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0185] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0186] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0187] e) evaluating the panel of mutated antibodies or antigen-binding portions thereof, relative to the parent antibody or antigen-portion thereof, for changes in at least one other property or characteristic;

[0188] f) repeating steps b) through e) for at least one other CDR position which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0189] g) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and not affecting at least one other property or characteristic, to form combination antibodies, or antigen-binding portions thereof; and

[0190] h) evaluating the activity and the retention of at least one other characteristic or property of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0191] In another embodiment the invention provides a method to improve the affinity of an antibody or antigen-binding portion thereof, comprising:

[0192] a) providing a parent antibody or antigen-binding portion thereof that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0193] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0194] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expression in a non-phage display system;

[0195] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0196] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other characteristic or property until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0197] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0198] a) providing a parent antibody or antigen-binding portion thereof;

[0199] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation at a position other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0200] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0201] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0202] e) evaluating the panel of mutated antibodies or antigen-binding portions thereof, relative to the parent antibody or antigen-portion thereof, for changes in at least one other property or characteristic;

[0203] f) repeating steps b) through e) for at least one other CDR position which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0204] g) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity but not affecting at least one other property or characteristic, to form combination antibodies, or antigen-binding portions thereof with at least one retained property or characteristic; and

[0205] h) evaluating the activity and the retention of at least one property of characteristic of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0206] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence

[0207] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, without affecting other characteristics, comprising:

[0208] a) providing a parent antibody or antigen-binding portion thereof;

[0209] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0210] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0211] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0212] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other property or characteristic until an antibody, or antigen-binding portion thereof, with an improved activity and retained other characteristic or property, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0213] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0214] a) providing a parent antibody or antigen-binding portion thereof that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0215] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0216] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expression in a non-phage display system;

[0217] d) evaluating the activity and retention of at least one other characteristic or property of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0218] e) repeating steps b) through d) for at least one other CDR position which is neither the position selected under b nor other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0219] f) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and not to affect at least one other characteristic or property, to form combination antibodies, or antigen-binding portions thereof; and

[0220] g) evaluating the activity and retention of at least one other characteristic or property of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof until an antibody, or antigen-binding portion thereof, with an improved activity and at least one other retained characteristic or property, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0221] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0222] In yet another aspect, the invention provides an isolated antibody (e.g., a human antibody) or antigen binding portion thereof that is capable of binding to the p40 subunit of IL-12 (e.g., the p40 subunit of human IL-12) and is capable of altering the conformational structure of said p40 subunit of IL-12.

[0223] In a related aspect, the invention provides an isolated antibody (e.g., a human antibody) or antigen binding portion thereof that is capable of binding to the p40 subunit of IL-12 (e.g., the p40 subunit of human IL-12) and is capable of altering the conformational structure of an interleukin, e.g., the p40 subunit of an interleukin. In one embodiment, the interleukin is IL-12. In another embodiment, the interleukin comprises a p40 subunit and a p19 subunit, e.g., the interleukin is IL-23. In yet another embodiment, the interleukin is a human interleukin such as human IL-12 or human IL-23.

[0224] In one embodiment, the antibody is Y61 or J695. In another embodiment, the antibody is not Y61 or J695.

[0225] In another embodiment, the isolated antibody, or antigen binding portion thereof, alters the conformational structure of the interleukin, e.g., IL-12 or IL-23, such that binding by the interleukin, e.g., IL-12 or IL-23, to an interleukin-interacting molecule is modulated.

[0226] In yet another embodiment, the interleukin-interacting molecule is an interleukin receptor, e.g., IL-12 or IL-23 receptor.

[0227] In a further embodiment, the isolated antibody, or antigen binding portion thereof, alters the conformational structure of an interleukin, e.g., IL-12 or IL-23, such that binding to the interleukin, e.g., IL-12 or IL-23, by a second antibody is inhibited.

[0228] In one embodiment, the interleukin is IL-12 and the second antibody binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of 1D4.7, C8.6.2 and C340 binds.

[0229] In another embodiment, the interleukin is IL-12 and the second antibody is selected from the group consisting of 1D4.7, C8.6.2 and C340.

[0230] In another aspect, the invention provides an isolated antibody, e.g., a human antibody, or antigen binding portion thereof, that binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of 1D4.7, C8.6.2, C340 and 7G3 does not bind.

[0231] In one embodiment, the antibody is Y61 or J695. In another embodiment, the antibody is not Y61 or J695.

[0232] In another embodiment, the isolated antibody, or antigen binding portion thereof, binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of Y61 and J695 binds.

[0233] In one embodiment, the antibody is not Y61 or J695.

[0234] In a further embodiment, the isolated antibody, or antigen binding portion thereof, inhibits the binding to IL-12 by a second antibody.

[0235] In yet a further embodiment, the second antibody binds to an epitope of IL-12 to which an antibody selected from the group consisting of 1D4.7, C8.6.2, C340 binds.

[0236] In one embodiment, the second antibody is selected from the group consisting of 1D4.7, C8.6.2 and C340.

[0237] In one embodiment, the isolated antibody, or antigen binding portion thereof, dissociates from the p40 subunit of human IL-12 with a K.sub.d of 1.times.10.sup.-10 M or less or a k.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by surface plasmon resonance.

[0238] In another embodiment, the isolated antibody, or antigen binding portion thereof, is a neutralizing antibody

[0239] In a further embodiment, the isolated antibody, or antigen binding portion thereof, inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less, or which inhibits human IFN.gamma. production with an IC.sub.50 of 1.times.10.sup.-10 M or less.

[0240] In one embodiment, the isolated antibody, or antigen binding portion thereof, is a human antibody.

[0241] In another aspect, the invention provides a pharmaceutical composition comprising an antibody of the invention, or an antigen binding portion thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise an additional therapeutic agent, e.g., a therapeutic agent selected from the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-1.beta. monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1.beta. converting enzyme inhibitors, TNF.alpha. converting enzyme inhibitors, T-cell signalling inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10, IL-11, IL-13 and TGF.beta..

[0242] In another embodiment, the therapeutic agent is selected from the group consisting of anti-TNF antibodies and antibody fragments thereof, TNFR-Ig constructs, TACE inhibitors, PDE4 inhibitors, corticosteroids, budenoside, dexamethasone, sulfasalazine, 5-aminosalicylic acid, olsalazine, IL-1.beta. converting enzyme inhibitors, IL-1ra, tyrosine kinase inhibitors, 6-mercaptopurines and IL-11.

[0243] In yet another embodiment, the therapeutic agent is selected from the group consisting of corticosteroids, prednisolone, methylprednisolone, azathioprine, cyclophosphamide, cyclosporine, methotrexate, 4-aminopyridine, tizanidine, interferon-.beta.1a, interferon-.beta.1b, Copolymer 1, hyperbaric oxygen, intravenous immunoglobulin, clabribine, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, PDGF, antibodies to CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands, methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38 or MAP kinase inhibitors, IL-1.beta. converting enzyme inhibitors, TACE inhibitors, T-cell signalling inhibitors, kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, sIL-13R, anti-P7s, p-selectin glycoprotein ligand (PSGL), antiinflammatory cytokines, IL-4, IL-10, IL-13 and TGF.beta..

[0244] In another aspect, the invention provides a composition comprising an antibody (e.g., a human antibody) or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, in an amount effective for altering the conformational structure of the interleukin. In one embodiment, the interleukin is IL-12. In another embodiment, the interleukin comprises a p40 subunit and a p19 subunit, e.g., the interleukin is IL-23.

[0245] In one embodiment, the amount of the antibody effective to alter the conformational structure of the p40 subunit of an interleukin, e.g., IL-12, is between about 0.1 and 2500 .mu.g/ml, preferably between about 1.0 and 250 .mu.g/ml, more preferably between about 2.0 and 125 .mu.g/ml, more preferably between about 4.0 and 65 .mu.g/ml, even more preferably between about 6.0 and 50 .mu.g/ml, preferably between about 8.0 and 40 .mu.g/ml, and most preferably between about 10 and 25 .mu.g/ml. In various embodiments, the amount of the antibody effective to alter the conformational structure of the p40 subunit of an interleukin, e.g., IL-12, is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60 .mu.g/ml or more. Ranges intermediate to the above recited amounts, e.g., between about 5.0 and 55 .mu.g/ml, between about 9.0 and 35 .mu.g/ml, and between about 15 and 20 .mu.g/ml, are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. Further, discrete amounts intermediate to any of the above recited amounts, e.g., 10.5, 11.5, and 12.5 .mu.g/ml, are also intended to be part of this invention.

[0246] In another aspect, the invention provides a composition comprising an antibody (e.g., a human antibody) or antigen binding portion thereof, that binds to the p40 subunit of IL-12 and is capable of altering the conformational structure of said p40 subunit packaged or promoted with instructions for use in altering the conformational structure of said p40 subunit.

[0247] In a related aspect, the invention provides a composition comprising an antibody (e.g., a human antibody) or antigen binding portion thereof, that binds to the p40 subunit of IL-12 and is capable of altering the conformational structure of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, packaged or promoted with instructions for use in altering the conformational structure of the interleukin, e.g., IL-12 or IL-23.

[0248] In one embodiment, the composition further comprises a means for determining whether the conformational structure of the interleukin, e.g., IL-12 or IL-23, has been altered.

[0249] In one embodiment, the interleukin is IL-12 and the antibody, or antigen binding portion thereof, binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of 1D4.7, C8.6.2, C340 and 7G3 does not bind.

[0250] In another embodiment, the antibody, or antigen binding portion thereof, binds an epitope to which an antibody selected from the group consisting of Y61 and J695 binds.

[0251] In yet another embodiment, the antibody, or antigen binding portion thereof, is not Y61 or J695.

[0252] In a further embodiment, the antibody, or antigen binding portion thereof, dissociates from the p40 subunit of human IL-12 with a K.sub.d of 1.times.10-1M or less or a k.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by surface plasmon resonance.

[0253] In one embodiment, the antibody, or antigen binding portion thereof, is a neutralizing antibody.

[0254] In one embodiment, the antibody, or antigen binding portion thereof, is a human antibody.

[0255] In yet another aspect, the invention provides a composition comprising a plurality of antibodies, or antigen binding portions thereof, wherein each antibody of the plurality of antibodies binds to a different epitope of the p40 subunit of IL-12.

[0256] In one embodiment, at least one antibody, or antigen binding portion thereof, binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of Y61 and J695 binds.

[0257] In another embodiment, at least one antibody, or antigen binding portion thereof, binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of 1D4.7, C8.6.2, C340 and 7G3 does not bind.

[0258] In one embodiment, at least one antibody, or antigen binding portion thereof, dissociates from the p40 subunit of human IL-12 with a K.sub.d of 1.times.10.sup.-10 M or less or a k.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by surface plasmon resonance.

[0259] In another embodiment, at least one antibody, or antigen binding portion thereof, is a neutralizing antibody.

[0260] In yet another embodiment, at least one antibody, or antigen binding portion thereof, inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less, or which inhibits human IFN.gamma. production with an IC.sub.50 of 1.times.10.sup.-10 M or less.

[0261] In a further embodiment, at least one antibody, or antigen binding portion thereof, is a human antibody.

[0262] In one embodiment, at least one antibody, or antigen binding portion thereof, has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25 and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0263] In another embodiment, the at least one antibody, or antigen binding portion thereof, further has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28.

[0264] In yet another embodiment, the at least one antibody, or antigen binding portion thereof, further has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29 and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30.

[0265] In one aspect, the invention provides a method for detecting the p40 subunit of an interleukin, e.g., IL-12 or IL-23, comprising contacting the p40 subunit of IL-12 with an antibody of the invention, or antigen-binding portion thereof, such that the p40 subunit is detected.

[0266] In a related aspect, the invention provides a method for detecting the p40 subunit of an interleukin, e.g., IL-12 or IL-23, comprising contacting the p40 subunit of the interleukin, e.g., IL-12 or IL-23, with an antibody of the invention, or antigen-binding portion thereof, such that the p40 subunit of the interleukin, e.g., IL-12 or IL-23, is detected.

[0267] In one embodiment, the p40 subunit of the interleukin, e.g., IL-12 or IL-23, is detected in vitro. In another embodiment, the p40 subunit of the interleukin, e.g., IL-12 or IL-23, is detected in a biological sample for diagnostic purposes.

[0268] In another aspect, the invention provides a method for inhibiting an activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, comprising contacting the interleukin, e.g., IL-12 or IL-23, with an antibody, or antigen-binding portion thereof, of the invention such that the activity is inhibited.

[0269] In a related aspect, the invention provides a method for inhibiting an activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, in a human subject suffering from a disorder in which the activity is detrimental, comprising administering to the human subject an antibody, or antigen-binding portion thereof, of the invention such that the activity of the interleukin, e.g., IL-12 or IL-23, in the human subject is inhibited.

[0270] In one embodiment, the interleukin is IL-12. In another embodiment, the interleukin comprises a p40 subunit and a p19 subunit, e.g., the interleukin is IL-23.

[0271] In one embodiment, the disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyoarthropathy, ankylosing spondylitis, systemic lupus erythematosis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, multiple sclerosis, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, thyroiditis, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, polyarteritis nodosa, Wegener's granulomatosis, Henoch-Schonlein purpura, microscopic vasculitis of the kidneys, chronic active hepatitis, Sjogren's syndrome, uveitis, sepsis, septic shock, sepsis syndrome, adult respiratory distress syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, myasthenia gravis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, fibrotic lung diseases, hemolytic anemia, malignancies, heart failure and myocardial infarction.

[0272] In one embodiment, the disorder is rheumatoid arthritis.

[0273] In one embodiment, the disorder is Crohn's disease.

[0274] In one embodiment, the disorder is multiple sclerosis.

[0275] In one embodiment, the disorder is psoriasis.

[0276] In yet another aspect, the invention provides a method for altering the conformational structure of the p40 subunit of IL-12, the method comprising contacting said subunit with an antibody, or antigen binding portion thereof, that is capable of binding said subunit and is capable of altering the conformational structure of said subunit, in an amount effective to alter the conformational structure of said subunit, thereby altering the conformational structure of said subunit.

[0277] In a related aspect, the invention provides a method for altering the conformational structure of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, the method comprising contacting the interleukin, e.g., IL-12 or IL-23, with an antibody, or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of the interleukin, e.g., IL-12 or IL-23, in an amount effective to alter the conformational structure of the interleukin, e.g., IL-12 or IL-23, thereby altering the conformational structure of the interleukin, e.g., IL-12 or IL-23.

[0278] In another aspect, the invention provides a method for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, the method comprising contacting the interleukin, e.g., IL-12 or IL-23, with an antibody, or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of the said subunit, in an amount effective to alter the conformational structure of said subunit, thereby inhibiting the activity of the interleukin, e.g., IL-12 or IL-23.

[0279] In a related aspect, the invention provides a method for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, the method comprising contacting the interleukin, e.g., IL-12 or IL-23, with an antibody, or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of the interleukin, e.g., IL-12 or IL-23, in an amount effective to alter the conformational structure of the interleukin, e.g., IL-12 or IL-23, thereby inhibiting the activity of the interleukin, e.g., IL-12 or IL-23.

[0280] In another aspect, the invention provides a method for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, in a human subject suffering from a disorder in which the activity of the interleukin, e.g., IL-12 or IL-23, is detrimental, the method comprising administering to the human subject an antibody, or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of said subunit, in an amount effective to alter the conformational structure of said subunit, thereby inhibiting the activity of the interleukin, e.g., IL-12 or IL-23, in the human subject.

[0281] In a related aspect, the invention provides a method for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, in a human subject suffering from a disorder in which the activity of the interleukin, e.g., IL-12 or IL-23, is detrimental, the method comprising administering to the human subject an antibody, or antigen binding portion thereof, that is capable of binding to the p40 subunit of IL-12 and is capable of altering the conformational structure of the interleukin, e.g., IL-12 or IL-23, in an amount effective to alter the conformational structure of the interleukin, e.g., IL-12 or IL-23, thereby inhibiting the activity of the interleukin, e.g., IL-12 or IL-23, in the human subject.

[0282] In one embodiment, the interleukin is IL-12. In another embodiment, the interleukin comprises a p40 subunit and a p19 subunit, e.g., the interleukin is IL-23.

[0283] In one embodiment, the disorder is selected from the group consisting of rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyoarthropathy, ankylosing spondylitis, systemic lupus erythematosis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, multiple sclerosis, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, thyroiditis, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, polyarteritis nodosa, Wegener's granulomatosis, Henoch-Schonlein purpura, microscopic vasculitis of the kidneys, chronic active hepatitis, Sjogren's syndrome, uveitis, sepsis, septic shock, sepsis syndrome, adult respiratory distress syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, myasthenia gravis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, fibrotic lung diseases, hemolytic anemia, malignancies, heart failure and myocardial infarction.

[0284] In one embodiment, the disorder is rheumatoid arthritis.

[0285] In one embodiment, the disorder is Crohn's disease.

[0286] In one embodiment, the disorder is multiple sclerosis.

[0287] In one embodiment, the disorder is psoriasis.

[0288] In one embodiment, the antibody is not Y61 or J695.

[0289] In another embodiment, the amount of the antibody effective to alter the conformational structure of the p40 subunit of an interleukin, e.g., IL-12, is between about 0.1 and 2500 .mu.g/ml, preferably between about 1.0 and 250 .mu.g/ml, more preferably between about 2.0 and 125 .mu.g/ml, more preferably between about 4.0 and 65 .mu.g/ml, even more preferably between about 6.0 and 50 .mu.g/ml, preferably between about 8.0 and 40 .mu.g/ml, and most preferably between about 10 and 25 .mu.g/ml. In various embodiments, the amount of the antibody effective to alter the conformational structure of the p40 subunit of an interleukin, e.g., IL-12, is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60 .mu.g/ml or more. Ranges intermediate to the above recited amounts, e.g., between about 5.0 and 55 .mu.g/ml, between about 9.0 and 35 .mu.g/ml, and between about 15 and 20 .mu.g/ml, are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. Further, discrete amounts intermediate to any of the above recited amounts, e.g., 10.5, 11.5, and 12.5 .mu.g/ml, are also intended to be part of this invention.

[0290] In one embodiment, the p40 subunit of an interleukin, e.g., IL-12 is contacted with the antibody, or antigen binding portion thereof, for a period of approximately 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2 hours, 5 hours or longer. In a preferred embodiment, the p40 subunit of an interleukin is contacted with the antibody, or antigen binding portion thereof, for a period of between 5 and 10 minutes, e.g., 5, 6, 7, 8, 9 or 10 minutes. Times intermediate to the above recited times, e.g., 2.5 minutes, 3.5 minutes, 4.5 minutes, 5.5 minutes, are also intended to be part of this invention.

[0291] In another aspect, the invention provides a method for identifying an antibody, or antigen binding portion thereof, suitable for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, the method comprising contacting the p40 subunit of an interleukin, e.g., IL-12 or IL-23, or a portion thereof, with an antibody, or antigen binding portion thereof, and determining whether the antibody, or antigen binding portion thereof, alters the conformational structure of said p40 subunit, thereby identifying an antibody, or antigen binding portion thereof, suitable for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23.

[0292] In a related aspect, the invention provides a method for identifying an antibody, or antigen binding portion thereof, suitable for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23, the method comprising contacting the p40 subunit of an interleukin, e.g., IL-12 or IL-23, or a portion thereof, with an antibody, or antigen binding portion thereof, and determining whether the antibody, or antigen binding portion thereof, alters the conformational structure of the interleukin, e.g., IL-12 or IL-23, thereby identifying an antibody, or antigen binding portion thereof, suitable for inhibiting the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23.

[0293] In one embodiment, the antibody, or antigen binding portion thereof, is suitable for inhibiting the activity of the interleukin, e.g., IL-12 or IL-23, in a human subject suffering from a disorder in which the activity of the interleukin, e.g., IL-12 or IL-23; is detrimental.

[0294] In another embodiment, the method further comprises determining whether binding by the interleukin, e.g., IL-12 or IL-23, to an interleukin-interacting molecule is modulated.

[0295] In one embodiment, the molecule that interacts with the interleukin, e.g., IL-12 or IL-23, is an interleukin receptor, e.g., IL-12 receptor or IL-23 receptor.

[0296] In one embodiment, the method further comprises determining whether binding to the interleukin, e.g., IL-12 or IL-23, by a second antibody is inhibited.

[0297] In one embodiment, the interleukin is IL-12 and the second antibody binds to an epitope of the p40 subunit of IL-12 to which an antibody selected from the group consisting of 1D4.7, C8.6.2 and C340 binds.

[0298] In one embodiment, the second antibody is selected from the group consisting of 1D4.7, C8.6.2 and C340.

BRIEF DESCRIPTION OF THE DRAWINGS

[0299] FIGS. 1A-1B show the heavy chain variable region amino acid sequence alignments of a series of human antibodies that bind human IL-12 compared to germline sequences Cos-3/JH3 and Dpl18 Lv1042. Kabat numbering is used to identify amino acid positions. For the Joe 9 wild type, the full sequence is shown. For the other antibodies, only those amino acids positions that differ from Joe 9 wild type are shown.

[0300] FIGS. 1C-1D show the light chain variable region amino acid sequence alignments of a series of human antibodies that bind human IL-12. Kabat numbering is used to identify amino acid positions. For the Joe 9 wild type, the full sequence is shown. For the other antibodies, only those amino acids positions that differ from Joe 9 wild type are shown.

[0301] FIGS. 2A-2E show the CDR positions in the heavy chain of the Y61 antibody that were mutated by site-directed mutagenesis and the respective amino acid substitutions at each position. The graphs at the right of the figures show the off-rates for the substituted antibodies (black bars) as compared to unmutated Y61 (open bar).

[0302] FIGS. 2F-2H show the CDR positions in the light chain of the Y61 antibody that were mutated by site-directed mutagenesis and the respective amino acid substitutions at each position. The graphs at the right of the figures show the off-rates for the substituted antibodies (black bars) as compared to unmutated Y61 (open bar).

[0303] FIG. 3 demonstrates the in vivo efficacy of the human anti-IL-12 antibody J695, on plasma neopterin levels in cynomolgus monkeys.

[0304] FIG. 4 shows a graph of mean arthritic score versus days after immunization of mice with collagen, demonstrating that treatment with C17.15 significantly decreases arthritis-related symptoms as compared to treatment with rat IgG.

DETAILED DESCRIPTION OF THE INVENTION

[0305] In order that the present invention may be more readily understood, certain terms are first defined.

[0306] The term "activity enhancing amino acid residue" includes an amino acid residue which improves the activity of the antibody. It should be understood that the activity enhancing amino acid residue may replace an amino acid residue at a contact, hypermutation or preferred selective mutagenesis position and, further, more than one activity enhancing amino acid residue can be present within one or more CDRs. An activity enhancing amino acid residue include, an amino acid residue that improves the binding specificity/affinity of an antibody, for example anti-human IL-12 antibody binding to human IL-12. The activity enhancing amino acid residue is also intended to include an amino acid residue that improves the neutralization potency of an antibody, for example, the human IL-12 antibody which inhibits human IL-12.

[0307] The term "antibody" includes an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0308] The term "antigen-binding portion" of an antibody (or "antibody portion") includes fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hIL-12). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab').sub.2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein. Preferred antigen binding portions are complete domains or pairs of complete domains.

[0309] The term "backmutation" refers to a process in which some or all of the somatically mutated amino acids of a human antibody are replaced with the corresponding germline residues from a homologous germline antibody sequence. The heavy and light chain sequences of the human antibody of the invention are aligned separately with the germline sequences in the VBASE database to identify the sequences with the highest homology. Differences in the human antibody of the invention are returned to the germline sequence by mutating defined nucleotide positions encoding such different amino acid. The role of each amino acid thus identified as candidate for backmutation should be investigated for a direct or indirect role in antigen binding and any amino acid found after mutation to affect any desirable characteristic of the human antibody should not be included in the final human antibody; as an example, activity enhancing amino acids identified by the selective mutagenesis approach will not be subject to backmutation. To minimize the number of amino acids subject to backmutation those amino acid positions found to be different from the closest germline sequence but identical to the corresponding amino acid in a second germline sequence can remain, provided that the second germline sequence is identical and colinear to the sequence of the human antibody of the invention for at least 10, preferably 12 amino acids, on both sides of the amino acid in question. Backmutation may occur at any stage of antibody optimization; preferably, backmutation occurs directly before or after the selective mutagenesis approach. More preferably, backmutation occurs directly before the selective mutagenesis approach.

[0310] The phrase "human interleukin 12" (abbreviated herein as hIL-12, or IL-12), as used herein, includes a human cytokine that is secreted primarily by macrophages and dendritic cells. The term includes a heterodimeric protein comprising a 35 kD subunit (p35) and a 40 kD subunit (p40) which are both linked together with a disulfide bridge. The heterodimeric protein is referred to as a "p70 subunit". The structure of human IL-12 is described further in, for example, Kobayashi, et al. (1989) J. Exp Med. 170:827-845; Seder, et al. (1993) Proc. Natl. Acad. Sci. 90:10188-10192; Ling, et al. (1995) J. Exp Med. 154:116-127; Podlaski, et al. (1992) Arch. Biochem. Biophys. 294:230-237. The term human IL-12 is intended to include recombinant human IL-12 (rh IL-12), which can be prepared by standard recombinant expression methods.

[0311] The phrase "interleukin comprising a p40 subunit" includes any interleukin, e.g., any human interleukin, that comprises a p40 subunit. Such interleukins are well known in the art and include IL-12, e.g., human IL-12, and IL-23, e.g., human IL-23.

[0312] The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[0313] The Kabat numbering is used herein to indicate the positions of amino acid modifications made in antibodies of the invention. For example, the Y61 anti-IL-12 antibody can be mutated from serine (S) to glutamic acid (E) at position 31 of the heavy chain CDR1 (H31S.fwdarw.E), or glycine (G) can be mutated to tyrosine (Y) at position 94 of the light chain CDR3 (L94G.fwdarw.Y).

[0314] The term "human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. The mutations preferably are introduced using the "selective mutagenesis approach" described herein. The human antibody can have at least one position replaced with an amino acid residue, e.g., an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence. The human antibody can have up to twenty positions replaced with amino acid residues which are not part of the human germline immunoglobulin sequence. In other embodiments, up to ten, up to five, up to three or up to two positions are replaced. In a preferred embodiment, these replacements are within the CDR regions as described in detail below. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0315] The phrase "recombinant human antibody" includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II, below), antibodies isolated from a recombinant, combinatorial human antibody library (described further in Section III, below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. In certain embodiments, however, such recombinant antibodies are the result of selective mutagenesis approach or backmutation or both.

[0316] An "isolated antibody" includes an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hIL-12 is substantially free of antibodies that specifically bind antigens other than hIL-12). An isolated antibody that specifically binds hIL-12 may bind IL-12 molecules from other species (discussed in further detail below). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[0317] A "neutralizing antibody" (or an "antibody that neutralized hIL-12 activity") includes an antibody whose binding to hIL-12 results in inhibition of the biological activity of hIL-12. This inhibition of the biological activity of hIL-12 can be assessed by measuring one or more indicators of hIL-12 biological activity, such as inhibition of human phytohemagglutinin blast proliferation in a phytohemagglutinin blast proliferation assay (PHA), or inhibition of receptor binding in a human IL-12 receptor binding assay (see Example 3-Interferon-gamma Induction Assay). These indicators of hIL-12 biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see Example 3).

[0318] The term "activity" includes activities such as the binding specificity/affinity of an antibody for an antigen, for example, an anti-hIL-12 antibody that binds to an IL-12 antigen and/or the neutralizing potency of an antibody, for example, an anti-hIL-12 antibody whose binding to hIL-12 inhibits the biological activity of hIL-12, e.g. inhibition of PHA blast proliferation or inhibition of receptor binding in a human IL-12 receptor binding assay (see Example 3).

[0319] The phrase "surface plasmon resonance" includes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Example 5 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0320] The term "K.sub.off", as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.

[0321] The term "K.sub.d", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.

[0322] The phrase "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0323] The phrase "isolated nucleic acid molecule", as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind hIL-12 including "isolated antibodies"), includes a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than hIL-12, which other sequences may naturally flank the nucleic acid in human genomic DNA. Thus, for example, an isolated nucleic acid of the invention encoding a VH region of an anti-IL-12 antibody contains no other sequences encoding other VH regions that bind antigens other than IL-12. The phrase "isolated nucleic acid molecule" is also intended to include sequences encoding bivalent, bispecific antibodies, such as diabodies in which VH and VL regions contain no other sequences other than the sequences of the diabody.

[0324] The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0325] The phrase "recombinant host cell" (or simply "host cell") includes a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0326] The term "modifying", as used herein, is intended to refer to changing one or more amino acids in the antibodies or antigen-binding portions thereof. The change can be produced by adding, substituting or deleting an amino acid at one or more positions. The change can be produced using known techniques, such as PCR mutagenesis.

[0327] The phrase "contact position" includes an amino acid position of in the CDR1, CDR2 or CDR3 of the heavy chain variable region or the light chain variable region of an antibody which is occupied by an amino acid that contacts antigen in one of the twenty-six known antibody-antigen structures. If a CDR amino acid in any of the 26 known solved structures of antibody-antigen complexes contacts the antigen, then that amino acid can be considered to occupy a contact position. Contact positions have a higher probability of being occupied by an amino acid which contact antigen than non-contact positions. Preferably a contact position is a CDR position which contains an amino acid that contacts antigen in greater than 3 of the 26 structures (>11.5%). Most preferably a contact position is a CDR position which contains an amino acid that contacts antigen in greater than 8 of the 25 structures (>32%).

[0328] The term "hypermutation position" includes an amino acid residue that occupies position in the CDR1, CDR2 or CDR3 region of the heavy chain variable region or the light chain variable region of an antibody that is considered to have a high frequency or probability for somatic hypermutation during in vivo affinity maturation of the antibody. "High frequency or probability for somatic hypermutation" includes frequencies or probabilities of a 5 to about 40% chance that the residue will undergo somatic hypermutation during in vivo affinity maturation of the antibody. It should be understood that all ranges within this stated range are also intended to be part of this invention, e.g., 5 to about 30%, e.g., 5 to about 15%, e.g., 15 to about 30%.

[0329] The term "preferred selective mutagenesis position" includes an amino acid residue that occupies a position in the CDR1, CDR2 or CDR3 region of the heavy chain variable region or the light chain variable region which can be considered to be both a contact and a hypermutation position.

[0330] The phrase "selective mutagenesis approach" includes a method of improving the activity of an antibody by selecting and individually mutating CDR amino acids at least one preferred selective mutagenesis position, hypermutation, and/or contact position. A "selectively mutated" human antibody is an antibody which contains a mutation at a position selected using a selective mutagenesis approach. In another embodiment, the selective mutagenesis approach is intended to provide a method of preferentially mutating selected individual amino acid residues in the CDR1, CDR2 or CDR3 of the heavy chain variable region (hereinafter H1, H2, and H3, respectively), or the CDR1, CDR2 or CDR3 of the light chain variable region (hereinafter referred to as L1, L2, and L3, respectively) of an antibody. Amino acid residues may be selected from preferred selective mutagenesis positions, contact positions, or hypermutation positions. Individual amino acids are selected based on their position in the light or heavy chain variable region. It should be understood that a hypermutation position can also be a contact position. In an embodiment, the selective mutagenesis approach is a "targeted approach". The language "targeted approach" is intended to include a method of preferentially mutating selected individual amino acid residues in the CDR1, CDR2 or CDR3 of the heavy chain variable region or the CDR1, CDR2 or CDR3 of the light chain variable region of an antibody in a targeted manner, e.g., a "Group-wise targeted approach" or "CDR-wise targeted approach". In the "Group-wise targeted approach", individual amino acid residues in particular groups are targeted for selective mutations including groups I (including L3 and H3), II (including H2 and L1) and III (including L2 and H1), the groups being listed in order of preference for targeting. In the "CDR-wise targeted approach", individual amino acid residues in particular CDRs are targeted for selective mutations with the order of preference for targeting as follows: H3, L3, H2, L1, H1 and L2. The selected amino acid residue is mutated, e.g., to at least two other amino acid residues, and the effect of the mutation on the activity of the antibody is determined. Activity is measured as a change in the binding specificity/affinity of the antibody, and/or neutralization potency of the antibody. It should be understood that the selective mutagenesis approach can be used for the optimization of any antibody derived from any source including phage display, transgenic animals with human IgG germline genes, human antibodies isolated from human B-cells. Preferably, the selective mutagenesis approach is used on antibodies which can not be optimized further using phage display technology. It should be understood that antibodies from any source including phage display, transgenic animals with human IgG germline genes, human antibodies isolated from human B-cells can be subject to backmutation prior to or after the selective mutagenesis approach.

[0331] The term "activity enhancing amino acid residue" includes an amino acid residue which improves the activity of the antibody. It should be understood that the activity enhancing amino acid residue may replace an amino acid residue at a preferred selective mutagenesis position, contact position, or a hypermutation position and, further, more than one activity enhancing amino acid residue can be present within one or more CDRs. An activity enhancing amino acid residue include, an amino acid residue that improves the binding specificity/affinity of an antibody, for example anti-human IL-12 antibody binding to human IL-12. The activity enhancing amino acid residue is also intended to include an amino acid residue that improves the neutralization potency of an antibody, for example, the human IL-12 antibody which inhibits human IL-12.

[0332] Various aspects of the invention are described in further detail in the following subsections.

I. Human Antibodies that Bind Human IL-12

[0333] This invention provides isolated human antibodies, or antigen-binding portions thereof, that bind to human IL-12. Preferably, the human antibodies of the invention are recombinant, neutralizing human anti-hIL-12 antibodies. Antibodies of the invention that bind to human IL-12 can be selected, for example, by screening one or more human V.sub.L and V.sub.H cDNA libraries with hIL-12, such as by phage display techniques as described in Example 1. Screening of human V.sub.L and V.sub.H cDNA libraries initially identified a series of anti-IL-12 antibodies of which one antibody, referred to herein as "Joe 9" (or "Joe 9 wild type"), was selected for further development. Joe 9 is a relatively low affinity human IL-12 antibody (e.g., a K.sub.off of about 0.1 sec.sup.-1), yet is useful for specifically binding and detecting hIL-12. The affinity of the Joe 9 antibody was improved by conducting mutagenesis of the heavy and light chain CDRs, producing a panel of light and heavy chain variable regions that were "mixed and matched" and further mutated, leading to numerous additional anti-hIL-12 antibodies with increased affinity for hIL-12 (see Example 1, Table 2 (see Appendix A) and the sequence alignments of FIGS. 1A-D).

[0334] Of these antibodies, the human anti-hIL-12 antibody referred to herein as Y61 demonstrated a significant improvement in binding affinity (e.g., a K.sub.off of about 2.times.10.sup.-4 sec.sup.-1). The Y61 anti-hIL-12 antibody was selected for further affinity maturation by individually mutating specific amino acids residues within the heavy and light chain CDRs. Amino acids residues of Y61 were selected for site-specific mutation (selective mutagenesis approach) based on the amino acid residue occupying a preferred selective mutagenesis position, contact and/or a hypermutation position. A summary of the substitutions at selected positions in the heavy and light chain CDRs is shown in FIGS. 2A-2H. A preferred recombinant neutralizing antibody of the invention, referred to herein as J695, resulted from a Gly to Tyr substitution at position 50 of the light chain CDR2 of Y61, and a Gly to Tyr substitution at position 94 of the light chain CDR3 of Y61.

[0335] Amino acid sequence alignments of the heavy and light chain variable regions of a panel of anti-IL-12 antibodies of the invention, on the lineage from Joe 9 wild type to J695, are shown in FIGS. 1A-1D. These sequence alignments allowed for the identification of consensus sequences for preferred heavy and light chain variable regions of antibodies of the invention that bind hIL-12, as well as consensus sequences for the CDR3, CDR2, and CDR1, on the lineage from Joe 9 to J695. Moreover, the Y61 mutagenesis analysis summarized in FIGS. 2A-2H allowed for the identification of consensus sequences for heavy and light chain variable regions that bind hIL-12, as well as consensus sequences for the CDR3, CDR2, and CDR1 that bind hIL-12 on the lineage from Y61 to J695 that encompasses sequences with modifications from Y61 yet that retain good hIL-12 binding characteristics. Preferred CDR, VH and VL sequences of the invention (including consensus sequences) as identified by sequence identifiers in the attached Sequence Listing, are summarized below.

TABLE-US-00001 SEQ ID ANTIBODY NO: CHAIN REGION SEQUENCE 1 Consensus CDR H3 (H/S)-G-S-(H/Y)-D-(N/T/Y) Joe 9 to J695 2 Consensus CDR L3 Q-(S/T)-Y-(D/E)-(S/R/K)-(S/G/Y)- Joe 9 to J695 (L/F/T/S)-(R/S/T/W/H)-(G/P)- (S/T/A/L)-(R/S/M/T/L)-(V/I/T/M/L) 3 Consensus CDR H2 F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G Joe 9 to J695 4 Consensus CDR L2 (G/Y)-N-(D/S)-(Q/N)-R-P-S Joe 9 to J695 5 Consensus CDR H1 F-T-F-S-(S/E)-Y-G-M-H Joe 9 to J695 6 Consensus CDR L1 (S/T)-G-(G/S)-(R/S)-S-N-I-(G/V)- Joe 9 to J695 (S/A)-(N/G/Y)-(T/D)-V-(K/H) 7 Consensus VH (full VH sequence; see Joe 9 to J695 sequence listing) 8 Consensus VL (full VL sequence; see Joe 9 to J695 sequence listing) 9 Consensus CDR H3 H-(G/V/C/H)-(S/T)-(H/T/V/R/I)- Y61 to J695 (D/S)-(N/K/A/T/S/F/W/H) 10 Consensus CDR L3 Q-S-Y-(D/S)-(Xaa)- Y61 to J695 (G/D/Q/L/F/R/H/N/Y)-T-H-P-A-L-L 11 Consensus CDR H2 (F/T/Y)-I-(R/A)-Y-(D/S/E/A)-(G/R)- Y61 to J695 S-(Xaa)-K-(Y/E)-Y-A-D-S-V-K-G 12 Consensus CDR L2 (G/Y/S/T/N/Q)-N-D-Q-R-P-S Y61 to J695 13 Consensus CDR H1 F-T-F-(Xaa)-(Xaa)-(Y/H)- Y61 to J695 (G/M/A/N/S)-M-H 14 Consensus CDR Ll S-G-G-R-S-N-I-G-(S/C/R/N/D/T)- Y61 to J695 (N/M/I)-(T/Y/D/H/K/P)-V-K 15 Consensus VH (full VH sequence; see Y61 to J695 sequence listing) 16 Consensus VL (full VL sequence; see Y61 to J695 sequence listing) 17 Y61 CDR H3 H-G-S-H-D-N 18 Y61 CDR L3 Q-S-Y-D-R-G-T-H-P-A-L-L 19 Y61 CDR H2 F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G 20 Y61 CDR L2 G-N-D-Q-R-P-S 21 Y61 CDR H1 F-T-F-S-S-Y-G-M-H 22 Y61 CDR L1 S-G-G-R-S-N-I-G-S-N-T-V-K 23 Y61 VH (full VH sequence; see sequence listing) 24 Y61 VL (full VL sequence; see sequence listing) 25 J695 CDR H3 H-G-S-H-D-N 26 J695 CDR L3 Q-S-Y-D-R-Y-T-H-P-A-L-L 27 J695 CDR H2 F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G 28 J695 CDR L2 Y-N-D-Q-R-P-S 29 J695 CDR H1 F-T-F-S-S-Y-G-M-H 30 J695 CDR L1 S-G-S-R-S-N-I-G-S-N-T-V-K 31 J695 VH (full VH sequence; see sequence listing) 32 J695 VL (full VL sequence; see sequence listing)

[0336] Antibodies produced from affinity maturation of Joe 9 wild type were functionally characterized by surface plasmon resonance analysis to determine the K.sub.d and K.sub.off rate. A series of antibodies were produced having a K.sub.off rate within the range of about 0.1 s.sup.-1 to about 1.times.10.sup.-5 s.sup.-1, and more preferably a K.sub.off of about 1.times.10.sup.-4 s.sup.-1 to 1.times.10.sup.-5 s.sup.-1 or less. Antibodies were also characterized in vitro for their ability to inhibit phytohemagglutinin (PHA) blast proliferation, as described in Example 3. A series of antibodies were produced having an IC.sub.50 value in the range of about 1.times.10.sup.-6 M to about 1.times.10.sup.-11 M, more preferably about 1.times.10.sup.-10 M to 1.times.10.sup.-11 M or less.

[0337] Accordingly, in one aspect, the invention provides an isolated human antibody, or antigen-binding portion thereof, that binds to human IL-12 and dissociates from human IL-12 with a K.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6 M or less. In preferred embodiments, the isolated human IL-12 antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-2 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-7 M or less. In more preferred embodiments, the isolated human IL-12 antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-8 M or less. In more preferred embodiments, the isolated human IL-12 antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-4 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less. In more preferred embodiments, the isolated human IL-12 antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-5 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-10 M or less. In even more preferred embodiments, the isolated human IL-12 antibody, or an antigen-binding portion thereof, dissociates from human IL-12 with a K.sub.off rate constant of 1.times.10.sup.-5 s.sup.-1 or less, or inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-11 M or less.

[0338] The dissociation rate constant (K.sub.off) of an IL-12 antibody can be determined by surface plasmon resonance (see Example 5). Generally, surface plasmon resonance analysis measures real-time binding interactions between ligand (recombinant human IL-12 immobilized on a biosensor matrix) and analyte (antibodies in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway, N.J.). Surface plasmon analysis can also be performed by immobilizing the analyte (antibodies on a biosensor matrix) and presenting the ligand (recombinant IL-12 in solution). Neutralization activity of IL-12 antibodies, or antigen binding portions thereof, can be assessed using one or more of several suitable in vitro assays (see Example 3).

[0339] It is well known in the art that antibody heavy and light chain CDRs play an important role in the binding specificity/affinity of an antibody for an antigen. Accordingly, the invention encompasses human antibodies having light and heavy chain CDRs of Joe 9, as well as other antibodies having CDRs that have been modified to improve the binding specificity/affinity of the antibody. As demonstrated in Example 1, a series of modifications to the light and heavy chain CDRs results in affinity maturation of human anti-hIL-12 antibodies. The heavy and light chain variable region amino acid sequence alignments of a series of human antibodies ranging from Joe 9 wild type to J695 that bind human IL-12 is shown in FIGS. 1A-1D. Consensus sequence motifs for the CDRs of antibodies can be determined from the sequence alignment (as summarized in the table above). For example, a consensus motif for the VH CDR3 of the lineage from Joe 9 to J695 comprises the amino acid sequence: (H/S)-G-S-(H/Y)-D-(N/T/Y) (SEQ ID NO: 1), which encompasses amino acids from position 95 to 102 of the consensus HCVR shown in SEQ ID NO: 7. A consensus motif for the VL CDR3 comprises the amino acid sequence: Q-(S/T)-Y-(D/E)-(S/R/K)-(S/G/Y)-(L/F/T/S)-(R/S/T/W/H)-(G/P)-(S/T/A/L)-(R/- S/M/T/L-V/I/T/M/L) (SEQ ID NO: 2), which encompasses amino acids from position 89 to 97 of the consensus LCVR shown in SEQ ID NO: 8.

[0340] Accordingly, in another aspect, the invention provides an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics:

[0341] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or less;

[0342] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 1; and

[0343] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 2.

[0344] In a preferred embodiment, the antibody further comprises a VH CDR2 comprising the amino acid sequence: F-I-R-Y-D-G-S-N-K-Y-Y-A-D-S-V-K-G (SEQ ID NO: 3) (which encompasses amino acids from position 50 to 65 of the consensus HCVR comprising the amino acid sequence SEQ ID NO: 7) and further comprises a VL CDR2 comprising the amino acid sequence: (G/Y)-N-(D/S)-(Q/N)-R-P-S (SEQ ID NO: 4) (which encompasses amino acids from position 50 to 56 of the consensus LCVR comprising the amino acid sequence SEQ ID NO: 8).

[0345] In another preferred embodiment, the antibody further comprises a VH CDR1 comprising the amino acid sequence: F-T-F-S-(S/E)-Y-G-M-H (SEQ ID NO: 5) (which encompasses amino acids from position 27 to 35 of the consensus HCVR comprising the amino acid sequence SEQ ID NO: 7) and further comprises a VL CDR1 comprising the amino acid sequence: (S/T)-G-(G/S)-(R/S)-S-N-I-(G/V)-(S/A)-(N/G/Y)-(T/D)-V-(K/H) (SEQ ID NO: 6) (which encompasses amino acids from position 24 to 34 of the consensus LCVR comprising the amino acid sequence SEQ ID NO: 8).

[0346] In yet another preferred embodiment, the antibody of the invention comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 7 and a LCVR comprising the amino acid sequence of SEQ ID NO: 8.

[0347] Additional consensus motifs can be determined based on the mutational analysis performed on Y61 that led to the J695 antibody (summarized in FIGS. 2A-2H). As demonstrated by the graphs shown in FIGS. 2A-2H, certain residues of the heavy and light chain CDRs of Y61 were amenable to substitution without significantly impairing the hIL-12 binding properties of the antibody. For example, individual substitutions at position 30 in CDR H1 with twelve different amino acid residues did not significantly reduce the K.sub.off rate of the antibody, indicating that is position is amenable to substitution with a variety of different amino acid residues. Thus, based on the mutational analysis (i.e., positions within Y61 that were amenable to substitution by other amino acid residues) consensus motifs were determined. The consensus motifs for the heavy and light chain CDR3s are shown in SEQ ID NOs: 9 and 10, respectively, consensus motifs for the heavy and light chain CDR2s are shown in SEQ ID NOs: 11 and 12, respectively, and consensus motifs for the heavy and light chain CDR1s are shown in SEQ ID NOs: 13 and 14, respectively. Consensus motifs for the VH and VL regions are shown in SEQ ID NOs: 15 and 16, respectively.

[0348] Accordingly, in one aspect, the invention features an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics:

[0349] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0350] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and

[0351] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0352] In a preferred embodiment, the antibody further comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 11 and further comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12.

[0353] In another preferred embodiment, the antibody further comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13 and further comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

[0354] In yet another preferred embodiment, the antibody of the invention comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 15 and a LCVR comprising the amino acid sequence of SEQ ID NO: 16.

[0355] A preferred antibody of the invention, the human anti-hIL-12 antibody Y61, was produced by affinity maturation of Joe 9 wild type by PCR mutagenesis of the CDR3 (as described in Example 1). Y61 had an improved specificity/binding affinity determined by surface plasmon resonance and by in vitro neutralization assays. The heavy and light chain CDR3s of Y61 are shown in SEQ ID NOs: 17 and 18, respectively, the heavy and light chain CDR2s of Y61 are shown in SEQ ID NOs: 19 and 20, respectively, and the heavy and light chain CDR1s of Y61 are shown in SEQ ID NOs: 21 and 22, respectively. The VH of Y61 has the amino acid sequence of SEQ ID NO: 23 and the VL of Y61 has the amino acid sequence of SEQ ID NO: 24 (these sequences are also shown in FIGS. 1A-1D, aligned with Joe9).

[0356] Accordingly, in another aspect, the invention features an isolated human antibody, or an antigen-binding portion thereof, which

[0357] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0358] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and

[0359] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

[0360] In a preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19 and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20.

[0361] In another preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21 and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22.

[0362] In yet another preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, comprising a the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24.

[0363] In certain embodiments, the full length antibody comprises a heavy chain constant region, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions, and any allotypic variant therein as described in Kabat (, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Preferably, the antibody heavy chain constant region is an IgG1 heavy chain constant region. Alternatively, the antibody portion can be an Fab fragment, an F(ab'.sub.2) fragment or a single chain Fv fragment.

[0364] Modifications of individual residues of Y61 led to the production of a panel of antibodies shown in FIGS. 2A-2H. The specificity/binding affinity of each antibody was determined by surface plasmon resonance and/or by in vitro neutralization assays.

[0365] Accordingly, in another aspect, the invention features an isolated human antibody, or an antigen-binding portion thereof, which

[0366] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0367] b) has a heavy chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 404-SEQ ID NO: 469; and

[0368] c) has a light chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 534-SEQ ID NO: 579.

[0369] In preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:335-SEQ ID NO: 403; and a light chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 506-SEQ ID NO: 533.

[0370] In another preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 288-SEQ ID NO: 334; and a light chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 470-SEQ ID NO: 505.

[0371] In yet another preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, comprising a the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24.

[0372] In certain embodiments, the full length antibody comprising a heavy chain constant region such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions and any allotypic variant therein as described in Kabat (, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Preferably, the antibody heavy chain constant region is an IgG1 heavy chain constant region. Alternatively, the antibody portion can be a Fab fragment, an F(ab'.sub.2) fragment or a single chain Fv fragment.

[0373] A particularly preferred recombinant, neutralizing antibody of the invention, J695, was produced by site-directed mutagenesis of contact and hypermutation amino acids residues of antibody Y61 (see Example 2 and section III below). J695 differs from Y61 by a Gly to Tyr substitution in Y61 at position 50 of the light chain CDR2 and by a Gly to Tyr substitution at position 94 of the light chain CDR3. The heavy and light chain CDR3s of J695 are shown in SEQ ID NOs: 25 and 26, respectively, the heavy and light chain CDR2s of J695 are shown in SEQ ID NOs: 27 and 28, respectively, and the heavy and light chain CDR1s of J695 are shown in SEQ ID NOs: 29 and 30, respectively. The VH of J695 has the amino acid sequence of SEQ ID NO: 31 and the VL of J695 has the amino acid sequence of SEQ ID NO: 32 (these sequences are also shown in FIGS. 1A-1D, aligned with Joe9).

[0374] Accordingly, in another aspect, the invention features an isolated human antibody, or an antigen-binding portion thereof, which

[0375] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0376] b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and

[0377] c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0378] In preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28.

[0379] In another preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30.

[0380] In yet another preferred embodiment, the isolated human antibody, or an antigen-binding portion thereof, has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32.

[0381] In certain embodiments, the full length antibody comprises a heavy chain constant region, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions and any allotypic variant therein as described in Kabat (, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Preferably, the antibody heavy chain constant region is an IgG1 heavy chain constant region. Alternatively, the antibody portion can be an Fab fragment, an F(ab'.sub.2) fragment or a single chain Fv fragment.

[0382] Additional mutations in the preferred consensus sequences for CDR3, CDR2, and CDR1 of antibodies on the lineage from Joe 9 to J695, or from the lineage Y61 to J695, can be made to provide additional anti-IL-12 antibodies of the invention. Such methods of modification can be performed using standard molecular biology techniques, such as by PCR mutagenesis, targeting individual contact or hypermutation amino acid residues in the light chain and/or heavy chain CDRs-, followed by kinetic and functional analysis of the modified antibodies as described herein (e.g., neutralization assays described in Example 3, and by BIAcore analysis, as described in Example 5).

[0383] Accordingly, in another aspect the invention features an isolated human antibody, or an antigen-binding portion thereof, which

[0384] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-6 M or less;

[0385] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5; and

[0386] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6.

[0387] In another aspect the invention features an isolated human antibody, or an antigen-binding portion thereof, which

[0388] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0389] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position, contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13; and

[0390] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position, contact position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

[0391] An ordinarily skilled artisan will also appreciate that additional mutations to the CDR regions of an antibody of the invention, for example in Y61 or in J695, can be made to provide additional anti-IL-12 antibodies of the invention. Such methods of modification can be performed using standard molecular biology techniques, as described above. The functional and kinetic analysis of the modified antibodies can be performed as described in Example 3 and Example 5, respectively. Modifications of individual residues of Y61 that led to the identification of J695 are shown in FIGS. 2A-2H and are described in Example 2.

[0392] Accordingly, in another aspect the invention features an isolated human antibody, or an antigen-binding portion thereof, which

[0393] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0394] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21; and

[0395] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 22.

[0396] In another aspect the invention features an isolated human antibody, or an antigen-binding portion thereof, which

[0397] a) inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay with an IC.sub.50 of 1.times.10.sup.-9 M or less;

[0398] b) comprises a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29; and

[0399] c) comprises a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, or a mutant thereof having one or more amino acid substitutions at a preferred selective mutagenesis position or a hypermutation position, wherein said mutant has a k.sub.off rate no more than 10-fold higher than the antibody comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30.

[0400] In yet another embodiment, the invention provides isolated human antibodies, or antigen-binding portions thereof, that neutralize the activity of human IL-12, and at least one additional primate IL-12 selected from the group consisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12, cynomolgus IL-12 and rhesus IL-12, but which do not neutralize the activity of the mouse IL-12.

II Selection of Recombinant Human Antibodies

[0401] Recombinant human antibodies of the invention can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP.TM. phage display kit, catalog no. 240612), examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, Kang et al. PCT Publication No. WO 92/18619; Winter et al. PCT Publication No. WO 92/20791; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

[0402] The antibody libraries used in this method are preferably scFv libraries prepared from human VL and VH cDNAs. The scFv antibody libraries are preferably screened using recombinant human IL-12 as the antigen to select human heavy and light chain sequences having a binding activity toward IL-12. To select for antibodies specific for the p35 subunit of IL-12 or the p70 heterodimer, screening assays were performed in the presence of excess free p40 subunit. Subunit preferences can be determined, for example by, micro-Friguet titration, as described in Example 1.

[0403] Once initial human VL and VH segments are selected, "mix and match" experiments, in which different pairs of the selected VL and VH segments are screened for IL-12 binding, are performed to select preferred VL/VH pair combinations (see Example 1). Additionally, to further improve the affinity and/or lower the off rate constant for hIL-12 binding, the VL and VH segments of the preferred VL/VH pair(s) can be randomly mutated, preferably within the CDR3 region of VH and/or VL, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complimentary to the VH CDR3 or VL CDR3, respectively, which primers have been "spiked" with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VH and VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be reselected and rescreened for binding to hIL-12 and sequences that exhibit high affinity and a low off rate for IL-12 binding can be selected. Table 2 (see Appendix A) shows antibodies that displayed altered binding specificity/affinity produced as a result of in vitro affinity maturation.

[0404] Following selection, isolation and screening of an anti-hIL-12 antibody of the invention from a recombinant immunoglobulin display library, nucleic acid encoding the selected antibody can be recovered from the phage particle(s) (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other antibody forms of the invention (e.g., linked to nucleic acid encoding additional immunoglobulin domains, such as additional constant regions). To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cells, as described in further detail in Section IV below.

[0405] Methods for selecting human IL-12 binding antibodies by phage display technology, and affinity maturation of selected antibodies by random or site-directed mutagenesis of CDR regions are described in further detail in Example 1.

[0406] As described in Example 1, screening of human VL and VH cDNA libraries identified a series of anti-IL-12 antibodies, of which the Joe 9 antibody was selected for further development. A comparison of the heavy chain variable region of Joe 9 with the heavy chain germline sequences selected from the VBASE database, revealed that Joe 9 was similar to the COS-3 germline sequence. COS-3 belongs to the V.sub.H3 family of germline sequences.

[0407] The V.sub.H3 family is part of the human VH germline repertoire which is grouped into seven families, V.sub.H1-V.sub.H7, based on nucleotide sequence homology (Tomlinson et al. (1992) J. Mol. Biol., 227, 776-798 and Cook et al. (1995) Immunology Today, 16, 237-242). The V.sub.H3 family contains the highest number of members and makes the largest contribution to the germline repertoire. For any given human V.sub.H3-germline antibody sequence, the amino acid sequence identity within the entire V.sub.H3 family is high (See e.g., Tomlinson et al. (1992) J. Mol. Biol., 227, 776-798 and Cook et al. (1995) Immunology Today, 16, 237-242). The range of amino acid sequence identity between any two germline VH sequences of the V.sub.H3 family varies from 69-98 residues out of approximately 100 VH residues, (i.e., 69-98% amino acid sequence homology between any two germline VH sequences). For most pairs of germline sequences there is at least 80 or more identical amino acid residues, (i.e., at least 80% amino acid sequence homology). The high degree of amino acid sequence homology between the V.sub.H3 family members results in certain amino acid residues being present at key sites in the CDR and framework regions of the VH chain. These amino acid residues confer structural features upon the CDRs.

[0408] Studies of antibody structures have shown that CDR conformations can be grouped into families of canonical CDR structures based on the key amino acid residues that occupy certain positions in the CDR and framework regions. Consequently, there are similar local CDR conformations in different antibodies that have canonical structures with identical key amino acid residues (Chothia et al. (1987) J. Mol. Biol., 196, 901-917 and Chothia et al. (1989) Nature, 342, 877-883). Within the V.sub.H3 family there is a conservation of amino acid residue identity at the key sites for the CDR1 and CDR2 canonical structures (Chothia et al. (1992) J. Mol. Biol., 227, 799-817).

[0409] The COS-3 germline VH gene, is a member of the V.sub.H3 family and is a variant of the 3-30 (DP-49) germline VH allele. COS-3, differs from Joe9 VH amino acid sequences at only 5 positions. The high degree of amino acid sequence homology between Joe9 VH and COS-3, and between Joe9 VH and the other V.sub.H3 family members also confers a high degree of CDR structural homology (Chothia et al. (1992) J. Mol. Biol., 227, 799-817; Chothia et al. (1987) J. Mol. Biol., 196, 901-917 and Chothia et al. (1989) Nature, 342, 877-883).

[0410] The skilled artisan will appreciate that based on the high amino acid sequence and canonical structural similarity to Joe 9, other V.sub.H3 family members could also be used to generate antibodies that bind to human IL-12. This can be performed, for example, by selecting an appropriate VL by chain-shuffling techniques (Winter et al. (1994) Annual Rev. Immunol., 12, 433-55), or by the grafting of CDRs from a rodent or other human antibody including CDRs from antibodies of this invention onto a V.sub.H3 family framework.

[0411] The human V lambda germline repertoire is grouped into 10 families based on nucleotide sequence homology (Williams et al. (1996) J. Mol. Biol., 264, 220-232). A comparison of the light chain variable region of Joe 9 with the light chain germline sequences selected from the VBASE database, revealed that Joe 9 was similar to the DPL8 lambda germline. The Joe9 VL differs from DPL8 sequence at only four framework positions, and is highly homologous to the framework sequences of the other V.sub..lamda.1 family members. Based on the high amino acid sequence homology and canonical structural similarity to Joe 9, other V.sub..lamda.1 family members may also be used to generate antibodies that bind to human IL-12. This can be performed, for example, by selecting an appropriate VH by chain-shuffling techniques (Winter et al. Supra, or by the grafting of CDRs from a rodent or other human antibody including CDRs from antibodies of this invention onto a V.sub..lamda.1 family framework.

[0412] The methods of the invention are intended to include recombinant antibodies that bind to hIL-12, comprising a heavy chain variable region derived from a member of the V.sub.H3 family of germline sequences, and a light chain variable region derived from a member of the V.sub..lamda.1 family of germline sequences. Moreover, the skilled artisan will appreciate that any member of the V.sub.H3 family heavy chain sequence can be combined with any member of the V.sub..lamda.1 family light chain sequence.

[0413] Those skilled in the art will also appreciate that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the germline may exist within a population (e.g., the human population). Such genetic polymorphism in the germline sequences may exist among individuals within a population due to natural allelic variation. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the a gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in germline sequences that are the result of natural allelic variation are intended to be within the scope of the invention.

[0414] Accordingly, in one aspect, the invention features an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0415] a) that binds to human IL-12 and dissociates from human IL-12 with a k.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0416] b) has a heavy chain variable region comprising an amino acid sequence selected from a member of the V.sub.H3 germline family, wherein the heavy chain variable region has a mutation at a contact or hypermutation position with an activity enhancing amino acid residue. [0417] c) has a light chain variable region comprising an amino acid sequence selected from a member of the V.sub..lamda.1 germline family, wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue.

[0418] In a preferred embodiment, the isolated human antibody, or antigen binding has mutation in the heavy chain CDR3.

[0419] In another preferred embodiment, the isolated human antibody, or antigen binding has mutation in the light chain CDR3.

[0420] In another preferred embodiment, the isolated human antibody, or antigen binding has mutation in the heavy chain CDR2.

[0421] In another preferred embodiment, the isolated human antibody, or antigen binding has mutation in the light chain CDR2.

[0422] In another preferred embodiment, the isolated human antibody, or antigen binding has mutation in the heavy chain CDR1.

[0423] In another preferred embodiment, the isolated human antibody, or antigen binding has mutation in the light chain CDR1.

[0424] An ordinarily skilled artisan will appreciate that based on the high amino acid sequence similarity between members of the V.sub.H3 germline family, or between members of the light chain V.sub..lamda.1 germline family, that mutations to the germlines sequences can provide additional antibodies that bind to human IL-12. Table 1 (see Appendix A) shows the germline sequences of the V.sub.H3 family members and demonstrates the significant sequence homology within the family members. Also shown in Table 1 are the germline sequences for V.sub..lamda.1 family members. The heavy and light chain sequences of Joe 9 are provided as a comparison. Mutations to the germline sequences of V.sub.H3 or V.sub..lamda.1 family members may be made, for example, at the same amino acid positions as those made in the antibodies of the invention (e.g. mutations in Joe 9). The modifications can be performed using standard molecular biology techniques, such as by PCR mutagenesis, targeting individual amino acid residues in the germline sequences, followed by kinetic and functional analysis of the modified antibodies as described herein (e.g., neutralization assays described in Example 3, and by BIAcore analysis, as described in Example 5).

[0425] Accordingly, in one aspect, the invention features isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0426] a) has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 595-667, wherein the heavy chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue. [0427] b) has a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 669-675, wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue.

[0428] An ordinarily skilled artisan will appreciate that based on the high amino acid sequence similarity between Joe 9 and COS-3 heavy chain germline sequence, and between Joe 9 and DPL8 lambda germline sequence, that other mutations to the CDR regions of these germlines sequences can provide additional antibodies that bind to human IL-12. Such methods of modification can be performed using standard molecular biology techniques as described above.

[0429] Accordingly, in one aspect, the invention features isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0430] a) that binds to human IL-12 and dissociates from human IL-12 with a k.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0431] b) has a heavy chain variable region comprising the COS-3 germline amino acid sequence, wherein the heavy chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue. [0432] c) has a light chain variable region comprising the DPL8 germline amino acid sequence, wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue.

[0433] Due to certain amino acid residues occupying key sites in the CDR and framework regions in the light and heavy chain variable region, structural features are conferred at these regions. In particular, the CDR2 and CDR1 regions are subject to canonical structural classifications. Since there is a high degree of amino acids sequence homology between family members, these canonical features are present between family members. The skilled artisan will appreciate that modifications at the amino acid residues that confer these canonical structures would produce additional antibodies that bind to IL-12. The modifications can be performed using standard molecular biology techniques as described above.

[0434] Accordingly, in another aspect, the invention features an isolated human antibody, or an antigen-binding portion thereof, which has the following characteristics: [0435] a) that binds to human IL-12 and dissociates from human IL-12 with a k.sub.off rate constant of 0.1 s.sup.-1 or less, as determined by surface plasmon resonance, or which inhibits phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin blast proliferation assay (PHA assay) with an IC.sub.50 of 1.times.10.sup.-6M or less. [0436] b) has a heavy chain variable region comprising an amino acid sequence selected from a member of the V.sub.H3 germline family, wherein the heavy chain variable region comprises a CDR2 that is structurally similar to CDR2s from other V.sub.H3 germline family members, and a CDR1 that is structurally similar to CDR1s from other V.sub.H3 germline family members, and wherein the heavy chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue; [0437] c) has a light chain variable region comprising an amino acid sequence selected from a member of the V.sub..lamda.1 germline family, wherein the light chain variable region comprises a CDR2 that is structurally similar to CDR2s from other V.sub..lamda.1 germline family members, and a CDR1 that is structurally similar to CDR1s from other V.sub..lamda.1 germline family members, and wherein the light chain variable region has a mutation at a preferred selective mutagenesis position, contact or hypermutation position with an activity enhancing amino acid residue.

[0438] Recombinant human antibodies of the invention have variable and constant regions which are homologous to human germline immunoglobulin sequences selected from the VBASE database. Mutations to the recombinant human antibodies (e.g., by random mutagenesis or PCR mutagenesis) result in amino acids that are not encoded by human germline immunoglobulin sequences. Also, libraries of recombinant antibodies which were derived from human donors will contain antibody sequences that differ from their corresponding germline sequences due to the normal process of somatic mutation that occurs during B-cell development. It should be noted that if the "germline" sequences obtained by PCR amplification encode amino acid differences in the framework regions from the true germline configuration (i.e., differences in the amplified sequence as compared to the true germline sequence), it may be desirable to change these amino acid differences back to the true germline sequences (i.e., "backmutation" of framework residues to the germline configuration). Thus, the present invention can optionally include a backmutation step. To do this, the amino acid sequences of heavy and light chain encoded by the germline (as found as example in VBASE database) are first compared to the mutated immunoglobulin heavy and light chain framework amino acid sequences to identify amino acid residues in the mutated immunoglobulin framework sequence that differ from the closest germline sequences. Then, the appropriate nucleotides of the mutated immunoglobulin sequence are mutated back to correspond to the germline sequence, using the genetic code to determine which nucleotide changes should be made. Mutagenesis of the mutated immunoglobulin framework sequence is carried out by standard methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis. The role of each amino acid identified as candidate for backmutation should be investigated for a direct or indirect role in antigen binding and any amino acid found after mutation to affect any desirable characteristic of the human antibody should not be included in the final human antibody; as an example, activity enhancing amino acids identified by the selective mutagenesis approach will not be subject to backmutation. Assays to determine the characteristics of the antibody resulting from mutagenesis can include ELISA, competitive ELISA, in vitro and in vivo neutralization assays and/or (see e.g. Example 3) immunohistochemistry with tissue sections from various sources (including human, primate and/or other species).

[0439] To minimize the number of amino acids subject to backmutation those amino acid positions found to be different from the closest germline sequence but identical to the corresponding amino acid in a second germline sequence can remain, provided that the second germline sequence is identical and colinear to the sequence of the human antibody of the invention for at least 10, preferably 12 amino acids, on both sides of the amino acid in question. This would assure that any peptide epitope presented to the immune system by professional antigen presenting cells in a subject treated with the human antibody of the invention would not be foreign but identical to a self-antigen, i.e. the immunoglobulin encoded by that second germline sequence. Backmutation may occur at any stage of antibody optimization; preferably, backmutation occurs directly before or after the selective mutagenesis approach. More preferably, backmutation occurs directly before the selective mutagenesis approach.

III. Modifications to Preferred Selective Mutagenesis Positions, Contact and/or Hypermutation Positions

[0440] Typically, selection of antibodies with improved affinities can be carried out using phage display methods, as described in section II above. This can be accomplished by randomly mutating combinations of CDR residues and generating large libraries containing antibodies of different sequences. However, for these selection methods to work, the antibody-antigen reaction must tend to equilibrium to allow, over time, preferential binding of higher affinity antibodies to the antigen. Selection conditions that would allow equilibrium to be established could not be determined (presumably due to additional non-specific interactions between the antigen and phage particle) when phage display methods were used to improve the affinity of selected anti-IL-12 antibodies, upon attaining a certain level of affinity achieved (i.e., that of antibody Y61). Accordingly, antibodies with even higher affinities could not be selected by phage display methods. Thus, for at least certain antibodies or antigens, phage display methods are limiting in their ability to select antibodies with a highly improved binding specificity/affinity. Accordingly, a method termed Selective Mutagenesis Approach which does not require phage display affinity maturation of antibodies, was established to overcome this limitation and is provided by the invention. Although this Selective Mutagenesis Approach was developed to overcome limitations using the phage display system, it should be noted that this method can also be used with the phage display system. Moreover, the selective mutagenesis approach can be used to improve the activity of any antibody.

[0441] To improve the activity (e.g., affinity or neutralizing activity) of an antibody, ideally one would like to mutate every CDR position in both the heavy and light chains to every other possible amino acid residue. However, since there are, on average, 70 CDR positions within an antibody, such an approach would be very time consuming and labor intensive. Accordingly, the method of the invention allows one to improve the activity of the antibody by mutating only certain selected residues within the heavy and/or light chain CDRs. Furthermore, the method of the invention allows improvement in activity of the antibody without affecting other desirable properties of the antibody.

[0442] Determining which amino acid residues of an antibody variable region are in contact with an antigen cannot be accurately predicted based on primary sequence or their positions within the variable region. Nevertheless, alignments of sequences from antibodies with different specificities conducted by Kabat et al. have identified the CDRs as local regions within the variable regions which differ significantly among antibodies (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-393, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Structural studies have shown that the antigen binding surface is formed by amino acid residues present in the CDRs. Other amino acid residues outside the CDR are also known to play structural roles or be directly involved in antigen binding. Therefore, for each antigen-antibody pair, amino acid residues within and outside of the CDRs may be important.

[0443] The sequence alignment studies by Tomlison et al identified a number of positions in the heavy and light chain CDR1 and CDR2, and in a portion of the kappa chain CDR3 which are frequent sites of somatic mutation. (Tomlison et al (1996) J. Mol. Biol. 256: 813-817). In particular, positions H31, H31B, H33, H33B, H52B, H56, H58, L30, L31, L31A, L50, L53, L91, L92, L93 and L94 were identified as frequent sites for somatic mutation. However, this analysis excludes the important heavy chain CDR3 regions, and sections of the light chain CDR3 which are known to lie in the center of an antibody binding site, and potentially provide important interactions with an antigen. Furthermore, Tomlison et al. propose that somatic diversity alone does not necessarily predict a role of a specific amino acid in antigen binding, and suggest conserved amino acid residues that contact the antigen, and diverse amino acid residues which do not contact the antigen. This conclusion is further supported by mutational studies on the role of somatic mutations to antibody affinity (Sharon, (1990), PNAS, 87:4814-7). Nineteen somatic mutations in a high-affinity anti-p-azophenylarsonate (Ars) antibody were simultaneously replaced with their corresponding germline residues, generating a germline version of the anti-Ars antibody which had a two-hundred fold loss in activity. The full affinity of the anti-Ars antibody could be recovered by restoring only three of the nineteen somatic mutations, demonstrating that many somatic mutations may be permitted that do not contribute to antigen binding activity.

[0444] The result can be explained in part by the nature of antibody diversity itself. Immature B-cells may produce initially low affinity antibodies that recognize a number of self or non-self antigens. Moreover, antibodies may undergo in the course of affinity maturation sequence variations that may cause self-reactivity. Hypermutation of such low affinity antibodies may serve to abolish self-reactivity ("negative selection") and increase affinity for the foreign antigen. Therefore, the analysis of primary and structural data of a large number of antibodies does not provide a method of predicting either (1) the role of somatic hyper-mutation sites in the affinity maturation process versus the process of decreasing affinity towards unwanted antigens, or (2) how a given amino acid contributes to the properties of a specific antigen-antibody pair.

[0445] Other attempts to address the role of specific amino acid residues in antigen recognition were made by analyzing a number of crystal structures of antigen-antibody complexes (MacCallum et al. (1996) J. Mol. Biol. 262: 732-745). The potential role of positions located within and outside the CDRs was indicated. Positions in CDRs involved in antigen binding in more than 10 of 26 analyzed structures included H31, H33, H50, H52, H53, H54, H56, H58, H95, H96, H97, H98 and H100 in the heavy chain and L30A, L32, L91, L92, L93, L94, L96 in the light chain. However, the authors noted that prediction of antigen contacts using these and other structural data may over and under predict contact positions, leading to the speculation that a different strategy may have to be applied to different antigens.

[0446] Pini et al. describe randomizing multiple residues in antibody CDR sequences in a large phage display library to rapidly increase antibody affinity (Pini et al. (1998) J. Biol Chem. 273: 21769-21776). However, the high affinity antibodies discussed by Pini et al. had mutations in a total of eight positions, and a reductionary analysis of which changes are absolutely required to improve affinity of the antibody becomes impractical because of the large number of possible combinations to be tested for the smallest number of amino acids required.

[0447] Furthermore, randomizing multiple residues may not necessarily preserve other desired properties of the antibody. Desirable properties or characteristics of an antibody are art-recognized and include for example, preservation of non-cross reactivity, e.g., with other proteins or human tissues and preservation of antibody sequences that are close to human germline immunoglobulin sequences improvement of neutralization potency. Other desirable properties or characteristics include ability to preserve species cross reactivity, ability to preserve epitope specificity and ability to preserve high expression levels of protein in mammalian cells. The desirable properties or characteristics can be observed or measured using art-recognized techniques including but not limited to ELISA, competitive ELISA, in vitro and in vivo neutralization assays (see e.g. Example 3), immunohistochemistry with tissue sections from different sources including human, primate or other sources as the need may be, and studies to expression in mammalian cells using transient expression or stable expression.

[0448] In addition, the method of Pini et al may introduce more changes than the minimal number actually required to improve affinity and may lead to the antibodies triggering anti-human-antibody (HAMA) formation in human subjects. Further, as discussed elsewhere, the phage display as demonstrated here, or other related method including ribosome display may not work appropriately upon reaching certain affinities between antibody and antigen and the conditions required to reach equilibrium may not be established in a reasonable time frame because of additional interactions including interactions with other phage or ribosome components and the antigen.

[0449] The ordinarily skilled artisan may glean interesting scientific information on the origin of antibody diversity from the teachings of the references discussed above. The present invention, however, provides a method for increasing antibody affinity of a specific antigen-antibody pair while preserving other relevant features or desirable characteristics of the antibody. This is especially important when considering the desirability of imparting a multitude of different characteristics on a specific antibody including antigen binding.

[0450] If the starting antibody has desirable properties or characteristics which need to be retained, a selective mutagenesis approach can be the best strategy for preserving these desirable properties while improving the activity of the antibody. For example, in the mutagenesis of Y61, the aim was to increase affinity for hIL-12, and to improve the neutralization potency of the antibody while preserving desired properties. Desired properties of Y61 included (1) preservation of non-cross reactivity with other proteins or human tissues, (2) preservation of fine epitope specificity, i.e. recognizing a p40 epitope preferably in the context of the p70 (p40/p35) heterodimer, thereby preventing binding interference from free soluble p40; and (3) generation of an antibody with heavy and light chain amino acid sequences that were as close as possible to their respective germline immunoglobulin sequences.

[0451] In one embodiment, the method of the invention provides a selective mutagenesis approach as a strategy for preserving the desirable properties or characteristics of the antibody while improving the affinity and/or neutralization potency. The term "selective mutagenesis approach" is as defined above and includes a method of individually mutating selected amino acid residues. The amino acid residues to be mutated may first be selected from preferred selective mutagenesis positions, then from contact positions, and then from hypermutation positions. The individual selected position can be mutated to at least two other amino acid residue and the effect of the mutation both on the desired properties of the antibody, and improvement in antibody activity is determined.

[0452] The Selective Mutagenesis approach comprises the steps of:

[0453] selecting candidate positions in the order 1) preferred selective mutagenesis positions; 2) contact positions; 3) hypermutation positions and ranking the positions based on the location of the position within the heavy and light chain variable regions of an antibody (CDR3 preferred over CDR2 preferred over CDR1);

[0454] individually mutating candidate preferred selective mutagenesis positions, hypermutation and/or contact positions in the order of ranking, to all possible other amino acid residues and analyzing the effect of the individual mutations on the activity of the antibody in order to determine activity enhancing amino acid residues;

[0455] if necessary, making stepwise combinations of the individual activity enhancing amino acid residues and analyzing the effect of the various combinations on the activity of the antibodies; selecting mutant antibodies with activity enhancing amino acid residues and ranking the mutant antibodies based on the location and identity of the amino acid substitutions with regard to their immunogenic potential. Highest ranking is given to mutant antibodies that comprise an amino acid sequence which nearly identical to a variable region sequence that is described in a germline database, or has an amino acid sequence that is comparable to other human antibodies. Lower ranking is given to mutant antibodies containing an amino acid substitution that is rarely encountered in either germline sequences or the sequences of other human antibodies. The lowest ranking is given to mutant antibodies with an amino acid substitution that has not been encountered in a germline sequence or the sequence of another human antibody. As set forth above, mutant antibodies comprising at least one activity enhancing amino acid residue located in CDR3 is preferred over CDR2 which is preferred over CDR1. The CDRs of the heavy chain variable regions are preferred over those of the light chain variable region.

[0456] The mutant antibodies can also be studied for improvement in activity, e.g. when compared to their corresponding parental antibody. The improvement in activity of the mutant antibody can be determined for example, by neutralization assays, or binding specificity/affinity by surface plasmon resonance analysis (see Example 3). Preferably, the improvement in activity can be at least 2-20 fold higher than the parental antibody. The improvement in activity can be at least "x.sub.1" to "x.sub.2" fold higher than the parental antibody wherein "x.sub.1" and "x.sub.2" are integers between and including 2 to 20, including ranges within the state range, e.g. 2-15, e.g. 5-10.

[0457] The mutant antibodies with the activity enhancing amino acid residue also can be studied to determine whether at least one other desirable property has been retained after mutation. For example, with anti-hIL-12 antibodies testing for, (1) preservation of non-cross reactivity with other proteins or human tissues, (2) preservation of epitope recognition, i.e. recognizing a p40 epitope preferably in the context of the p70 (p40/p35) heterodimer, thereby preventing binding interference from free soluble p40; and (3) generation of antibodies with heavy and light chain amino acid sequences that were as close as possible to their respective germline immunoglobulin sequences, and determining which would be least likely to elicit a human immune response based on the number of differences from the germline sequence. The same observations can be made on an antibody having more than one activity enhancing amino acid residues, e.g. at least two or at least three activity enhancing amino acid residues, to determine whether retention of the desirable property or characteristic has occurred.

[0458] An example of the use of a "selective mutagenesis approach", in the mutagenesis of Y61 is described below. The individual mutations H31S.fwdarw.E, L50.fwdarw.Y, or L94G.fwdarw.Y each improved neutralization activity of the antibody. However, when combination clones were tested, the activity of the combined clone H31S.fwdarw.E+L50.fwdarw.Y+L94G.fwdarw.Y was no better than L50.fwdarw.Y+L94G.fwdarw.Y (J695). Therefore, changing the germline amino acid residue Ser to Glu at position 31 of CDR1 was unnecessary for the improved activity of J695 over Y61. The selective mutagenesis approach therefore, identified the minimal number of changes that contributed to the final activity, thereby reducing the immunogenic potential of the final antibody and preserving other desired properties of the antibody.

[0459] Isolated DNA encoding the VH and VL produced by the selected mutagenesis approach can be converted into full length antibody chain genes, to Fab fragment genes as to a scFV gene, as described in section IV. For expression of VH and VL regions produced by the selected mutagenesis approach, expression vectors encoding the heavy and light chain can be transfected into variety host cells as described in detail in section IV. Preferred host cells include either prokaryotic host cells, for example, E coli, or eukaryotic host cells, for example, yeast cells, e.g., S. cerevisae. Most preferred eukaryotic host cells are mammalian host cells, described in detail in section IV.

[0460] The selective mutagenesis approach provides a method of producing antibodies with improved activities without prior affinity maturation of the antibody by other means. The selective mutagenesis approach provides a method of producing antibodies with improved affinities which have been subject to back mutations. The selective mutagenesis approach also provides a method of improving the activity of affinity matured antibodies.

[0461] The skilled artisan will recognize that the selective mutagenesis approach can be used in standard antibody manipulation techniques known in the art. Examples include, but are not limited to, CDR grafted antibodies, chimeric antibodies, scFV fragments, Fab fragments of a full length antibodies and human antibodies from other sources, e.g., transgenic mice.

[0462] Rapid large scale mutational analysis of antibodies include in vitro transcription and translation using ribosome display technology (see e.g., Hanes et al., (1997) Proc. Natl. Acad. Sci. 94: 4937-4942; Dall Acqua et al., (1998) Curr. Opin. Struc. Biol. 8: 443-450; He et al., (1997) Nucleic Acid Res. 25: 5132-5134), and U.S. Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. The selective mutagenesis approach also provides a method of producing antibodies with improved activities that can be selected using ribosomal display techniques.

[0463] In the methods of the invention, antibodies or antigen binding portions thereof are further modified by altering individual positions in the CDRs of the HCVR and/or LCVR. Although these modifications can be made in phage-displayed antibodies, the method is advantageous in that it can be performed with antibodies that are expressed in other types of host systems, such as bacterial, yeast or mammalian cell expression systems. The individual positions within the CDRs selected for modification are based on the positions being a contact and/or hypermutation position.

[0464] Preferred contact positions and hypermutation positions as defined herein are shown in Table 3 (see Appendix A) and their modification in accordance with the method of the invention is described in detail in Example 2. Preferred contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96. Preferred hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93. More preferred amino acid residues (referred to as "preferred selective mutagenesis positions") are both contact and hypermutation positions and are selected from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94. Particularly preferred contact positions are selected from the group consisting of L50 and L94.

Preferred activity enhancing amino acid residues replace amino acid residues located at positions selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94, and L96. More preferred activity enhancing amino acid residues replace amino acid residues located at positions H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94. Particularly, preferred activity enhancing amino acid residues replace amino acid residues located at positions selected from the group consisting of L50 and L94.

[0465] In general, the method of the invention involves selecting a particular preferred selective mutagenesis position, contact and/or hypermutation position within a CDR of the heavy or light chain of a parent antibody of interest, or antigen binding portion thereof, randomly mutagenizing that individual position (e.g., by genetic means using a mutagenic oligonucleotide to generate a "mini-library" of modified antibodies), or mutating a position to specific desired amino acids, to identify activity enhancing amino acid residues expressing, and purifying the modified antibodies (e.g., in a non-phage display host system), measuring the activity of the modified antibodies for antigen (e.g., by measuring k.sub.off rates by BIAcore analysis), repeating these steps for other CDR positions, as necessary, and combining individual mutations shown to have improved activity and testing whether the combination(s) generate an antibody with even greater activity (e.g., affinity or neutralizing potency) than the parent antibody, or antigen-binding portion thereof.

[0466] Accordingly, in one embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0467] a) providing a parent antibody or antigen-binding portion thereof;

[0468] b) selecting in order a 1) preferred selective mutagenesis position, 2) contact position, or 3) hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected preferred selective mutagenesis position, contact or hypermutation position;

[0469] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0470] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

[0471] e) optionally, repeating steps a) through d) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0472] f) combining, in the parent antibody, or antigen-binding portion thereof, individual mutations shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0473] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained. Preferably, the selected antibody or antibodies have an improved activity without loss or with retention of at least one desirable characteristic or property of the parental antibody as described above. The desirable characteristic or property can be measured or observed by the ordinarily skilled artisan using art-recognized techniques.

[0474] Preferred contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96. Preferred hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93. More preferred selective mutagenesis positions are selected from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93 and L94. Particularly preferred contact positions are selected from the group consisting of L50 and L94.

[0475] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0476] a) providing a parent antibody or antigen-binding portion thereof;

[0477] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation;

[0478] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0479] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0480] e) optionally, repeating steps a) through d) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0481] f) combining, in the parent antibody, or antigen-binding portion thereof, two individual activity enhancing amino acid residues shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0482] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof; until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0483] Preferred contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96. Preferred hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93. More preferred selective mutagenesis positions are selected from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93 and L94. Particularly preferred contact positions are selected from the group consisting of L50 and L94.

[0484] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0485] a) providing a parent antibody or antigen-binding portion thereof;

[0486] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation;

[0487] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0488] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0489] e) optionally, repeating steps a) through d) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0490] f) combining, in the parent antibody, or antigen-binding portion thereof, three individual activity enhancing amino acid residues shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0491] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0492] Preferably, the activity enhancing amino acid residue replaces amino acid residues located at positions selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96.

[0493] Following mutagenesis of individual selected positions, mutated clones can be sequenced to identify which amino acid residues have been introduced into the selected position in each clone. A small number of clones (e.g., about 24) can be selected for sequencing, which statistically should yield 10-15 unique antibodies, whereas larger numbers of clones (e.g., greater than 60) can be sequenced to ensure that antibodies with every possible substitution at the selected position are identified.

[0494] In one embodiment, contact and/or hypermutation positions within the CDR3 regions of the heavy and/or light chains are first selected for mutagenesis. However, for antibodies that have already been affinity matured in vitro by random mutagenesis of the CDR3 regions via phage display selection, it may be preferably to first select contact and/or hypermutation positions within CDR1 or CDR2 of the heavy and/or light chain.

[0495] In a more preferred embodiment, preferred selective mutagenesis positions within the CDR3 regions of the heavy and/or light chains are first selected for mutagenesis. However, for antibodies that have already been affinity matured in vitro by random mutagenesis of the CDR3 regions via phage display selection, it may be preferably to first select preferred selective mutagenesis positions within CDR1 or CDR2 of the heavy and/or light chain.

[0496] In another preferred embodiment, the optimization of a selected antibody by the selective mutagenesis approach is done sequentially as follows: preferred selective mutagenesis positions selected from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 are mutated first to at least 2 other amino acids each (preferably 5-14 other amino acids) and the resulting antibodies are characterized for increased affinity, neutralization potency (and possibly also for at least one other retained characteristic or property discussed elsewhere). If a mutation of a single preferred selective mutagenesis position does not increase the affinity or neutralization potency at all or sufficiently and if even the combination of multiple activity enhancing amino acids replacing amino acids in preferred selective mutagenesis positions does not result in an combination antibody which meets the target activity (including affinity and/or neutralization potency), additional amino acid residues will be selected for selective mutagenesis from the group consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 are mutated to at least 2 other amino acids each (preferably 5-14 other amino acids) and the resulting antibodies are characterized for increased affinity, neutralization potency (and possibly also for at least one other retained characteristic or property discussed elsewhere).

[0497] If a mutation of a single amino acid residue selected from the group consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 does not increase the activity (including affinity and/or neutralization potency) at all or not sufficiently and if even the combination of multiple activity enhancing amino acids replacing amino acids in those positions does not result in an combination antibody which meets the targeted activity (including affinity and/or target neutralization potency), additional amino acid residues will be selected for selective mutagenesis from the group consisting of H33B, H52B, L31A and are mutated to at least 2 other amino acids each (preferably 5-14 other amino acids) and the resulting antibodies are characterized for increased affinity, neutralization potency (and possibly also for at least one other retained characteristic or property discussed elsewhere).

[0498] It should be understood that the sequential selective mutagenesis approach may end at any of the steps outline above as soon as an antibody with the desired activity (including affinity and neutralization potency) has been identified. If mutagenesis of the preselected positions has identified activity enhancing amino acids residues but the combination antibody still do not meet the targets set for activity (including affinity and neutralization potency) and/or if the identified activity enhancing amino acids also affect other desired characteristics and are therefore not acceptable, the remaining CDR residues may be subjected to mutagenesis (see section IV).

[0499] The method of the invention can be used to improve activity of an antibody, or antigen binding portion thereof, to reach a predetermined target activity (e.g. a predetermined affinity and/or neutralization potency, and/or a desired property or characteristic).

[0500] Accordingly, the invention provides a method of improving the activity of an antibody, or antigen-binding portion thereof, to attain a predetermined target activity, comprising:

[0501] a) providing a parent antibody a antigen-binding portion thereof;

[0502] b) selecting a preferred selective mutagenesis position selected from group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94.

[0503] c) individually mutating the selected preferred selective mutagenesis position to at least two other amino acid residues to hereby create a first panel of mutated antibodies, or antigen binding portions thereof;

[0504] d) evaluating the activity of the first panel of mutated antibodies, or antigen binding portions thereof to determined if mutation of a single selective mutagenesis position produces an antibody or antigen binding portion thereof with the predetermined target activity or a partial target activity;

[0505] e) combining in a stepwise fashion, in the parent antibody, or antigen binding portion thereof, individual mutations shown to have an improved activity, to form combination antibodies, or antigen binding portions thereof.

[0506] f) evaluating the activity of the combination antibodies, or antigen binding portions thereof to determined if the combination antibodies, or antigen binding portions thereof have the predetermined target activity or a partial target activity.

[0507] g) if steps d) or f) do not result in an antibody or antigen binding portion thereof having the predetermined target activity, or result an antibody with only a partial activity, additional amino acid residues selected from the group consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 are mutated to at least two other amino acid residues to thereby create a second panel of mutated antibodies or antigen-binding portions thereof;

[0508] h) evaluating the activity of the second panel of mutated antibodies or antigen binding portions thereof, to determined if mutation of a single amino acid residue selected from the group consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96 results an antibody or antigen binding portion thereof, having the predetermined target activity or a partial activity;

[0509] i) combining in stepwise fashion in the parent antibody, or antigen-binding portion thereof, individual mutations of step g) shown to have an improved activity, to form combination antibodies, or antigen binding portions thereof;

[0510] j) evaluating the activity of the combination antibodies or antigen binding portions thereof, to determined if the combination antibodies, or antigen binding portions thereof have the predetermined target activity or a partial target activity;

[0511] k) if steps h) or j) do not result in an antibody or antigen binding portion thereof having the predetermined target activity, or result in an antibody with only a partial activity, additional amino acid residues selected from the group consisting of H33B, H52B and L31A are mutated to at least two other amino acid residues to thereby create a third panel of mutated antibodies or antigen binding portions thereof;

[0512] l) evaluating the activity of the third panel of mutated antibodies or antigen binding portions thereof, to determine if a mutation of a single amino acid residue selected from the group consisting of H33B, H52B and L31A resulted in an antibody or antigen binding portion thereof, having the predetermined target activity or a partial activity;

[0513] m) combining in a stepwise fashion in the parent antibody, or antigen binding portion thereof, individual mutation of step k) shown to have an improved activity, to form combination antibodies, or antigen binding portions, thereof;

[0514] n) evaluating the activity of the combination antibodies or antigen-binding portions thereof, to determine if the combination antibodies, or antigen binding portions thereof have the predetermined target activity to thereby produce an antibody or antigen binding portion thereof with a predetermined target activity.

[0515] A number of mutagenesis methods can be used, including PCR assembly, Kunkel (dut-ung-) and thiophosphate (Amersham Sculptor kit) oligonucleotide-directed mutagenesis.

[0516] A wide variety of host expression systems can be used to express the mutated antibodies, including bacterial, yeast, baculoviral and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC119(Sfi). Other antibody expression systems are known in the art and/or are described below in section IV.

[0517] The modified antibodies, or antigen binding portions thereof, produced by the method of the invention can be identified without the reliance on phage display methods for selection. Accordingly, the method of the invention is particularly advantageous for improving the activity of a recombinant parent antibody or antigen-binding portion thereof, that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in the phage-display system.

[0518] Accordingly, in another embodiment, the invention provides a method for improving the affinity of an antibody, or antigen-binding portion thereof, comprising:

[0519] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0520] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected contact or hypermutation position;

[0521] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0522] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

[0523] e) optionally repeating steps b) through d) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0524] f) combining, in the parent antibody, or antigen-binding portion thereof, individual mutations shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0525] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0526] Preferred contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96. Preferred hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93. More preferred selective mutagenesis positions are selected from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93 and L94. Particularly preferred contact positions are selected from the group consisting of L50 and L94.

[0527] With available methods it is not possible or it is extremely laborious to derive an antibody with increased binding affinity and neutralization potency while retaining other properties or characteristics of the antibodies as discussed above. The method of this invention, however, can readily identify such antibodies. The antibodies subjected to the method of this invention can come from any source.

[0528] Therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0529] a) providing a recombinant parent antibody or antigen-binding portion thereof;

[0530] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected preferred selective mutagenesis position, contact or hypermutation position;

[0531] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expressing said panel in an appropriate expression system;

[0532] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0533] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristics, wherein the property or characteristic is one that needs to be retained in the antibody;

until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0534] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0535] In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0536] In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0537] In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0538] If therefore, the affinity of an antibody for a specific antigen should be improved, but where the phage display (or related system including ribosome display) method is no longer applicable, and other desirable properties or characteristics should be retained, the method of the invention can be used. Accordingly, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0539] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0540] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected preferred selective mutagenesis position, contact or hypermutation position;

[0541] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0542] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0543] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristic, wherein the property or characteristic is one that needs to be retained, until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0544] f) optionally, repeating steps a) through e) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0545] g) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and at least one retained property or characteristic, to form combination antibodies, or antigen-binding portions thereof; and

[0546] h) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained other property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0547] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0548] In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0549] In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0550] In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0551] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0552] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0553] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected contact or hypermutation position;

[0554] c) individually mutating said selected preferred selective mutagenesis position, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0555] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0556] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristic, wherein the property or characteristic is one that needs to be retained, until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0557] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0558] In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0559] In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0560] In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0561] In another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0562] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0563] b) selecting a preferred selective mutagenesis position, contact or hypermutation position within a complementarity determining region (CDR) for mutation, thereby identifying a selected contact or hypermutation position;

[0564] c) individually mutating said selected preferred selective mutagenesis positions, contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0565] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0566] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof for at least one other property or characteristic, wherein the property or characteristic is one that needs to be retained, until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0567] f) optionally, repeating steps a) through e) for at least one other preferred selective mutagenesis position, contact or hypermutation position;

[0568] g) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and at least on retained other characteristic, to form combination antibodies, or antigen-binding portions thereof; and

[0569] h) evaluating the activity of the combination antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0570] In a preferred embodiment, the contact positions are selected from the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0571] In another preferred embodiment, the hypermutation positions are selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and L93 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0572] In a more preferred embodiment the residues for selective mutagenesis are selected from the preferred selective mutagenesis positions from the group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0573] In a more preferred embodiment, the contact positions are selected from the group consisting of L50 and L94 and the other characteristic is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

IV. Modifications of Other CDR Residues

[0574] Ultimately, all CDR residues in a given antibody-antigen pair identified by any means to be required as activity enhancing amino acid residues and/or required directly or indirectly for binding to the antigen and/or for retaining other desirable properties or characteristics of the antibody. Such CDR residues are referred to as "preferred selective mutagenesis positions". It should be noted that in specific circumstances that preferred selective mutagenesis residues can be identified also by other means including co-crystallization of antibody and antigen and molecular modeling.

[0575] If the preferred attempts to identify activity enhancing amino acids focussing on the preferred selective mutagenesis positions, contact or hypermutation positions described above are exhausted, or if additional improvements are required, the remaining CDR residues may be modified as described below. It should be understood that the antibody could already be modified in any one or more contact or hypermutation positions according to the embodiments discussed above but may require further improvements. Therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0576] a) providing a parent antibody or antigen-binding portion thereof;

[0577] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0578] c) individually mutating said selected position e.g., to at least two other amino acid residues to thereby create a mutated antibody or a panel of mutated antibodies, or antigen-binding portions thereof;

[0579] d) evaluating the activity of the mutated antibody or the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0580] e) evaluating the mutated antibody or the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other property or characteristic until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0581] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence

[0582] If mutagenesis of a single residue is not sufficient other residues can be included; therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0583] a) providing a parent antibody or antigen-binding portion thereof;

[0584] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0585] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0586] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0587] e) repeating steps b) through d) for at least one other CDR position which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0588] f) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0589] g) evaluating the activity of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0590] If the preferred attempts to identify activity enhancing amino acids focussing on the contact or hypermutation positions described above are exhausted, or if additional improvements are required, and the antibody in question can not further be optimized by mutagenesis and phage display (or related ribosome display) methods the remaining CDR residues may be modified as described below. It should be understood that the antibody could already be modified in any one or more preferred selective mutagenesis position, contact or hypermutation positions according to the embodiments discussed above but may require further improvements.

[0591] Therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0592] a) providing a recombinant parent antibody or antigen-binding portion thereof; that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0593] b) selecting a selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and;

[0594] c) individually mutating said selected contact or hypermutation position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof, and expressing said panel in a non-phage display system;

[0595] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0596] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other property or characteristic, until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0597] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence.

[0598] If a single mutagenesis is not sufficient to increase the affinity of the antibody other residues may be included in the mutagenesis. Therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0599] a) providing a parent antibody or antigen-binding portion thereof that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0600] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0601] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expression in a non-phage display system;

[0602] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0603] e) repeating steps b) through d) for at least one other position which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94;

[0604] g) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity, to form combination antibodies, or antigen-binding portions thereof; and

[0605] h) evaluating the activity and other property or characteristic of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof;

until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0606] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence

[0607] The preferred attempts to identify activity enhancing amino acids focussing on the preferred selective mutagenesis positions, contact or hypermutation positions described may be exhausted, or additional improvements may be required, and it is important to retain other properties or characteristics of the antibody.

[0608] Therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, without affecting other characteristics, comprising:

[0609] a) providing a parent antibody or antigen-binding portion thereof;

[0610] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0611] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0612] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0613] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other property or characteristic until an antibody, or antigen-binding portion thereof, with an improved activity and retained other property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0614] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence

[0615] If mutagenesis of a single residue is not sufficient other residues can be included; therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0616] a) providing a parent antibody or antigen-binding portion thereof;

[0617] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0618] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof;

[0619] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0620] e.) evaluating the panel of mutated antibodies or antigen-binding portions thereof, relative to the parent antibody or antigen-portion thereof, for changes in at least one other characteristic or property;

[0621] e) repeating steps b) through e) for at least one other CDR position which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0622] f) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and not affecting at least one other property or characteristic, to form combination antibodies, or antigen-binding portions thereof; and

[0623] g) evaluating the activity and the retention of at least one other property or characteristic of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof until an antibody, or antigen-binding portion thereof, with an improved activity and at least one retained other property or characteristic, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0624] Mutagenesis of the preferred selective mutagenesis position, contact and hypermutation residues may not have increased the affinity of the antibody sufficiently, and mutagenesis and the phage display method (or related ribosome display method) may no longer be useful and at least one other characteristic or property of the antibody should be retained.

[0625] Therefore, in another embodiment the invention provides a method to improve the affinity of an antibody or antigen-binding portion thereof, comprising:

[0626] a) providing a parent antibody or antigen-binding portion thereof that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0627] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0628] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expression in a non-phage display system;

[0629] d) evaluating the activity of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof thereby identifying an activity enhancing amino acid residue;

[0630] e) evaluating the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, for changes in at least one other property or characteristic until an antibody, or antigen-binding portion thereof, with an improved activity, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

[0631] Preferably, the other characteristic or property is selected from 1) preservation of non-crossreactivity with other proteins or human tissues, 2) preservation of epitope recognition, i.e. recognizing p40 epitope preferably in the context of the p70 p40/p35 heterodimer preventing binding interference from free, soluble p40 and/or 3) to produce an antibody with a close to germline immunoglobulin sequence

[0632] If mutagenesis of a single residue is not sufficient other residues can be included; therefore, in another embodiment, the invention provides a method for improving the activity of an antibody, or antigen-binding portion thereof, comprising:

[0633] a) providing a parent antibody or antigen-binding portion thereof that was obtained by selection in a phage-display system but whose activity cannot be further improved by mutagenesis in said phage-display system;

[0634] b) selecting an amino acid residue within a complementarity determining region (CDR) for mutation other than H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0635] c) individually mutating said selected position to at least two other amino acid residues to thereby create a panel of mutated antibodies, or antigen-binding portions thereof and expression in a non-phage display system;

[0636] d) evaluating the activity and retention of at least one other property or characteristic of the panel of mutated antibodies, or antigen-binding portions thereof, relative to the parent antibody or antigen-binding portion thereof, thereby identifying an activity enhancing amino acid residue;

[0637] e) repeating steps b) through d) for at least one other CDR position which is neither the position selected under b) nor a position at H30, H31, H31B, H32, H33, H35, H50, H52, H52A, H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50, L52, L53, L55, L91, L92, L93, L94 and L96;

[0638] f) combining, in the parent antibody, or antigen-binding portion thereof, at least two individual activity enhancing amino acid residues shown to have improved activity and not to affect at least one other property or characteristic, to form combination antibodies, or antigen-binding portions thereof; and

[0639] g) evaluating the activity and retention of at least one property or characteristic of the combination antibodies, or antigen-binding portions thereof with two activity enhancing amino acid residues, relative to the parent antibody or antigen-binding portion thereof until an antibody, or antigen-binding portion thereof, with an improved activity and at least one other retained characteristic or property, relative to the parent antibody, or antigen-binding portion thereof, is obtained.

V. Expression of Antibodies

[0640] An antibody, or antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.

[0641] To obtain a DNA fragment encoding the heavy chain variable region of Joe 9 wt or a Joe 9 wt-related antibody, antibodies specific for human IL-12 were screened from human libraries and mutated, as described in section II. Once DNA fragments encoding Joe 9 wt or Joe 9 wt-related VH and VL segments are obtained, mutagenesis of these sequences is carried out by standard methods, such as PCR site directed mutagenesis (PCR-mediated mutagenesis in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or other site-directed mutagenesis methods. Human IL-12 antibodies that displayed a level of activity and binding specificity/affinity that was desirable, for example J695, were further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0642] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region and any allotypic variant therein as described in Kabat (, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), but most preferably is an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0643] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but most preferably is a lambda constant region.

[0644] To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly.sub.4-Ser).sub.3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

[0645] To express the antibodies, or antibody portions of the invention, DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the J695 or J695-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting the J695 or J695-related VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0646] In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., U.S. Pat. No. 5,464,758 by Bujard et al. and U.S. Pat. No. 5,654,168 by Bujard et al.

[0647] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

[0648] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0649] Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to hIL-12 The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than hIL-12 by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.

[0650] In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are culture to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Antibodies or antigen-binding portions thereof of the invention can be expressed in an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res. 20: 6287-6295). Plant cells can also be modified to create transgenic plants that express the antibody or antigen binding portion thereof, of the invention.

[0651] In view of the foregoing, another aspect of the invention pertains to nucleic acid, vector and host cell compositions that can be used for recombinant expression of the antibodies and antibody portions of the invention. Preferably, the invention features isolated nucleic acids that encode CDRs of J695, or the full heavy and/or light chain variable region of J695. Accordingly, in one embodiment, the invention features an isolated nucleic acid encoding an antibody heavy chain variable region that encodes the J695 heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25. Preferably, the nucleic acid encoding the antibody heavy chain variable region further encodes a J695 heavy chain CDR2 which comprises the amino acid sequence of SEQ ID NO: 27. More preferably, the nucleic acid encoding the antibody heavy chain variable region further encodes a J695 heavy chain CDR1 which comprises the amino acid sequence of SEQ ID NO: 29. Even more preferably, the isolated nucleic acid encodes an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 (the full VH region of J695).

[0652] In other embodiments, the invention features an isolated nucleic acid encoding an antibody light chain variable region that encodes the J695 light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 26. Preferably, the nucleic acid encoding the antibody light chain variable region further encodes a J695 light chain CDR2 which comprises the amino acid sequence of SEQ ID NO: 28. More preferably, the nucleic acid encoding the antibody light chain variable region further encodes a J695 light chain CDR1 which comprises the amino acid sequence of SEQ ID NO: 30. Even more preferably, the isolated nucleic acid encodes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 32 (the full VL region of J695).

[0653] The invention also provides recombinant expression vectors encoding both an antibody heavy chain and an antibody light chain. For example, in one embodiment, the invention provides a recombinant expression vector encoding: [0654] a) an antibody heavy chain having a variable region comprising the amino acid sequence of SEQ ID NO: 31; and [0655] b) an antibody light chain having a variable region comprising the amino acid sequence of SEQ ID NO: 32.

[0656] The invention also provides host cells into which one or more of the recombinant expression vectors of the invention have been introduced. Preferably, the host cell is a mammalian host cell, more preferably the host cell is a CHO cell, an NS0 cell or a COS cell. Still further the invention provides a method of synthesizing a recombinant human antibody of the invention by culturing a host cell of the invention in a suitable culture medium until a recombinant human antibody of the invention is synthesized. The method can further comprise isolating the recombinant human antibody from the culture medium.

VI. Pharmaceutical Compositions and Pharmaceutical Administration

[0657] The antibodies and antibody-portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.

[0658] The antibodies and antibody-portions of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. Preferably, the antibody or antibody-portions will be prepared as an injectable solution containing 0.1-250 mg/ml antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule or pre-filled syringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants.

[0659] In a preferred embodiment, the pharmaceutical composition includes the antibody at a dosage of about 0.01 mg/kg-10 mg/kg. More preferred dosages of the antibody include 1 mg/kg administered every other week, or 0.3 mg/kg administered weekly.

[0660] The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

[0661] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0662] The antibodies and antibody-portions of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0663] In certain embodiments, an antibody or antibody portion of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[0664] Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an antibody or antibody portion of the invention is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which IL-12 activity is detrimental. For example, an anti-hIL-12 antibody or antibody portion of the invention may be coformulated and/or coadministered with one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules). Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies. It will be appreciated by the skilled practitioner that when the antibodies of the invention are used as part of a combination therapy, a lower dosage of antibody may be desirable than when the antibody alone is administered to a subject (e.g., a synergistic therapeutic effect may be achieved through the use of combination therapy which, in turn, permits use of a lower dose of the antibody to achieve the desired therapeutic effect).

[0665] Interleukin 12 plays a critical role in the pathology associated with a variety of diseases involving immune and inflammatory elements. These diseases include, but are not limited to, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, atopic dermatitis, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, idiopathic leucopenia, autoimmune neutropenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), insulin-dependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis and vitiligo. The human antibodies, and antibody portions of the invention can be used to treat autoimmune diseases, in particular those associated with inflammation, including, rheumatoid spondylitis, allergy, autoimmune diabetes, autoimmune uveitis.

[0666] Preferably, the antibodies of the invention or antigen-binding portions thereof, are used to treat rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin dependent diabetes mellitus and psoriasis, as described in more detail in section VII.

[0667] A human antibody, or antibody portion, of the invention also can be administered with one or more additional therapeutic agents useful in the treatment of autoimmune and inflammatory diseases.

[0668] Antibodies of the invention, or antigen binding portions thereof can be used alone or in combination to treat such diseases. It should be understood that the antibodies of the invention or antigen binding portion thereof can be used alone or in combination with an additional agent, e.g., a therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art-recognized as being useful to treat the disease or condition being treated by the antibody of the present invention. The additional agent also can be an agent which imparts a beneficial attribute to the therapeutic composition e.g., an agent which effects the viscosity of the composition.

[0669] It should further be understood that the combinations which are to be included within this invention are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations which are part of this invention can be the antibodies of the present invention and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.

[0670] Preferred combinations are non-steroidal anti-inflammatory drug(s) also referred to as NSAIDS which include drugs like ibuprofen. Other preferred combinations are corticosteroids including prednisolone; the well known side-effects of steroid use can be reduced or even eliminated by tapering the steroid dose required when treating patients in combination with the anti-IL-12 antibodies of this invention. Non-limiting examples of therapeutic agents for rheumatoid arthritis with which an antibody, or antibody portion, of the invention can be combined include the following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands including CD154 (gp39 or CD40L).

[0671] Preferred combinations of therapeutic agents may interfere at different points in the autoimmune and subsequent inflammatory cascade; preferred examples include TNF antagonists like chimeric, humanized or human TNF antibodies, D2E7, (U.S. application Ser. No. 08/599,226 filed Feb. 9, 1996), cA2 (Remicade.TM.), CDP 571, anti-TNF antibody fragments (e.g., CDP870), and soluble p55 or p75 TNF receptors, derivatives thereof, (p75TNFR1gG (Enbrel.TM.) or p55TNFR1gG (Lenercept), soluble IL-13 receptor (sIL-13), and also TNF.alpha. converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors (e.g., Interleukin-1-converting enzyme inhibitors, such as Vx740, or IL-1RA etc.) may be effective for the same reason. Other preferred combinations include Interleukin 11, anti-P7s and p-selectin glycoprotein ligand (PSGL). Yet another preferred combination are other key players of the autoimmune response which may act parallel to, dependent on or in concert with IL-12 function; especially preferred are IL-18 antagonists including IL-18 antibodies or soluble IL-18 receptors, or IL-18 binding proteins. It has been shown that IL-12 and IL-18 have overlapping but distinct functions and a combination of antagonists to both may be most effective. Yet another preferred combination are non-depleting anti-CD4 inhibitors. Yet other preferred combinations include antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2) including antibodies, soluble receptors or antagonistic ligands.

[0672] The antibodies of the invention, or antigen binding portions thereof, may also be combined with agents, such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine, olsalazine chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate (intramuscular and oral), azathioprine, cochicine, corticosteroids (oral, inhaled and local injection), beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting enzyme inhibitors (e.g., Vx740), anti-P7s, p-selectin glycoprotein ligand (PSGL), TNF.alpha. converting enzyme (TACE) inhibitors, T-cell signalling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel.TM.) and p55TNFRIgG (Lenercept), sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13)) and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGF.beta.). Preferred combinations include methotrexate or leflunomide and in moderate or severe rheumatoid arthritis cases, cyclosporine.

[0673] Non-limiting examples of therapeutic agents for inflammatory bowel disease with which an antibody, or antibody portion, of the invention can be combined include the following: budenoside; epidermal growth factor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1.beta. monoclonal antibodies; anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. The antibodies of the invention, or antigen binding portions thereof, may also be combined with agents, such as methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting enzyme inhibitors (e.g., Vx740), anti-P7s, p-selectin glycoprotein ligand (PSGL), TNF.alpha. converting enzyme inhibitors, T-cell signalling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13)) and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGF.beta.).

[0674] Preferred examples of therapeutic agents for Crohn's disease in which an antibody or an antigen binding portion can be combined include the following: TNF antagonists, for example, anti-TNF antibodies, D2E7 (U.S. application Ser. No. 08/599,226, filed Feb. 9, 1996), cA2 (Remicade.TM.), CDP 571, anti-TNF antibody fragments (e.g., CDP870), TNFR-Ig constructs (p75TNFRIgG (Enbrel.TM.) and p55TNFRIgG (Lenercept)), anti-P7s, p-selectin glycoprotein ligand (PSGL), soluble IL-13 receptor (sIL-13), and PDE4 inhibitors. Antibodies of the invention or antigen binding portions thereof, can be combined with corticosteroids, for example, budenoside and dexamethasone. Antibodies of the invention or antigen binding portions thereof, may also be combined with agents such as sulfasalazine, 5-aminosalicylic acid and olsalazine, and agents which interfere with synthesis or action of proinflammatory cytokines such as IL-1, for example, IL-1.beta. converting enzyme inhibitors (e.g., Vx740) and IL-1ra. Antibodies of the invention or antigen binding portion thereof may also be used with T cell signaling inhibitors, for example, tyrosine kinase inhibitors 6-mercaptopurines. Antibodies of the invention or antigen binding portions thereof, can be combined with IL-11.

[0675] Non-limiting examples of therapeutic agents for multiple sclerosis with which an antibody, or antibody portion, of the invention can be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-.beta.1a (Avonex; Biogen); interferon-.beta.1b (Betaseron; Chiron/Berlex); Copolymer 1 (Cop-1; Copaxone; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigen binding portions thereof, can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. The antibodies of the invention, or antigen binding portions thereof, may also be combined with agents, such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adensosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents which interfere with signalling by proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting enzyme inhibitors (e.g., Vx740), anti-P7s, p-selectin glycoprotein ligand (PSGL), TACE inhibitors, T-cell signalling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13)) and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-13 and TGF.beta.).

[0676] Preferred examples of therapeutic agents for multiple sclerosis in which the antibody or antigen binding portion thereof can be combined to include interferon-.beta., for example, IFN.beta.1a and IFN.beta.1b; copaxone, corticosteroids, IL-1 inhibitors, TNF inhibitors, and antibodies to CD40 ligand and CD80.

[0677] The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0678] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0679] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.01-20 mg/kg, more preferably 1-10 mg/kg, even more preferably 0.3-1 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

VII. Uses of the Antibodies of the Invention

[0680] Given their ability to bind to hIL-12, the anti-hIL-12 antibodies, or portions thereof, of the invention can be used to detect hIL-12 (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method for detecting hIL-12 in a biological sample comprising contacting a biological sample with an antibody, or antibody portion, of the invention and detecting either the antibody (or antibody portion) bound to hIL-12 or unbound antibody (or antibody portion), to thereby detect hIL-12 in the biological sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.

[0681] Alternative to labeling the antibody, hIL-12 can be assayed in biological fluids by a competition immunoassay utilizing rhIL-12 standards labeled with a detectable substance and an unlabeled anti-hIL-12 antibody. In this assay, the biological sample, the labeled rhIL-12 standards and the anti-hIL-12 antibody are combined and the amount of labeled rhIL-12 standard bound to the unlabeled antibody is determined. The amount of hIL-12 in the biological sample is inversely proportional to the amount of labeled rhIL-12 standard bound to the anti-hIL-12 antibody.

[0682] The Y61 and J695 antibodies of the invention can also be used to detect IL-12 from species other than humans, in particular IL-12 from primates. For example, Y61 can be used to detect IL-12 in the cynomolgus monkey and the rhesus monkey. J695 can be used to detect IL-12 in the cynomolgus monkey, rhesus monkey, and baboon. However, neither antibody cross reacts with mouse or rat IL-12 (see Example 3, subsection F).

[0683] The antibodies and antibody portions of the invention are capable of neutralizing hIL-12 activity in vitro (see Example 3) and in vivo (see Example 4). Accordingly, the antibodies and antibody portions of the invention can be used to inhibit IL-12 activity, e.g., in a cell culture containing hIL-12, in human subjects or in other mammalian subjects having IL-12 with which an antibody of the invention cross-reacts (e.g. primates such as baboon, cynomolgus and rhesus). In a preferred embodiment, the invention provides an isolated human antibody, or antigen-binding portion thereof, that neutralizes the activity of human IL-12, and at least one additional primate IL-12 selected from the group consisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12, cynomolgus IL-12 and rhesus IL-12, but which does not neutralize the activity of the mouse IL-12. Preferably, the IL-12 is human IL-12. For example, in a cell culture containing, or suspected of containing hIL-12, an antibody or antibody portion of the invention can be added to the culture medium to inhibit hIL-12 activity in the culture.

[0684] In another embodiment, the invention provides a method for inhibiting IL-12 activity in a subject suffering from a disorder in which IL-12 activity is detrimental. IL-12 has been implicated in the pathophysiology of a wide variety of disorders (Windhagen et al., (1995) J. Exp. Med. 182: 1985-1996; Morita et al. (1998) Arthritis and Rheumatism. 41: 306-314; Bucht et al., (1996) Clin. Exp. Immunol. 103: 347-367; Fais et al. (1994) J. Interferon Res. 14:235-238; Parronchi et al., (1997) Am. J. Path. 150:823-832; Monteleone et al., (1997) Gastroenterology. 112:1169-1178, and Berrebi et al., (1998) Am. J. Path 152:667-672; Parronchi et al (1997) Am. J. Path. 150:823-832). The invention provides methods for inhibiting IL-12 activity in a subject suffering from such a disorder, which method comprises administering to the subject an antibody or antibody portion of the invention such that IL-12 activity in the subject is inhibited. Preferably, the IL-12 is human IL-12 and the subject is a human subject. Alternatively, the subject can be a mammal expressing a IL-12 with which an antibody of the invention cross-reacts. Still further the subject can be a mammal into which has been introduced hIL-12 (e.g., by administration of hIL-12 or by expression of an hIL-12 transgene). An antibody of the invention can be administered to a human subject for therapeutic purposes (discussed further below). Moreover, an antibody of the invention can be administered to a non-human mammal expressing a IL-12 with which the antibody cross-reacts for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration).

[0685] As used herein, the phrase "a disorder in which IL-12 activity is detrimental" is intended to include diseases and other disorders in which the presence of IL-12 in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which IL-12 activity is detrimental is a disorder in which inhibition of IL-12 activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of IL-12 in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of IL-12 in serum, plasma, synovial fluid, etc. of the subject), which can be detected, for example, using an anti-IL-12 antibody as described above. There are numerous examples of disorders in which IL-12 activity is detrimental. In one embodiment, the antibodies or antigen binding portions thereof, can be used in therapy to treat the diseases or disorders described herein. In another embodiment, the antibodies or antigen binding portions thereof, can be used for the manufacture of a medicine for treating the diseases or disorders described herein. The use of the antibodies and antibody portions of the invention in the treatment of a few non-limiting specific disorders is discussed further below:

A. Rheumatoid Arthritis:

[0686] Interleukin-12 has been implicated in playing a role in inflammatory diseases such as rheumatoid arthritis. Inducible IL-12p40 message has been detected in synovia from rheumatoid arthritis patients and IL-12 has been shown to be present in the synovial fluids from patients with rheumatoid arthritis (see e.g., Morita et al., (1998) Arthritis and Rheumatism 41: 306-314). IL-12 positive cells have been found to be present in the sublining layer of the rheumatoid arthritis synovium. The human antibodies, and antibody portions of the invention can be used to treat, for example, rheumatoid arthritis, juvenile rheumatoid arthritis, Lyme arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis. Typically, the antibody, or antibody portion, is administered systemically, although for certain disorders, local administration of the antibody or antibody portion may be beneficial. An antibody, or antibody portion, of the invention also can be administered with one or more additional therapeutic agents useful in the treatment of autoimmune diseases.

[0687] In the collagen induced arthritis (CIA) murine model for rheumatoid arthritis, treatment of mice with an anti-IL-12 mAb (rat anti-mouse IL-12 monoclonal antibody, C17.15) prior to arthritis profoundly suppressed the onset, and reduced the incidence and severity of disease. Treatment with the anti-IL-12 mAb early after onset of arthritis reduced severity, but later treatment of the mice with the anti-IL-12 mAb after the onset of disease had minimal effect on disease severity.

B. Crohn's Disease

[0688] Interleukin-12 also plays a role in the inflammatory bowel disease, Crohn's disease. Increased expression of IFN-.gamma. and IL-12 occurs in the intestinal mucosa of patients with Crohn's disease (see e.g., Fais et al., (1994) J. Interferon Res. 14: 235-238; Parronchi et al., (1997) Amer. J. Pathol. 150: 823-832; Monteleone et al., (1997) Gastroenterology 112: 1169-1178; Berrebi et al., (1998) Amer. J. Pathol. 152: 667-672). Anti-IL-12 antibodies have been shown to suppress disease in mouse models of colitis, e.g., TNBS induced colitis IL-2 knockout mice, and recently in IL-10 knock-out mice. Accordingly, the antibodies, and antibody portions, of the invention, can be used in the treatment of inflammatory bowel diseases.

C. Multiple Sclerosis

[0689] Interleukin-12 has been implicated as a key mediator of multiple sclerosis. Expression of the inducible IL-12 p40 message or IL-12 itself can be demonstrated in lesions of patients with multiple sclerosis (Windhagen et al., (1995) J. Exp. Med. 182: 1985-1996, Drulovic et al., (1997) J. Neurol. Sci. 147: 145-150). Chronic progressive patients with multiple sclerosis have elevated circulating levels of IL-12. Investigations with T-cells and antigen presenting cells (APCs) from patients with multiple sclerosis revealed a self-perpetuating series of immune interactions as the basis of progressive multiple sclerosis leading to a Th1-type immune response. Increased secretion of IFN-.gamma. from the T cells led to increased IL-12 production by APCs, which perpetuated the cycle leading to a chronic state of a Th1-type immune activation and disease (Balashov et al., (1997) Proc. Natl. Acad. Sci. 94: 599-603). The role of IL-12 in multiple sclerosis has been investigated using mouse and rat experimental allergic encephalomyelitis (EAE) models of multiple sclerosis. In a relapsing-remitting EAE model of multiple sclerosis in mice, pretreatment with anti-IL-12 mAb delayed paralysis and reduced clinical scores. Treatment with anti-IL-12 mAb at the peak of paralysis or during the subsequent remission period reduced clinical scores. Accordingly, the antibodies or antigen binding portions thereof of the invention may serve to alleviate symptoms associated with multiple sclerosis in humans.

D. Insulin-Dependent Diabetes Mellitus

[0690] Interleukin-12 has been implicated as an important mediator of insulin-dependent diabetes mellitus (IDDM). IDDM was induced in NOD mice by administration of IL-12, and anti-IL-12 antibodies were protective in an adoptive transfer model of IDDM. Early onset IDDM patients often experience a so-called "honeymoon period" during which some residual islet cell function is maintained. These residual islet cells produce insulin and regulate blood glucose levels better than administered insulin. Treatment of these early onset patients with an anti-IL-12 antibody may prevent further destruction of islet cells, thereby maintaining an endogenous source of insulin.

E. Psoriasis

[0691] Interleukin-12 has been implicated as a key mediator in psoriasis. Psoriasis involves acute and chronic skin lesions that are associated with a TH1-type cytokine expression profile. (Hamid et al. (1996) J. Allergy Clin. Immunol. 1:225-231; Turka et al. (1995) Mol. Med. 1:690-699). IL-12 p35 and p40 mRNAs were detected in diseased human skin samples. Accordingly, the antibodies or antigen binding portions thereof of the invention may serve to alleviate chronic skin disorders such psoriasis.

[0692] The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including literature references, issued patents, and published patent applications, as cited throughout this application are hereby expressly incorporated by reference. It should further be understood that the contents of all the tables attached hereto (see Appendix A) are incorporated by reference.

TABLE-US-00002 TABLE 1 VH3 Family Germline Amino Acid Sequences Numbering according to Kabat (Joe9 VH included for comparison) SEQ ID germline NO: VH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 594 dp-29 E V Q L V E S G G G L V Q P G G S L R L S C A A S 595 DP-30 E V Q L V E S G G G L V Q P G G S L R L S C A A S 596 HC15-7 E V Q L V E S G G G L V Q P G G S L R L S C A A S 597 VHD26 E V Q L L E S G G G L V Q P G G S L R L S C A A S 598 DP-31 E V Q L V E S G G G L V Q P G B S L R L S C A A S 599 DP-32 E V Q L V E S G G G V V R P G G S L R L S C A A S 600 DP-33 E V Q L V E S G G V V V Q P G G S L R L S C A A S 601 dp-35 Q V Q L V E S G G G L V K P G G S L R L S C A A S 602 VH3-8 E V Q L L E S G G G L V K P G G S L R L S C A A S 603 yac-9 E V Q L V E S G G G L V Q P G G S L K L S C A A S 604 dp-38 E V Q L V E S G G G L V K P G G S L R L S C A A S 605 LSG2 E V Q L V E S G G G L V K P G G S L R L S C A A S 606 LSG3 E V Q L V E S G G G L V K P G G S L R L S C A A S 607 LSG4 E V Q L V E S G G G L V K P G G S L R L S C A A S 608 LSG6 E V Q L V E S G G G L V K P G G S L R L S C A A S 609 v3-15 E V Q L V E S G G A L V K P G G S L R L S C A A S 610 dp-39 E V Q L V E S G G G L V Q P G G S L R L S C P A S 611 dp-40 E V Q L V E S G G G L V Q P G G S L R L S C A A S 612 dp-59 E V Q L V E S G G G L V Q P G G S L R L S C A A S 613 v3-16p E V Q L V E S G G G L V Q P G G S L R L S C A A S 614 v3-19p T V Q L V E S G G G L V E P G G S L R L S C A A S 615 v3-13 E V H L V E S G G G L V Q P G G A L R L S C A A S 616 DP-42 E V Q L V E T G G G L I Q P G G S L R L S C A A S 617 dp-44 E V Q L V Q S G G G L V H P G G S L R L S C A G S 618 DP-45 E V Q L V Q S G G G L V Q P G G S L R L S C A G S 619 dp-47 E V Q L L E S G G G L V Q P G G S L R L S C A A S 620 flm E V Q L V E S G G G L V Q P G G S L R L S C S A S 621 P1 E V Q L V E S G G G L V Q P G G S L R L S C S A S 622 v3-64 E V Q L V E S G G G L V Q P G G S L R L S C A A S 623 vh26 E V Q L L E S G G G L V Q P G G S L R L S C A A S 624 B25 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 625 b32e Q V Q L V E S G G G V V Q P G R S L R L S C A A S 626 B37 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 627 B43 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 628 B48 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 629 B52 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 630 B54 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 631 cos-8 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 632 cp-46 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 633 F2M Q V Q L V E S G G G L V Q P G G S L R L S C S A S 634 F3 Q V Q L V E S G G G L V Q P G G S L R L S C S A S 635 F7 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 636 hv3005 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 637 P2 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 638 dp-48 E V Q L V E S G G G L V Q P G G S L R L S C A A S 639 dp-58 E V Q L V E S G G G L V Q P G G S L R L S C A A S 640 B1 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 641 B13 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 642 B18 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 643 B26 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 644 B28E Q V Q L V E S G G G V V Q P G R S L R L S C A A S 645 B29E Q V Q L V E S G G G V V Q P G R S L R L S C A A S 646 B29M Q V Q L V E S G G G V V Q P G R S L R L S C A A S 647 B30 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 648 B32M Q V Q L V E S G G G V V Q P G R S L R L S C A A S 649 cos-3 Q V Q L V E S G G G V V Q P G G S L R L S C A A S 650 dp-49 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 651 dP-50 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 652 P6 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 653 P9E Q V Q L V E S G G G V V Q P G R S L R L S C A A S 654 v3-30 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 655 v3-33 Q V Q L V E S G G G V V Q P G R S L R L S C A A S 656 dp-51 E V Q L V E S G G G L V Q P G G S L R L S C A A S 657 dp-77 E V Q L V E S G G G L V K P G G S L R L S C A A S 658 HHG4 E V Q L V E S G G G L V K P G G S L R L S C A A S 659 v3-21 E V Q L V E S G G G L V K P G G S L R L S C A A S 660 v3-48 E V Q L V E S G G G L V Q P G G S L R L S C A A S 661 DP-52 E D Q L V E S G G G L V Q P G G S L R P S C A A S 662 cos-6 E V Q L V E S G G G L V Q P G G S L R L S C A A S 663 dp-53 E V Q L V E S G G G L V Q P G G S L R L S C A A S 664 dp-54 E V Q L V E S G G G L V Q P G G S L R L S C A A S 665 dp-87 E V Q L V E S G G G L V Q P G G S L R L S C A A S 666 VH3-11 E V Q L V E S G G G L V Q P G G S L R L S C A A S 667 JOE9 VH Q V Q L V Q S G G G V V Q P G R S L R L S C A A S VH3 Family Germline Amino Acid Sequences Numbering according to Kabat (Joe9 VH included for comparison) SEQ ID germline CDR H1 NO: VH 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 594 dp-29 G F T F S D H Y M D W V R Q A P G K G L E W V G 595 DP-30 G F T F S D H Y M S W V R Q A Q G K G L E L V G 596 HC15-7 G F T F S D H Y M S W V R Q A Q G K G L E L V G 597 VHD26 G F T F S D H Y M S W V R Q A Q G K G L E L V G 598 DP-31 G F T F D D Y A M H W V R Q A P G K G L E W V S 599 DP-32 G F T F D D Y G M S W V R Q A P G K G L E W V S 600 DP-33 G F T F D D Y T M H W V R Q A P G K G L E W V S 601 dp-35 G F T F S D Y Y M S W I R Q A P G K G L E W V S 602 VH3-8 G F T F S D Y Y M S W I R Q A P G K G L E W V S 603 yac-9 G F T F S G S A M H W V R Q A S G K G L E W V G 604 dp-38 G F T F S N A W M S W V R Q A P G K G L E W V G 605 LSG2 G F T F S N A W M S W V R Q A P G K G L E W V G 606 LSG3 G F T F S N A W M S W V R Q A P G K G L E W V G 607 LSG4 G F T F S N A W M S W V R Q A P G K G L E W V G 608 LSG6 G F T F S N A W M N W V R Q A P G K G L E W V G 609 v3-15 G F T F S N A W M S W V R Q A P G K G L E W V G 610 dp-39 G F T F S N H Y M S W V R Q A P G K G L E W V S 611 dp-40 G F T F S N H Y T S W V R Q A P G K G L E W V S 612 dp-59 G F T F S N S D M N W V H Q A P G K G L E W V S 613 v3-16p G F T F S N S D M N W A R K A P G K G L E W V S 614 v3-19p G F T F S N S D M N W V R Q A P G K G L E W V S 615 v3-13 G F T F S N Y D M H W V R Q A T G K G L E W V S 616 DP-42 G F T V S S N Y M S W V R Q A P G K G L E W V S 617 dp-44 G F T F S S Y A M H W V R Q A P G K G L E W V S 618 DP-45 G F T F S S Y A M H W V R Q A P G K G L E W V S 619 dp-47 G F T F S S Y A M S W V R Q A P G K G L E W V S 620 flm G F T F S S Y A M H W V R Q A P G K G L E Y V S 621 P1 G F T F S S Y A M H W V R Q A P G K G L E Y V S 622 v3-64 G F T F S S Y A M H W V R Q A P G K G L E Y V S 623 vh26 G F T F S S Y A M S W V R Q A P G K G L E W V S 624 B25 G F T F S S Y A M H W V R Q A P G K G L E W V A 625 b32e G F T F S S Y A M H W V R Q A P G K G L E W V A 626 B37 G F T F S S Y A M H W V R Q A P G K G L E W V A 627 B43 G F T F S S Y A M H W V R Q A P G K G L E W V A 628 B48 G F T F S S Y A M H W V R Q A P G K G L E W V A 629 B52 G F T F S S Y A M H W V R Q A P G K G L E W V A 630 B54 G F T F S S Y A M H W V R Q A P G K G L E W V A 631 cos-8 G F T F S S Y A M H W V R Q A P G K G L E W V A 632 dp-46 G F T F S S Y A M H W V R Q A P G K G L E W V A 633 F2M G F T F S S Y A M H W V R Q A P G K G L E Y V S 634 F3 G F T F S S Y A M H W V R Q A P G K G L E Y V S 635 F7 G F T F S S Y A M H W V R Q A P G K G L E W V A 636 hv3005 G F T F S S Y A M H W V R Q A P G K G L E W V A

637 P2 G F T F S S Y A M H W V R Q A P G K G L E W V A 638 dp-48 G F T F S S Y D M H W V R Q A T G K G L E W V S 639 dp-58 G F T F S S Y E M N W V R Q A P G K G L E W V S 640 B1 G F T F S S Y G M H W V R Q A P G K G L E W V A 641 B13 G F T F S S Y G M H W V R Q A P G K G L E W V A 642 B18 G F T F S S Y G M H W V R Q A P G K G L E W V A 643 B26 G F T F S S Y G M H W V R Q A P G K G L E W V A 644 B28E G F T F S S Y G M H W V R Q A P G K G L E W V A 645 B29E G F T F S S Y G M H W V R Q A P G K G L E W V A 646 B29M G F T F S S Y G M H W V R Q A P G K G L E W V A 647 B30 G F T F S S Y G M H W V R Q A P G K G L E W V A 648 B32M G F T F S S Y G M H W V R Q A P G K G L E W V A 649 cos-3 G F T F S S Y G M H W V R Q A P G K G L E W V A 650 dp-49 G F T F S S Y G M H W V R Q A P G K G L E W V A 651 dp-50 G F T F S S Y G M H W V R Q A P G K G L E W V A 652 P6 G F T F S S Y G M H W V R Q A P G K G L E W V A 653 P9E G F T F S S Y G M H W V R Q A P G K G L E W V A 654 v3-30 G F T F S S Y G M H W V R Q A P G K G L E W V A 655 v3-33 G F T F S S Y G M H W V R Q A P G K G L E W V A 656 dp-51 G F T F S S Y S M N W V R Q A P G K G L E W V S 657 dp-77 G F T F S S Y S M N W V R Q A P G K G L E W V S 658 HHG4 G F T F S S Y S M N W V R Q A P G K G L E W V S 659 v3-21 G F T F S S Y S M N W V R Q A P G K G L E W V S 660 v3-48 G F T F S S Y S M N W V R Q A P G K G L E W V S 661 DP-52 G F T F S S Y V L H W V R R A P G K G P E W V S 662 cos-6 G F T F S S Y W M H W V R Q A P G K G L V W V S 663 dp-53 G F T F S S Y W M H W V R Q A P G K G L V W V S 664 dp-54 G F T F S S Y W M S W V R Q A P G K G L E W V A 665 dp-87 G F T F S S Y W M H W V R Q A P G K G L V W V S 666 VH3-11 G F T F S S Y G M H W V R Q A P G K G L E W V A 667 JOE9 VH G F T F S S Y G M H W V R Q A P G K G L E W V A VH3 Family Germline Amino Acid Sequences Numbering according to Kabat (Joe9 VH included for comparison) SEQ ID germline CDR H2 NO: VH 50 51 52 52A 52B 52C 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 594 dp-29 R T R N K A N S Y T T E Y A A S V K G R F T I S R 595 DP-30 L I R N K A N S Y T T E Y A A S V K G R L T I S R 596 HC15-7 L I R N K A N S Y T T E Y A A S V K G R L T I S R 597 VHD26 L I R N K A N S Y T T E Y A A S V K G R L T I S R 598 DP-31 G I S W . . N S G S I G Y A D S V K G R F T I S R 599 DP-32 G I N W . . N G G S T G Y A D S V K G R F T I S R 600 DP-33 L I S W . . D G G S T Y Y A D S V K G R F T I S R 601 dp-35 Y I . . S S S G S T I Y Y A D S V K G R F T I S R 602 VH3-8 Y I . . S S S S S Y T N Y A D S V K G R F T I S R 603 yac-9 R I R S K A N S Y A T A Y A A S V K G R F T I S R 604 dp-38 R I K S K T D G G T T D Y A A P V K G R F T I S R 605 LSG2 R I E S K T D G G T T D Y A A P V K G R F T I S R 606 LSG3 R I K S K T D G G T T D Y A A P V K G R F T I S R 607 LSG4 R I K S K T D G G T T N Y A A P V K G R F T I S R 608 LSG6 R I K S K T D G G T T D Y A A P V K G R F T I S R 609 v3-15 R I K S K T D G G T T D Y A A P V K G R F T I S R 610 dp-39 Y I . . S G D S G Y T N Y A D S V K G R F T I S R 611 dp-40 Y S . . S G N S G Y T N Y A D S V K G R F T I S R 612 dp-59 G V . . S W N G S R T H Y A D S V K G R F I I S R 613 v3-16p G V . . S W N G S R T H Y V D S V K R R F I I S R 614 v3-19p G V . . S W N G S R T H Y A D S V K G R F I I S R 615 v3-13 A N . . G T A G . D T Y Y P G S V K G R F T I S R 616 DP-42 V I . Y . . S G G S T Y Y A D S V K G R F T I S R 617 dp-44 A I . . . G T G G G T Y Y A D S V K G R F T I S R 618 DP-45 A I . . . G T G G G T Y Y A D S V K G R F T I S R 619 dp-47 A I . . S G S G G S T Y Y A D S V K G R F T I S R 620 flm A I . . S S N G G S T Y Y A D S V K G R F T I S R 621 P1 A I . . S S N G G S T Y Y A D S V K G R F T I S R 622 v3-64 A I . . S S N G G S T Y Y A N S V K G R F T I S R 623 vh26 A I . . S G S G G S T Y Y A D S V K G R F T I S R 624 B25 V I . . S Y D G S N K Y Y T D S V K G R F T I S R 625 b32e V I . . S Y D G S N K Y Y A D S V K G R F T I S R 626 B37 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 627 B43 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 628 B48 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 629 B52 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 630 B54 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 631 cos-8 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 632 dp-46 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 633 F2M A I . . S S N G G S T Y Y A D S V K G R F T I S R 634 F3 A I . . S S N G G S T Y Y A D S V K G R F T I S R 635 F7 V I . . S Y D G S N K Y Y A D S V K G R F A I S R 636 hv3005 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 637 P2 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 638 dp-48 A I . . G T A G . D T Y Y P G S V K G R F T I S R 639 dp-58 Y I . . S S S G S T I Y Y A D S V K G R F T I S R 640 B1 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 641 B13 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 642 B18 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 643 B26 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 644 B28E V I . . S Y D G S N K Y Y A D S V K G R F T I S R 645 B29E V I . . S Y D G S N K Y Y A D S V K G R F T I S R 646 B29M V I . . S Y D G S N K Y Y A D S V K G R F T I S R 647 B30 V I . . W Y D G S N K Y Y A D S V K G R F T I S R 648 B32M V I . . S Y D G S N K Y Y A D S V K G R F T I S R 649 cos-3 F I . . R Y D G S N K Y Y A D S V K G R F T I S R 650 dp-49 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 651 dp-50 V I . . W Y D G S N K Y Y A D S V K G R F T I S R 652 P6 V I . . W Y D G S N K Y Y A D S V K G R F T I S R 653 P9E V I . . S Y D G S N K Y Y A D S V K G R F T I S R 654 v3-30 V I . . S Y D G S N K Y Y A D S V K G R F T I S R 655 v3-33 V I . . W Y D G S N K Y Y A D S V K G R F T I S R 656 dp-51 V I . . S S S S S T I Y Y A D S V K G R F T I S R 657 dp-77 S I . . S S S S S Y I Y Y A D S V K G R F T I S R 658 HHG4 S I . . . S S S S Y I Y Y A D S V K G R F T I S R 659 v3-21 S I . . S S S S S Y I Y Y A D S V K G R F T I S R 660 v3-48 Y I . . S S S S S T I Y Y A D S V K G R F T I S R 661 DP-52 A I G . . . T G G D T Y Y A D S V M G R F T I S R 662 cos-6 R I . . N S D G S S T S Y A D S V K G R F T I S R 663 dp-53 R I . . N S D G S S T S Y A D S V K G R F T I S R 664 dp-54 N I . . K Q D G S E K Y Y A D S V K G R F T I S R 665 dp-87 R I . . N S D G S S T S Y A D S M K G Q F T I S R 666 VH3-11 N I . . K Q D G S E K Y Y V D S V K G R F T I S R 667 JOE9 VH F I . . R Y D G S N K Y Y A D S V K G R F T I S R VH3 Family Germline Amino Acid Sequences Numbering according to Kabat (Joe9 VH included for comparison) SEQ ID germline NO: VH 72 73 74 75 76 77 78 79 80 81 82 82A 82B 82C 83 84 85 86 87 88 89 90 91 92 93 94 594 dp-29 D D S K N S L Y L Q M N S L K T E D T A V Y Y C A R 595 DP-30 E D S K N T L Y L Q M S S L K T E D L A V Y Y C A R 596 HC15-7 E D S K N T M Y L Q M S N L K T E D L A V Y Y C A R 597 VHD26 E D S K N T L Y L Q M S S L K T E D L A V Y Y C A R 598 DP-31 D N A K N S L Y L Q M N S L R A E D T A L Y Y C A K 599 DP-32 D N A K N S L Y L Q M N S L R A E D T A L Y H C A R 600 DP-33 D N S K N S L Y L Q M N S L R T E D T A L Y Y C A K 601 dp-35 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 602 VH3-8 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 603 yac-9 D D S K N T A Y L Q M N S L K T E D T A V Y Y C T R 604 dp-38 D D S K N T L Y L Q M N S L K T E D T A V Y Y C T T 605 LSG2 D D S K N T L Y L Q M N S L K T E D T A V Y Y C T T

606 LSG3 D D S K N T L Y L Q M N S L K T E D T A V Y Y C T T 607 LSG4 D D S K N T L Y L Q M N S L K T E D T A V Y Y C T T 608 LSG6 D D S K N T L Y L Q M N S L K T E D T A V Y Y C T T 609 v3-15 D D S K N T L Y L Q M N S L K T E D T A V Y Y C T T 610 dp-39 D N A N N S P Y L Q M N S L R A E D T A V Y Y C V K 611 dp-40 D N A K N S L Y L Q M N S L R A E D T A V Y Y C V K 612 dp-59 D N S R N T L Y L Q T N S L R A E D T A V Y Y C V R 613 v3-16p D N S R N S L Y L Q K N R R R A E D M A V Y Y C V R 614 v3-19p D N S R N F L Y Q Q M N S L R P E D T A V Y Y C V R 615 v3-13 E N A K N S L Y L Q M N S L R A G D T A V Y Y C A R 616 DP-42 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 617 dp-44 D N A K N S L Y L Q M N S L R A E D M A V Y Y C A R 618 DP-45 D N A K N S L Y L Q M N S L R A E D M A V Y Y C A R 619 dp-47 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 620 flm D N S K N T L Y V Q M S S L R A E D T A V Y Y C V K 621 P1 D N S K N T L Y V Q M S S L R A E D T A V Y Y C V K 622 v3-64 D N S K N T L Y L Q M G S L R A E D M A V Y Y C A R 623 vh26 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 624 B25 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 625 b32e D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 626 B37 D N S K N T L Y L Q M S S L R A E D T A V Y Y C A R 627 B43 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 628 B48 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 629 B52 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 630 B54 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 631 cos-8 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 632 dp-46 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 633 F2M D N S K N T L Y L Q M S S L R A E D T A V Y Y C V K 634 F3 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 635 F7 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 636 hv3005 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 637 P2 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 638 dp-48 E N A K N S L Y L Q M N S L R A G D T A V Y Y C A R 639 dp-58 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 640 B1 D N S K N T L Y L Q M N S L R L R A R L C I T V R E 641 B13 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 642 B18 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 643 B26 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 644 B28E D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 645 B29E D N S K N R L Y L Q M N S L R A E D T A V Y Y C A R 646 B29M D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 647 B30 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 648 B32M D N S K N T L Y L Q M N S L R A E G T A V Y Y C A R 649 cos-3 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 650 dp-49 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 651 dp-50 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 652 P6 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K 653 P9E D N S K N T L Y L Q M N S L R A E D T A V R E - - - 654 v3-30 D N S K N T L Y L Q M N S L R A E D T A V Y Y C A R 655 v3-33 D N S T N T L F L Q M N S L R A E D T A V Y Y C A R 656 dp-51 D N A K N S L Y L Q M N S L R D E D T A V Y Y C A R 657 dp-77 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 658 HHG4 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 659 v3-21 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 660 v3-48 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 661 DP-52 D N A K K S L Y L Q M N S L I A E D M A V Y Y C A R 662 cos-6 D N A K N T L Y L Q M N S L R A E D T A V Y Y C A R 663 dp-53 D N A K N T L Y L Q M N S L R A E D T A V Y Y C A R 664 dp-54 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 665 dp-87 D N A K N T L Y L Q M N S L R A E D M A V Y Y C T R 666 VH3-11 D N A K N S L Y L Q M N S L R A E D T A V Y Y C A R 667 JOE9 VH D N S K N T L Y L Q M K S L R A E D T A V Y Y C T T V.lamda.1 Family Germline Amino Acid Sequences Numbering according to Kabat. (Joe9 VL included for comparison) SEQ ID NO: gene* VL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 668 1b DPL5 Q S V L T Q P P S V S A A P G Q K V T I S C 669 1d DPL4 Q S V L T Q P P S V S A A P G Q K V T I S C 670 1c DPL2 Q S V L T Q P P S A S G T P G Q R V T I S C 671 1g DPL3 Q S V L T Q P P S A S G T P G Q R V T I S C 672 1a DPL1 Q S V L T Q P P S V S E A P R Q R V T I S C 673 1f DPL9 Q S V L T Q P P S V S G A P G Q R V T I S C 674 1e DPL8 Q S V V T Q P P S V S G A P G Q R V T I S C 675 JOE9 Q Y V L T Q P P S V S G T P G Q R V T I S C VL V.lamda.1 Family Germline Amino Acid Sequences Numbering according to Kabat. (Joe9 VL included for comparison) SEQ ID CDR L1 NO: gene* VL 24 25 26 27 27A 27B 27C 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 668 1b DPL5 S G S S S N I G N N Y . V S W Y Q Q L P G T A P K 669 1d DPL4 S G S S S D M G N Y A . V S W Y Q Q L P G T A P K 670 1c DPL2 S G S S S N I G S N T . V N W Y Q Q L P G T A P K 671 1g DPL3 S G S S S N I G S N Y . V Y W Y Q Q L P G T A P K 672 1a DPL1 S G S S S N I G N N . A V N W Y Q Q L P G K A P K 673 1f DPL9 T G S S S N I G A G Y V V H W Y Q Q L P G T A P K 674 1e DPL8 T G S S S N I G A G Y D V H W Y Q Q L P G T A P K 675 JOE9 S G G R S N I G S N T . V K W Y Q Q L P G T A P K VL V.lamda.1 Family Germline Amino Acid Sequences Numbering according to Kabat. (Joe9 VL included for comparison) SEQ ID CDR L2 NO: gene* VL 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 668 1b DPL5 L L I Y D N N K R P S G I P D R F S G S K S G T S A 669 1d DPL4 L L I Y E N N K R P S G I P D R F S G S K S G T S A 670 1c DPL2 L L I Y S N N Q R P S G V P D R F S G S K S G T S A 671 1g DPL3 L L I Y R N N Q R P S G V P D R F S G S K S G T S A 672 1a DPL1 L L I Y Y D D L L P S G V S D R F S G S K S G T S A 673 1f DPL9 L L I Y G N S N R P S G V P D Q F S G S K S G T S A 674 1e DPL8 L L I Y G N S N R P S G V P D R F S G S K S G T S A 675 JOE9 L L I Y G N D Q R P S G V P D R F S G S K S G T S A VL V.lamda.1 Family Germline Amino Acid Sequences Numbering according to Kabat. (Joe9 VL included for comparison) SEQ ID CDR L3 NO: gene* VL 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 95A 95B 668 1b DPL5 T L G I T G L Q T G D E A D Y Y C G T W D S S L S A 669 1d DPL4 T L G I T G L W P E D E A D Y Y C L A W D T S P R A 670 1c DPL2 S L A I S G L Q S E D E A D Y Y C A A W D D S L N G 671 1g DPL3 S L A I S G L R S E D E A D Y Y C A A W D D S L S G 672 1a DPL1 S L A I S G L Q S E D E A D Y Y C A A W D D S L N G 673 1f DPL9 S L A I T G L Q S E D E A D Y Y C K A W D N S L N A 674 1e DPL8 S L A I T G L Q A E D E A D Y Y C Q S Y D S S L S G 675 JOE9 S L A I T G V Q A E D E A D Y Y C Q S Y D S S L R G VL *Williams, J M B, 1996, 264, 220-232

TABLE-US-00003 TABLE 2 H3 L3 IFN SEQ SEQ PHA gamma ID ID RB assay assay IC50 Clone NO: H3 NO: L3 koff IC50 (M) IC50 (M) (M) Joe9 wt 77 SGSYDY 110 QSYDSSLRGSRV 1.00E-01 1.50E-06 1.00E-06 Joe9 wt 77 SGSYDY 110 QSYDSSLRGSRV 5.00E-07 IgG1 70-1 78 HGSHDN 110 Joe9 wt 1.34 e-2 2.00E-07 70-1 78 HGSHDN 110 Joe9 wt 2.00E-07 IgG1 70-2 79 HGSYDY 110 Joe9 wt 3.30E-02 3-5.0E-7 70-7 80 RRRSNY 110 Joe9 wt 1.29E-01 3-5.0E-7 70-13 81 SGSIDY 110 Joe9 wt 7.20E-02 3-5.0E-7 78-34 77 wt 111 QSYDRGFTGSRV 1.64 e-2 2.00E-07 6.00E-07 78-25 77 wt 112 QSYDSSLRGSRV 5.00E-02 78-28 77 wt 112 QSYDSSLRGSRV 4.66E-02 78-35 77 wt 113 OSYDSSLTGSRV 4.99E-02 4.00E-07 79-1 77 wt 114 QSYDSSLWGSRV 2.00E-07 6.00E-07 101-14 79 70-2 111 78-34 7.52E-03 101-9 79 70-2 113 78-35 8.54E-03 101-19 81 70-13 111 78-34 4.56E-02 101-8 81 70-13 111 78-34 1.01E-02 101-4 81 70-13 113 78-35 9.76E-03 101-5 81 70-13 113 78-35 4.45E-02 101-11 78 70-1 111 78-34 4.5 e-3 3.00E-08 (12) 101-11 78 70-1 111 78-34 1.60E-09 IgG1 26-1 78 70-1 114 79-1 7.4 e-3 6.00E-08 (2, 3) 136-9 82 HGSHDD 115 QTYDISESGSRV 3.20E-03 136-10 82 HGSHDD 116 QSYDRGFTGSRV 1.40E-03 2.00E-09 136-14 83 HGSHDN 117 QTYDRGFTGSRV 1.10E-03 3.00E-10 1.00E-07 136-15 83 HGSHDN 118 QTYDKGFTGSSV 7.4 e-4 1.00E-10 2.00E-09 136-15 83 HGSHDN 118 QTYDKGFIGSSV 4.60E-04 6.00E-09 germline 136-16 83 HGSHDN 119 QSYDRRFTGSRV 6.10E-04 3.00E-10 5.00E-09 136-17 83 HGSHDN 120 QSYDWNFTGSRV 2.90E-05 2.00E-09 7.00E-09 136-18 83 HGSHDN 121 QSYDRGFTGSRV 1.10E-03 8.00E-10 136-21 83 HGSHDN 122 QSYDNGFTGSRV 4.20E-04 2.00E-09 136-24 83 HGSHDN 123 QSYDNAVTASKV 8.90E-04 1.00E-09 101-11 84 TT HGSHDN WGQG 124 QSYDRGFTGSRV 4.5 .times. 10 - 3 2 .times. 10 - 9 2.00E-08 136-15M1 85 AK ...... .... 124 QSYDRGFTGSRV 4.00E-10 149-4 86 .. ...... .S.. 124 ............ 1.37 .times. 10 - 3 8 .times. 10 - 11 3.00E-09 149-5 87 .. .....T .... 125 QSYDSSLWGTRV 1.02 .times. 10 - 3 1.2 .times. 10 - 10 3.00E-09 149-6 84 .. ...... .... 124 ............ 2.73 .times. 10 - 3 6 .times. 10 - 10 2.00E-09 149-7 84 .. ...... .... 126 .....D...... 1.13 .times. 10 - 3 9 .times. 10 - 10 3.00E-09 149-8 88 K. ...... .... 2.33 .times. 10 - 3 3 .times. 10 - 9 149-9 89 K. ...... ..H. 127 ...E......M. 3.54 .times. 10 - 3 1.8 .times. 10 - 10 149-11 90 .. ...... .S.. 128 ....N....A.. 1.43 .times. 10 - 2 2 .times. 10 - 10 4.00E-09 149-12 84 .. ...... .... 3.73 .times. 10 - 3 neutralising 149-13 84 .. ...... .... 2.22 .times. 10 - 3 5 .times. 10 - 10 149-14 91 .. .R..N. .... 1.5 .times. 10 - 10 6.00E-09 92 TT HGSHDN 124 QSYDRGFTGSRV 156-1 93 .. .....T 126 .....D...... 5.00E-03 156-2 93 .. .....T 129 .....R...... 156-3 93 .. .....T 128 ....N....A.. 9.00E-03 156-4 93 .. .....T 127 ...E.....SM. 156-5 93 .. .....T 130 .T..K.....S. 156-6 92 .. ...... 126 .....D...... 3.00E-03 156-7 92 .. ...... 129 .....R...... 156-8 92 .. ...... 128 ....N....A.. 156-9 92 .. ...... 127 ...E.....SM. 156-10 92 .. ...... 130 .T..K.....S. 156-11 94 .K ...... 126 .....D...... 156-12 94 .K ...... 129 .....R...... 156-13 94 .K ...... 128 ....N....A.. 156-14 94 .K ...... 127 ...E.....SM. 156-15 94 .K ...... 130 .T..K.....S. 156-16 93 .. .....T 124 ............ 156-17 92 .. ...... 125 ....SSLW.T.. 6.00E-03 156-18 93 .. .....T 125 ....SSLW.T.. 92 TT HGSRDN 124 QSYDRGFTGSRV 103-1 95 .. Q.R... 124 ............ 2.9 .times. 10 - 3 103-2 96 K. R.R... 130 .T..K.....S. 7.3 .times. 10 - 4 7.00E-11 1.00E-09 103-3 97 .. .....K 124 ............ 2.5 .times. 10 - 3 103-6 131 .....D...T.. 4.5 .times. 10 - 4 103-7 98 .. .....D 131 .....D...T.. 3.7 .times. 10 - 4 1.40E-10 1.00E-09 103-8 99 K. ...... 130 .T..K.....S. 3.3 .times. 10 - 4 6.00E-11 1.50E-09 103-14 & 100 KT HGSHDN 132 QSYDRGFTGSMV 6.7 e-4 4.00E-11 1.20E-09 9 103-8 & 100 KT HGSHDN 133 QTYDKGFTGSSV 5.3 e-4 1.50E-09 2 103-4 101 TT HGSHDN 134 QSYDRGFTGARV 1.6 e-4 8.60E-11 9.00E-10 103-152 101 TT HGSHDN 135 QSYERGFTGARV 8.60E-11 102 TT SGSYDY 136 QSYDRGFTGSRVF 170-1 102 .. ...... 137 .........FK.. 2.35E-03 170-2 102 .. ...... 138 .......VSAY.. 8.80E-04 170-3 102 .. ...... 139 ......L.VTK.. 1.11E-03 170-4 102 .. ...... 140 ......Y.A.... 8.11E-04 170-7 102 .. ...... 141 ..........K.. 5.30E-04 170-11 102 .. ...... 142 ......L..F... 4.40E-04 170-13 102 .. ...... 143 .........YK.. 1.59E-03 170-15 102 .. ...... 144 ......L..Y.L. 4.43E-03 170-19 103 .. H..H.N 145 ........DYK.. 1.00E-03 170-21 104 .. H..Q.N 146 .........P.L. 3.89E-03 170-22 102 .. ...... 147 ......L...... 5.60E-04 170-23 103 .. H..H.N 148 .........A..W 1.00E-03 2.00E-10 170-24 104 .. H..Q.N 149 .........Y... 2.80E-04 5.00E-10 170-35 105 A. H..Q.N 136 ............. 1.00E-05 170-38 150 .........P... 2.10E-04 170-39 151 ......M.S.... 2.79E-03 170-36 83 HGSHDN 152 QSYDRDSTGSRVF 4.00E-04 2.00E-10 170-25 106 HGSQDT 153 QSYDSSLRGSRVF 5.00E-04 5.00E-11 106 SGSYDY 136 QSYDRGFTGSRVF 73-B1 107 SGSYDY 154 H...SD....... 3.25E-03 >1E-8 73-B2 107 SGSTDY 155 H.SES........ 2.07E-03 73-B6 107 SGSYDY 156 H...NR....... 2.51E-03 >1E-8 73-C1 107 SGSYDY 157 H...SR....... 2.71E-03 >1E-8 73-C2 107 SGSYDY 158 ....SE....... 3.79E-03 73-C6 107 SGSYDY 159 ....T........ 3.96E-03 73-D1 107 SGSYDY 160 H...S........ 3.99E-03 73-D2 107 SGSYDY 161 ....T........ 3.56E-03 73-D4 107 SGSYDY 162 H...TK....... 5.36E-03 73-D5 107 SGSYDY 163 H.S.S........ 3.57E-03 73-E3 107 SGSYDY 164 ....SD....... 4.98E-03 73-E6 107 SGSYDY 165 H..ES........ 4.17E-03 73-F3 107 SGSYDY 166 ....APWS..... 7.08E-03 73-F5 107 SGSYDY 167 ...DSD....K.. 3.74E-03 73-G2 107 SGSYDY 168 HTN.S........ 3.98E-03 73-G3 107 SGSYDY 169 H...TR....... 3.50E-03 73-G4 107 SGSYDY 170 ....MR....... 6.58E-03 73-G5 107 SGSYDY 171 H.S.SDS...... 6.01E-03 73-G6 107 SGSYDY 172 ...NTD....... 6.30E-03 73-H2 107 SGSYDY 173 ....S........ 5.93E-03 73-F6 107 SGSYDY 174 H...M........ 5.87E-03

73-H3 107 SGSYDY 175 H...N........ 6.85E-03 73-C5 107 SGSYDY 176 H.H..D....... 4.84E-03 73-B7 108 HGSQDN 177 QSYDSSLRGSRV 2.50E-03 7.00E-09 136 QSYDRGFTGSRVF M2 A2 83 HGSHDN 178 ......IH..... 4.00E-02 M2 A4 83 HGSHDN 179 ....S..P..... 8.49E-03 M2 A5 83 HGSHDN 180 ....I.S...... 4.01E-02 M2 B1 83 HGSHDN 181 ....S.L...... 7.97E-03 M2 B3 83 HGSHDN 182 ....I.M...... 4.60E-02 M2 B4 83 HGSHDN 183 ....I.L...... 4.42E-02 M2 B5 83 HGSHDN 184 ....S.V...... 8.38E-03 M2 B6 83 HGSHDN 185 ......L.A.... 2.81E-02 M2 C2 83 HGSHDN 181 ....S.L...... 4.85E-02 M2 C3 83 HGSHDN 186 ....T.L...... 4.62E-02 M2 C4 83 HGSHDN 181 ....S.L...... 8.16E-03 M2 C5 83 HGSHDN 187 ....TAL...... 4.71E-02 M2 Dl 83 HGSHDN 188 ....IR....... 3.71E-02 M2 D2 83 HGSHDN 189 ....IRS...... 3.85E-02 M2 D3 83 HGSHDN 190 ....NRL...... 3.33E-02 M2 D4 83 HGSHDN 191 ...ETS....... 5.81E-02 M2 D5 83 HGSHDN 192 ....SSS...... 5.18E-02 M2 D6 83 HGSHDN 193 ....S...A.... 5.01E-02 M2 El 83 HGSHDN 194 .T..K.....S.. 5.32E-02 M2 E2 83 HGSHDN 195 ....N........ 4.77E-02 M2 E6 83 HGSHDN 196 ....T...K.... 9.77E-03 M2 Fl 83 HGSHDN 197 ....SDV...... 6.16E-02 M2 H5 83 HGSHDN 198 ....A........ 9.90E-03 124 QSYDRGFTGSRV A5 83 HGSHDN 199 ......THPSML 1.12E-03 Al2 83 HGSHDN 200 ......TTPRPM 1.43E-03 A4 83 HGSHDN 201 ......RNPALT 1.47E-03 A6 83 HGSHDN 202 ......THPWLH 1.87E-03 A10 83 HGSHDN 203 ......NSPATV 1.87E-03 All 83 HGSHDN 204 ......TFPSPQ 2.07E-03 C2 83 HGSNDN 205 ......LNPSAT 2.23E-03 A8 83 HGSHDN 206 ......KSNKML 2.37E-03 B8 83 HGSHDN 207 ......HTAHLY 2.40E-03 C6 83 HGSHDN 208 ......QTPSIT 2.42E-03 A3 83 HGSHDN 209 ......YPRNIL 2.51E-03 B11 83 HGSHDN 210 ......ITPGLA 2.95E-03 B5 83 HGSHDN 211 ......QPHAVL 3.04E-03 C10 83 HGSHDN 212 ......NSPIPT 3.10E-03 C4 83 HGSHDN 213 ......TPNNSF 3.23E-03 C3 83 HGSHDN 214 ....S.VDPGPY 3.34E-03 B2 83 HGSHDN 215 ......RPRHAL 3.61E-03 A2 83 HGSHDN 216 ......PYHPIR 3.80E-03 C5 83 HGSHDN 217 ......PHTQPT 3.91E-03 A7 83 HGSHDN 218 ......HNNFSP 3.95E-03 C9 83 HGSHDN 219 ......PTHLPH 3.97E-03 B3 83 HGSHDN 220 ......TPSYPT 4.12E-03 C8 83 HGSHDN 221 ....S.TSNLLP 5.36E-03 B7 83 HGSHDN 222 ......DSNHDL 5.45E-03 Al 83 HGSHDN 223 ......LPRLTH 5.66E-03 C7 83 HGSHDN 224 ......IPTSYL 5.83E-03 C12 83 HGSHDN 225 ......LRVQAP 5.85E-03 B10 83 HGSHDN 226 ......LSDSPL 6.04E-03 B6 83 HGSHDN 227 ....S.SLRRIL 7.58E-03 A9 83 HGSHDN 228 ......PARTSP 7.98E-03 B9 83 HGSHDN 229 ......RAAHPQ 8.66E-03 124 QSYDRGFTGSRV 177-D7 83 HGSHDN 230 ......TQPABI 4.07E-04 177-G6 83 HGSHDN 231 ......THPTMI 5.50E-04 177-D9 83 HGSHDN 232 ......RIPABT 6.32E-04 177-C6 83 HGSHDN 233 ......THPVPA 7.94E-04 177-H5 83 HGSHDN 234 ......SBPIPA 1.32E-03 177-H9 83 HGSHDN 235 ......THPVPA 1.58E-03 177-H10 83 HGSHDN 236 ......THPTMY 3.44E-03 144-F1 83 HGSHDN 237 ......HHYTTF 5.80E-04 43-E3 83 HGSHDN 238 ......SHPAAE 8.00E-04 43-E9 83 HGSHDN 239 ......TIPSIE 8.00E-04 43-G2 83 HGSHDN 240 ......SSPAIM 7.00E-04 43-G3 83 HGSHDN 241 ......IWPNLN 9.00E-04 31-A6 83 HGSHDN 242 ......THPNLN 5.00E-04 31-B5 83 HGSHDN 243 ......THPSIS 5.00E-04 124 QSYDRGFTGSRV Y17 83 HGSHDN 244 QSYDRGSAPMIN 8.90E-05 4.50E-10 >1E-8 Y19 83 HGSHDN 245 QSYDRGHHPAMS 2.26E-04 3.00E-11 >1E-8 Y38 83 HGSHDN 246 ......THPSIT 5.08E-04 5.50E-11 2.60E-09 Y45 83 HGSHDN 247 ......TDPAIV 6.17E-04 4.00E-11 4.30E-09 Y61 83 HGSHDN 248 ......THPALL 2.75 e-4 4E-11 1.40E-10 Y61 IgG 83 HGSHDN 248 ......THPALL 1.50E-04 1.60E-11 1.30E-10 Y61 IgG 83 HGSHDN 248 ......THPALL 1.50E-04 1.60E-11 1.30E-10 1.60E- germline 10 Y139 83 HGSHDN 249 ......SHPALT 5.92E-04 3E-11 4.50E-10 Y139 83 HGSHDN 249 ......SHPALT 1.00E-09 IgG1 Y174 83 HGSHDN 250 ......TTPAPE 7.55E-04 6E-11 2.00E-09 Y177 83 HGSHDN 251 ......SHPTLI 6.61E-04 5E-11 1.00E-09 A5 83 HGSHDN 252 ......THPSML 4.50E-04 6.60E-11 Al2 83 HGSHDN 253 ......TTPRPN 5.57E-04 2.50E-10 D9 83 HGSHDN 254 ......RLPAQT 8.21E-04 3.5E-09 >> G6 83 HGSHDN 255 ......THPLTI 5.08E-04 1E-10 1.00E-09 G6 IgG1 83 HGSHDN 255 ......THPLTI 1.00E-09 C6 83 HGSHDN 256 QSYDRGQTPSIT 1.07E-03 3.5E-10 1.00E-08 Y55 83 HGSHDN 257 QSYDRGTHFQMY 1.06E-03 1.40E-10 >1E-8 A4 83 HGSHDN 258 QSYDRGRNPALT 6.30E-04 2.50E-10 A03 83 HGSHDN 259 QSYDRGTHPLTM 3.04E-04 3.00E-11 4.00E-10 A03 IgG1 83 HGSHDN 260 QSYDRGTHPLTM 3.04 e-4 2.90E-11 3.80E-10 A03 IgG 83 HGSHDN 260 QSYDRGTHPLTM 2.50E-04 3.50E-11 1.75E-10 germline 99-B11 83 HGSHDN 261 QSYDSGYTGSRV 5.40E-03 99-C11 83 HGSHDN 262 QSYDSGFTGSRV 5.70E-03 99-H4 83 HGSHDN 263 QSYDSRFTGSRV 4.80E-03 99-E9 83 HGSHDN 262 QSYDSGFTGSRV 5.40E-03 99-H7 83 HGSHDN 264 QSYPDGTPASRV 3.30E-03 99-H11 83 HGSHDN 265 QSYSTHMPISRV 4.90E-03 99-F6 83 HGSHDN 266 QSYDSGSTGSRV 4.90E-03 99-F7 83 HGSHDN 267 QSYPNSYPISRV 4.80E-03 99-F8 83 HGSHDN 268 QSYIRAPQQV 3.70E-03 99-F11 83 HGSHDN 262 QSYDSGFTGSRV 5.40E-03 99-G7 83 HGSHDN 269 QSYLKSRAFSRV 4.80E-03 99-G11 83 HGSHDN 270 QSYDSRFTGSRV 4.30E-03 124 QSYDRGFTGSRV L3.3R3M-B1 83 HGSHDN 271 ......FTGSMV 5.46E+00 L3.3R3M-B3 83 HGSHDN 272 ......FTGSMV 5.51E+00 L3.3R3M-C6 83 HGSHDN 273 ......FTGFDG 6.17E+00 L3.3R3M-F9 83 HGSHDN 274 ......TAPALS 4.99E+00 L3.3R3M-G8 83 HGSHDN 275 ......SYPALR 5.55E+00 L3.3R3M-H6 83 HGSHDN 276 ......NWPNSN 5.69E+00 L3.3R3M-H10 83 HGSHDN 277 ......TAPSLL 5.35E+00 L3.3R3M-A3 83 HGSHDN 278 ......FTGSMV 5.37E+00 L3.3R3M-F8 83 HGSHDN 279 ......TTPRIR 4.99E+00 L3.3R3M-G1 83 HGSHDN 280 ......FTGSMV 4.21E+00 L3.3R3M-G7 83 HGSHDN 281 ......FTGSMV 4.24E+00 L3.3R3M-H11 83 HGSHDN 282 ......MIPALT 3.95E+00 Y61-L94N 109 CKT HGSHDN 283 QSYDRNTHPALL 8.00E-11 Y61-L94F 109 CKT HGSHDN 284 QSYDRFTHPALL 6.00E-11

Y61-L94Y 109 CKT HGSHDN 285 QSYDRYTHPALL 2.00E-11 2.00E-11 Y61-L94Y 109 CKT HGSHDN 285 QSYDRYTHPALL 1.27E-04 6.00E-11 5.00E-11 4.00E- IgG 11 Y61-L50Y 109 CKT HGSHDN 286 QSYDRGTHPALL 2.00E-11 2.00E- 11 Y61-L50Y* 109 CKT HGSHDN 286 QSYDRGTHPALL 6.98E-05 2.00E-11 3.00E- IgG 11 Y61-L50Y- 109 CKT HGSHDN 286 QSYDRGTHPALL 2.99E-05 6.00E-11 2.00E- H31E** 11 IgG Y61-L50Y- 109 CKT HGSHDN 287 QSYDRYTHPALL 4.64E-05 1.00E-11 1.00E- H31E- 11 L94Y** IgG J695 (Y61- 109 CKT HGSHDN 287 QSYDRYTHPALL 5.14E-05 5.00E-11 1.00E-11 5.00E- L94Y- 12 L50Y IgG*) *CDR L2: L50G to Y **CDR L2: L50G to Y; CDR H1: H31S to E

TABLE-US-00004 TABLE 3 CDR H1 CDR H2 Kabat Number 27 28 29 30 31 32 33 34 35 50 51 52 52A 53 54 55 Y61 VH F T F S S Y G M H F I R Y D G S Contact x x x x x x x x x x Positions Hypermutation x x x x Positions CDR H2 CDR H3 Kabat Number 56 57 58 59 60 61 62 63 64 65 95 96 97 98 101 102 Y61 VH N K Y Y A D S V K G H G S H D N Contact x x x x x x x Positions Hypermutation x x Positions CDR L1 CDR L2 Kabat number 24 25 26 27 27A 27B 28 29 30 31 32 33 34 50 51 52 Y61 VL S G G R S N I G S N T V K G N D Contact x x x x x x Positions Hypermutation x x x Positions CDR L2 CDR L3 Kabat number 53 54 55 56 89 90 91 92 93 94 95 95A 95B 95C 96 97 Y61 VL Q R P S Q S Y D R G T H P A L L Contact x x x x x x x Positions Hypermutation x x Positions x contact and/or hypermutation position x contact and/or hypermutation position Mutated in Y61

TABLE-US-00005 TABLE 4 Neutralization Activity in the Presence of Excess Free IL-12 p40 PHA assay IC50 PHA assay IC50 (M) PHA assay IC50 (M) SEQ ID NO: Clone (M) p70:p40 1:0 p70:p40 1:20 p70:p40 1:50 VH: 47 136-15 2.00E-09 5.00E-09 4.00E-09 VL: 48 VH: 51 149-5 6.50E-09 7.00E-09 4.00E-09 VL: 52 VH: 53 149-6 9.00E-10 1.00E-09 1.00E-09 VL: 54 VH: 84 149-7 3.50E-09 2.50E-09 4.00E-09 VL: 126 VH: 23 Y61 IgG 1.80E-10 1.80E-10 VL: 24 VH: 65 AO3 IgG1 2.50E-10 2.20E-10 VL: 66 VH: 31 J695 1.00E-11 3.50E-11 VL: 32

EXAMPLES

Example 1

Isolation of Anti-IL-12 Antibodies

A. Screening for IL-12 Binding Antibodies

[0693] Antibodies to hIL-12 were isolated by screening three separate scFv phage display libraries prepared using human VL and VH cDNAs from mRNA derived from human tonsils (referred to as scFv 1), tonsil and peripheral blood lymphocytes (PBL) (referred to as scFv 2), and bone marrow-derived lymphocytes (referred to as BMDL). Construction of the library and methods for selection are described in Vaughan et al. (1996) Nature Biotech. 14: 309-314.

[0694] The libraries were screened using the antigens, human IL-12 p70 subunit, human IL-12 p40 subunit, chimaeric IL-12 (mouse p40/human p35), mouse IL-12, biotinylated human IL-12 and biotinylated chimaeric IL-12. IL-12 specific antibodies were selected by coating the antigen onto immunotubes using standard procedures (Marks et al., (1991) J. Mol. Biol. 222: 581-597). The scFv library 2 was screened using either IL-12, or biotinylated-IL-12, and generated a significant number of IL-12 specific binders. Five different clonotypes were selected, determined by BstN1 enzymatic digestion patterns, and confirmed by DNA sequencing. The main clonotypes were VHDP58/VLDPL11, VHDP77/VLDPK31, VHDP47/VL and VHDP77/VLDPK31, all of which recognized the p40 subunit of IL-12.

[0695] Screening of the BMDL library with IL-12 p70 generated 3 different clonotypes. Two of these were found to be cross-reactive clones. The dominant clone was sequenced and consisted of VHDP35/VLDP. This clone recognizes the p40 subunit of IL-12. Screening of the scFv library 1, using IL-12 p70, did not produce specific IL-12 antibodies.

[0696] In order to identify IL-12 antibodies which preferentially bind to the p70 heterodimer or the p35 subunit of IL-12, rather than the p40 subunit, the combined scFv 1+2 library, and the BMDL library were used. To select IL-12 antibodies that recognized the p70 heterodimer or p35 subunit, phage libraries were preincubated and selected in the presence of free p40. Sequencing of isolated clones revealed 9 different antibody lineages. Subunit preferences were further analyzed by `micro-Friguet` titration. The supernatant containing scFv was titrated on biotin-captured IL-12 in an ELISA and the ED.sub.50 determined. The concentration of scFv producing 50% ED was preincubated with increasing concentrations of free p70 or p40 (inhibitors). A decrease in the ELISA signal on biotin-IL-12 coated plates was measured and plotted against the concentration of free p70 or p40. This provided the IC.sub.50 for each clone with respect to p70 and p40. If the titrations for both subunits overlaps, then the scFv binds to both p40 and p70. Any variation from this gives the degree of preference of p70 over p40.

B. Affinity Maturation of Antibody Lineage Specific for IL-12 (Joe 9)

[0697] The clones were tested for their ability to inhibit IL-12 binding to its receptor in an IL-12 receptor binding assay (referred to as RBA), and for their ability to inhibit IL-12 induced proliferation of PHA stimulated human blast cells (PHA assay), described in Example 3. Clone Joe 9 had the lowest IC.sub.50 value in both the RBA and the PHA assay, with an IC.sub.50 value of 1.times.10.sup.-6 M in both assays. In addition the heavy chain variable region (VH) of Joe 9 had the least number of changes compared to the closest germline sequence COS-3, identified from the VBASE database. Table 1 (see Appendix A) shows the V.sub.H3 family of germline sequences, of which COS-3 is a member, as well as members of V.sub..lamda.1 family of germline sequences. Therefore, Joe 9 was selected for affinity maturation. The amino acids sequences of VH and VL of the Joe9 wild type (Joe9 wt) antibody are shown in FIG. 1A-1D.

[0698] In order to increase the affinity of Joe 9, various mutations of the complementarity determining region 3 (CDR3) of both the heavy and light chains were made. The CDR3 variants were created by site-directed PCR mutagenesis using degenerate oligonucleotides specific for either the heavy chain CDR3 (referred to as "H3") or the light chain CDR3 (referred to as "L3"), with an average of three base substitutions in each CDR3 (referred to as "spike"). PCR mutagenesis of the heavy chain CDR3 was performed using the degenerate heavy chain oligonucleotide containing a random mixture of all four nucleotides, 5' TGTCCCTTGGCCCCA(G)(T)(A)(G)(T)(C)(A)(T)(A)(G)(C)(T)(C)(C)(C)(A)(C)(T) GGTCGTACAGTAATA 3' (SEQ ID NO: 580), and oligonucleotide pUC Reverse Tag GAC ACC TCG ATC AGC GGA TAA CAA TTTCAC ACA GG (SEQ ID NO: 581) to generate a repertoire of heavy chain CDR3 mutants. The parent light chain was amplified using Joe 9 reverse oligonucleotide (5'TGG GGC CAA GGG ACA3' (SEQ ID NO:582) and the fdteteseq 24+21 oligonucleotide (5'-ATT CGT CCT ATA CCG TTC TAC TTT GTC GTC TTT CCA GAC GTT AGT-3' (SEQ ID NO: 583).

[0699] Complementarity between the two PCR products was used to drive annealing of the two fragments in a PCR assembly reaction and the full length recombined scFv library was amplified with pUC Reverse Tag (SEQ ID NO: 581) and fdTag 5'-ATT CGT CCT ATA CCG TTC-3' (SEQ ID NO: 584). PCR mutagenesis of the light chain was performed using the light chain oligonucleotide containing a mixture of all four nucleotides 5'GGTCCCAGTTCCGAAGACCCTCGAACC(C)(C)(T)(C)(A)(G)(G)(C)(T) (G)(C)(T)(G)(T)(C)ATATGACTGGCAGTAATAGTCAGC 3' (SEQ ID NO: 585), and Joe 9 reverse oligonucleotide 5'TGG GGC CAA GGG ACA3' (SEQ ID NO: 586) to produce a repertoire of light chain CDR3 mutants. The parent heavy chain was amplified with pUC Reverse Tag (SEQ ID NO: 581) and HuJH3FOR oligonucleotide 5'TGAAGAGACGGTGACCATTGTCCC3' (SEQ ID NO: 587). Complementarity between the two PCR products was used to drive annealing of the two fragments in a PCR assembly reaction and the full length recombined scFv library was amplified with Reverse Tag GAC ACC TCG ATC AGC G (SEQ ID NO: 588) and HuJ.lamda. 2-3 FOR NOT oligonucleotide 5'GAG TCA TTC TCG ACT TGC GGC CGC ACC TAG GAC GGT CAG CTT GGT CCC 3' (SEQ ID NO: 589).

[0700] Heavy chain CDR3 mutants were selected using 1 nM biotinylated IL-12, and washed for 1 h at room temperature in PBS containing free IL-12 or p40 at a concentration of 7 nM. Clones were analyzed by phage ELISA and those that bound to IL-12 were tested in BIAcore kinetic binding studies using a low density IL-12 chip (see procedure for BIAcore analysis in Example 5). Generally, BIAcore analysis measures real-time binding interactions between ligand (recombinant human IL-12 immobilized on a biosensor matrix) and analyte (antibodies in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway, N.J.). The system utilizes the optical properties of SPR to detect alterations in protein concentrations within a dextran biosensor matrix. Proteins are covalently bound to the dextran matrix at known concentrations. Antibodies are injected through the dextran matrix and specific binding between injected antibodies and immobilized ligand results in an increased matrix protein concentration and resultant change in the SPR signal. These changes in SPR signal are recorded as resonance units (RU) and are displayed with respect to time along the y-axis of a sensorgram. To determine the off rate (k.sub.off), on rate (k.sub.on), association rate (Ka) and dissociation rate (Kd) constants, BIAcore kinetic evaluation software (version 2.1) was used. Clones that demonstrated an improvement in the k.sub.off rate were analyzed by neutralization assays which included inhibition by antibody of IL-12 binding to its receptor (RBA assay), inhibition of IL-12-induced proliferation in PHA stimulated human blast cells (PHA assay), and inhibition of IL-12-induced interferon gamma production by human blast cells (IFN gamma assay). A summary of the dissociation rates and/or IC.sub.50 values from neutralization assays of heavy chain CDR3 spiked clones 70-1 through 70-13 is presented in Table 2 (see Appendix A). Clone 70-1 displayed a k.sub.off rate that was better than the parent Joe 9 clone, and had the lowest IC.sub.50 value of 2.0.times.10.sup.-7 M. Therefore clone 70-1 was selected for conversion to complete IgG1.

[0701] Light chain CDR3 mutants were selected using 1 nM biotin-IL-12 and washed with PBS containing 7 nM free p40. Clones were screened in phage ELISA and those that bound to IL-12 were tested in BIAcore binding analysis using low density IL-12 chips. Clones that displayed an off rate which was better than the parent Joe 9 clone were tested in neutralization assays which measured either, inhibition of IL-12 receptor binding, or inhibition of PHA blast cell proliferation. A summary of the dissociation rates and/or IC.sub.50 values from neutralization assays of light chain CDR3 mutant clones, 78-34 through 79-1, is presented in Table 2 (see Appendix A).

[0702] Based on the k.sub.off rate, clones 78-34 and 78-35 displayed an improved k.sub.off rate compared to the parent Joe 9. Both of these clones were selected for combination analysis with heavy chain mutants.

C. Combination Clones

[0703] Mutant light and heavy chain clones that exhibited the best binding characteristics were used for combination and assembly of scFvs. Mutant clones with improved potency characteristics were combined by PCR overlap extension and pull-through of the mutated VH and VL segments as described above. Clones 101-14 through 26-1, shown in Table 2 (see Appendix A), were produced from the combination of heavy chain mutants (70-2, 70-13 and 70-1) with light chain mutants (78-34, 78-35 and 79-1). The k.sub.off rates and/or IC.sub.50 values from neutralization assays for these clones are presented in Table 2.

[0704] BIAcore binding analysis identified clone 101-11, produced from the combination of the heavy chain CDR3 mutant clone 70-1 with the light chain CDR3 mutant clone 78-34, as having an off rate of 0.0045 .sup.s-1. This k.sub.off rate was a significant improvement compared to the k.sub.off rates for either the heavy chain CDR3 mutant clone 70-1 (0.0134 .sup.s-1), or for the light chain CDR3 mutant clone 78-34 (0.0164 .sup.s-1) alone. Furthermore, clone 101-11 showed a significant improvement in neutralization assays. Accordingly, clone 101-11 was selected for affinity maturation as described below.

D. Affinity Maturation of Clone 101-11

[0705] Further affinity maturation of clone 101-11 consisted of repeat cycles of PCR mutagenesis of both the heavy and light chain CDR3s of 101-11 using spiked oligonucleotide primers. The clones were selected with decreasing concentrations of biotinylated IL-12 (bio-IL-12). The binding characteristics of the mutated clones was assessed by BIAcore binding analysis and RBA, PHA neutralization assays. The k.sub.off rates and/or IC.sub.50 values for clones 136-9 through 170-25 are presented in Table 2 (see Appendix A). Clone 103-14 demonstrated an improved IC.sub.50 value in both the receptor binding assay and the PHA blast assay. Clone 103-14 also demonstrated a low k.sub.off rate, and accordingly was selected for further affinity maturation.

E. Generation and Selection of Randomized Libraries of Clone 103-14 Light CDR3

[0706] The light chain CDR3 of clone 103-14 (QSYDRGFTGSMV (SEQ ID NO: 590)) was systematically randomized in 3 segments using 3 different libraries as outlined below, where X is encoded by a randomized codon of sequence NNS with N being any nucleotide and S being either deoxycytosine or deoxyguanidine.

TABLE-US-00006 L3.1 = XXXXXXFTGSMV (SEQ ID NO: 591) L3.2 = QSYXXXXXXSMV (SEQ ID NO: 592) L3.3 = QSYDRGXXXXXX (SEQ ID NO: 593)

[0707] Randomized mutagenesis of all three light chain CDRs (referred to as L3.1, L3.2, and L3.3) of clone 103-14 was performed. The heavy chain CDR3 (referred to as H3) of clone 103-14 was not mutated. Four randomized libraries based on clone 103-14 (H3 and L3.1, L3.2 & L3.3) were constructed and subjected to a large variety of selection conditions that involved using limiting antigen concentration and the presence or absence of excess free antigen (p40 and p70). The outputs from selections (clones 73-B1 through 99-G11) were screened primarily by BIAcore, and on occasion with RBA and are shown in Table 2 (see Appendix A).

[0708] Random mutagenesis of the light chain CDR of 103-14 generated clone Y61, which exhibited a significant improvement in IC.sub.50 value compared to the parent clone 103-14. Y61 was selected for conversion to a whole IgG1. Whole Y61-IgG1 has an IC.sub.50 value of approximately 130 pM determined by the PHA assay. The IC.sub.50 value was not affected by a 50 fold molar excess of free p40, demonstrating that free p40 did not cross-react with Y61 anti-IL-12 antibody to thereby decrease the antibody binding to the heterodimer. The full length sequences of Y61 heavy chain variable region and light chain variable region are shown below.

Y61 Heavy Chain Variable Region Peptide Sequence

TABLE-US-00007 [0709] (SEQ ID NO: 23) CDR H1 QVQLVESGGGVVQPGRSLRLSCAASFTFS SYGMH WVRQAPGKGLEWVA CDR H2 FIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCKT CDR H3 HGSHDN WGQGTMVTVSS

Y61 Light Chain Variable Region Peptide Sequence

TABLE-US-00008 [0710] (SEQ ID NO: 24) CDR L1 QSVLTQPPSVSGAPGQRVTISC SGGRSNIGSNTVK WYQQLPGTAPKLL IY CDR L2 GNDQRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYC CDR L3 QSYDRGTHPALL FGTGTKVTVLG

CDR residues are assigned according to the Kabat definitions.

Example 2

Mutation of Y61 at Hypermutation and Contact Positions

[0711] Typically selection of recombinant antibodies with improved affinities can be carried out using phage display methods. This is accomplished by randomly mutating combinations of CDR residues to generate large libraries containing single-chain antibodies of different sequences. Typically, antibodies with improved affinities are selected based on their ability to reach an equilibrium in an antibody-antigen reaction. However, when Y61 scFV was expressed on phage surface and incubated with IL-12, selection conditions could not be found that would allow the system to reach normal antibody-antigen equilibrium. The scFV-phage remained bound to IL-12, presumably due to a non-specific interaction, since purified Y61 scFv exhibits normal dissociation kinetics. Since the usual methods of phage-display affinity maturation to Y61 (i.e. library generation and selections by mutagenesis of multiple CDR residues) could not be utilized, a new strategy was developed in which individual CDR positions were mutated.

[0712] This strategy involves selection of appropriate CDR positions for mutation and is based on identification and selection of amino acids that are preferred selective mutagenesis positions, contact positions, and/or hypermutation positions. Contact positions are defined as residues that have a high probability of contact with an antigen when the antigen interacts with the antibody, while hypermutation positions are defined as residues considered to have a high probability for somatic hypermutation during in vivo affinity maturation of the antibody. Preferred selective mutagenesis positions are CDR positions that are both contact and hypermutation positions. The Y61 antibody was already optimized in the CDR3 regions using the procedure described in Example 1, therefore it was difficult to further improve the area which lies at the center of the antibody binding site using phage-display selection methods. Greater improvements in activity were obtained by mutation of potential contact positions outside the CDR3 regions by either removing a detrimental antigen-antibody contact or, engineering a new contact.

[0713] Amino acids residues of Y61 which were considered contact points with antigen, and those CDR positions which are sites of somatic hypermutations during in vivo affinity maturation, are shown in Table 3 (see Appendix A). For Y61 affinity maturation, 15 residues outside CDR3, 3 residues within the L3 loop, and 5 residues in the H3 loop were selected for PCR mutagenesis.

[0714] Y61 scFv gene was cloned into the pUC119(Sfi) plasmid vector for mutagenesis. Oligonucleotides were designed and synthesized with randomized codons to mutate each selected position. Following PCR mutagenesis, a small number of clones (.about.24) were sequenced and expressed in a host cell, for example, in a bacterial, yeast or mammalian host cell. The expressed antibody was purified and the k.sub.off measured using the BIAcore system. Clones with improved off-rates, as compared to Y61, were then tested in neutralization assays. This procedure was repeated for other CDR positions. Individual mutations shown to have improved neutralization activity were combined to generate an antibody with even greater neutralization potency.

[0715] The Y61 CDR positions that were mutated in order to improve neutralization potency, and the respective amino-acid substitutions at each position are shown in FIGS. 2A-2H. Off-rates, as determined by BIAcore analysis, are given. These off rates are also shown in the histograms to the right of each table.

[0716] Results of these substitutions at positions H30, H32, H33, H50, H53, H54, H58, H95, H97, H101, L50, L92, L93, demonstrated that all amino-acid substitutions examined resulted in antibodies with poorer off-rates than Y61. At positions H52, L32, and L50, only a one amino acid substitution was found to improve the off-rate of Y61, all other changes adversely affected activity. For L50, this single Gly.fwdarw.Tyr change significantly (5-10 times) improved the neutralization potency of Y61. The results demonstrated the importance of these positions to Y61 activity, and suggest that in most cases phage-display was able to select for the optimal residues. However, at positions H31, H56, L30, and L94, several substitutions were found to improve Y61 off-rate, suggesting that these positions were also important for antigen binding, although the phage display approach did not allow selection of the optimal residues.

[0717] Selective mutation of contact and hypermutation positions of Y61 identified amino acid residue L50 in the light chain CDR2, and residue L94 of the light chain CDR3, which improved the neutralization ability of Y61. A combination of these mutations produced an additive effect, generating an antibody, J695, that exhibited a significant increase in neutralization ability. The full length sequence of J695 heavy and light chain variable region sequences is shown below.

J695 Heavy Chain Variable Region Peptide Sequence

TABLE-US-00009 [0718] (SEQ ID NO: 31) CDR H1 QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWV A CDR H2 FIRYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCKT CDR H3 HGSHDN WGQGTMVTVSS

J695 Light Chain Variable Region Peptide Sequence

TABLE-US-00010 [0719] (SEQ ID NO: 32) CDR L1 QSVLTQPPSVSGAPGQRVTISC SGSRSNIGSNTVK WYQQLPGTAPKLL IY CDR L2 YNDQRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYC CDR L3 QSYDRYTHPALL FGTGTKVTVLG

CDR residues are assigned according to the Kabat definitions.

[0720] A summary of the heavy and light chain variable region sequence alignments showing the lineage development of clones that were on the path from Joe9 to J695 is shown in FIGS. 1A-1D. The CDRs and residue numbering are according to Kabat.

Example 3

Functional Activity of Anti-hIL-12 Antibodies

[0721] To examine the functional activity of the human anti-human IL-12 antibodies of the invention, the antibodies were used in several assays that measure the ability of an antibody to inhibit IL-12 activity.

A. Preparation of Human PHA-Activated Lymphoblasts

[0722] Human peripheral blood mononuclear cells (PBMC) were isolated from a leukopac collected from a healthy donor by Ficoll-Hypaque gradient centrifugation for 45 minutes at 1500 rpm as described in Current Protocols in Immunology, Unit 7.1. PBMC at the interface of the aqueous blood solution and the lymphocyte separation medium were collected and washed three times with phosphate-buffered saline (PBS) by centrifugation for 15 minutes at 1500 rpm to remove Ficoll-Paque particles.

[0723] The PBMC were then activated to form lymphoblasts as described in Current Protocols in Immunology, Unit 6.16. The washed PBMC were resuspended at 0.5-1.times.10.sup.6 cells/ml in RPMI complete medium (RPMI 1640 medium, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 .mu.g/ml streptomycin), supplemented with 0.2% (v/v) PHA-P (Difco, Detroit, Mich.) and cultured for four days at 37.degree. C. in a 5% CO.sub.2 atmosphere. After four days, cell cultures were split 1:1 by volume in RPMI complete medium, plus 0.2% (v/v) PHA-P and 50 U/ml recombinant human IL-2. Recombinant human IL-2 was produced by transfection of an expression vector carrying the human IL-2 cDNA into COS cells (see Kaufman et al., (1991) Nucleic Acids Res. 19, 4484-4490), and purified as described in PCT/US96/01382. Cell cultures were then incubated for an additional one to three days. PHA blast cells were harvested, washed twice with RPMI complete medium and frozen in 95% FBS, 5% DMSO at 10.times.10.sup.6 cells/ml.

[0724] PHA blast cells to be used for the IL-12 receptor binding assay (see section B) were collected after one day culture in the presence of IL-2, whereas PHA blast cells to be used for the PHA blast proliferation assay (see section C) and the interferon-gamma induction assay (see section D) were collected after three day culture in the presence of IL-2.

B. IL-12 Receptor Binding Assay

[0725] The ability of anti-IL-12 antibodies to inhibit binding of radiolabelled IL-12 to IL-12 receptors on PHA blasts were analyzed as follows. Various concentrations of anti-IL-12 antibody were preincubated for 1 hour at 37.degree. C. with 50-100 pM .sup.125I-hIL-12 (iodinated hIL-12 was prepared using the Bolton-Hunter labeling method to a specific activity of 20-40mCi/mg from NEN-Dupont) in binding buffer (RPMI 1640, 5% FBS, 25 mM Hepes pH 7.4). PHA blast cells isolated as described above, were washed once and resuspended in binding buffer to a cell density of 2.times.10.sup.7 cells/ml. PHA blasts (1.times.10.sup.6 cells) were added to the antibody .sup.125I-hIL-12 mixture and incubated for two hours at room temperature. Cell bound radioactivity was separated from free 1-hIL-12 by centrifugation of the assay mixture for 30 seconds at room temperature, aspiration of the liquid and a wash with 0.1 ml binding buffer, followed by centrifugation at 4.degree. C. for 4 min at 10,000.times.g. The cell pellet was examined for cell bound radioactivity using a gamma counter. Total binding was determined in the absence of antibody and non-specific binding was determined by inclusion of 25 nM unlabeled IL-12 in the assay. Incubations were carried out in duplicate.

[0726] In the IL-12 receptor binding assay using the Y61 and J695 human anti-IL-12 antibodies, both antibodies demonstrated a comparable inhibition of IL-12 receptor binding. Y61 inhibited IL-12 receptor binding with an IC.sub.50 value of approximately 1.6.times.10.sup.-11M, while J695 had an IC.sub.50 value of approximately 1.1.times.10.sup.-11M.

C. Human PHA Blast Proliferation Assay

[0727] Anti-IL-12 antibodies were evaluated for their ability to inhibit PHA blast proliferation (which proliferation is stimulated by IL-12). Serial dilutions of anti-IL-12 antibody were preincubated for 1 hour at 37.degree. C., 5% CO.sub.2 with 230 pg/ml hIL-12 in 100 ml RPMI complete medium in a microtiter plate (U-bottom, 96-well, Costar, Cambridge, Mass.). PHA blast cells isolated as described above, were washed once and resuspended in RPMI complete medium to a cell density of 3.times.10.sup.5 cells/ml. PHA blasts (100 ml, 3.times.10.sup.4 cells) were added to the antibody/hIL-12 mixture, incubated for 3 days at 37.degree. C., 5% CO.sub.2 and labeled for 4-6 hours with 0.5 mCi/well (3H)-Thymidine (Amersham, Arlington Heights, Ill.). The culture contents were harvested onto glass fiber filters by means of a cell harvester (Tomtec, Orange, CT) and (3H)-Thymidine incorporation into cellular DNA was measured by liquid scintillation counting. All samples were assayed in duplicate.

[0728] The results of neutralization in the presence of varying concentrations of p70:p40 (i.e. the ratio of IL-12 heterodimer to free p40 subunit) is shown in Table 4 (see Appendix A).

[0729] Analysis of the Y61 human anti-IL-12 antibody in the PHA blast proliferation assay demonstrated that the antibody inhibited PHA blast proliferation with an IC.sub.50 value of approximately 1.8.times.10.sup.-10 M in the presence of IL-12 p70 alone, without any excess p40 (p70:p40 ratio of 1:0). In the presence of a 50-fold excess of free p40 (p70:p40 at a ratio of 1:50), the Y61 antibody inhibited PHA blast proliferation with an IC.sub.50 value of approximately 1.8.times.10.sup.-10M. This result demonstrates that the ability of Y61 to inhibit blast proliferation is not compromised by the presence of excess p40.

[0730] The human anti-IL-12 antibody, J695 inhibited PHA blast proliferation with an IC.sub.50 value of approximately 1.0.times.10.sup.-11M in the presence of p70:p40 at a ratio of 1:0. In the presence of a p70:p40 ratio of 1:50, this antibody inhibited PHA blast proliferation with an IC.sub.50 value of approximately 5.8.+-.2.8.times.10.sup.-12 M (n=2), demonstrating that the excess p40 had only a slight inhibitory effect on the antibody. Overall results demonstrate the improved neutralization activity of J695 in comparison with Y61 due to the mutations at L50 and L94.

D. Interferon-Gamma Induction Assay

[0731] The ability of anti-IL-12 antibodies to inhibit the production of IFN.gamma. by PHA blasts (which production is stimulated by IL-12) was analyzed as follows. Various concentrations of anti-IL-12 antibody were preincubated for 1 hour at 37.degree. C., 5% CO.sub.2 with 200-400 pg/ml hIL-12 in 100 ml RPMI complete medium in a microtiter plate (U-bottom, 96-well, Costar). PHA blast cells isolated as described above, were washed once and resuspended in RPMI complete medium to a cell density of 1.times.10.sup.7 cells/ml. PHA blasts (100 .mu.l of 1.times.10.sup.6 cells) were added to the antibody/hIL-12 mixture and incubated for 18 hours at 37.degree. C. and 5% CO.sub.2. After incubation, 150 .mu.l of cell free supernatant was withdrawn from each well and the level of human IFN.gamma. produced was measured by ELISA (Endogen Interferon gamma ELISA, Endogen, Cambridge, Mass.). Each supernatant was assayed in duplicate.

[0732] Analysis of human anti-hIL-12 antibody, Y61 in this assay demonstrated that Y61 inhibited human IFN.gamma. production with an IC.sub.50 value of approximately 1.6.times.10.sup.-10M, while the human anti-IL-12 antibody, J695, inhibited human IFN.gamma. production with an IC.sub.50 value of approximately 5.0.+-.2.3.times.10.sup.-12 M (n=3). The result demonstrates the substantial improvement in the affinity of J695 as a result of the modifications at L50 and L94.

E. Induction of Non-Human IL-12 from Isolated PBMC

[0733] To examine the cross-reactivity of the human anti-hIL-12 antibodies with IL-12 from other species, non-human IL-12 was produced as follows. PBMC were separated from fresh heparinized blood by density gradient centrifugation as described above using lymphoprep (Nycomed, Oslo, Norway) for cynomolgus monkey, baboon, and dog, PBMC, Accu-paque (Accurate Chemical & Sci. Corp., Westbury, N.Y.) for dog PBMC or Lympholyte-rat (Accurate Chemical & Sci. Corp., Westbury, N.Y.) for rat PBMC.

[0734] The PBMC were then induced to produce IL-12 as described (D'Andrea et al., (1992) J. Exp. Med 176, 1387-1398, Villinger et al., (1995) J. Immunol. 155, 3946-3954, Buettner et al., (1998) Cytokine 10, 241-248). The washed PBMC were resuspended at 1.times.10.sup.6 cells/ml in RPMI complete medium, supplemented with 0.0075% (wt/vol) of SAC (Pansorbin; Calbiochem-Behring Co., La Jolla, Calif.) or 1-5 mg/ml ConA (Sigma Chemical Co., St. Louis, Mo.) plus 0.0075% SAC and incubated for 18 hours at 37.degree. C. in a 5% CO.sub.2 atmosphere. Cell-free and SAC-free medium was collected by centrifugation and filtering through 0.2 mm filters.

[0735] IL-12 from the rhesus monkey was obtained as recombinant rhesus IL-12 from Emory University School of Medicine, Atlanta, Ga.

F. Murine 2D6 Cell Proliferation Assay

[0736] The murine T cell clone 2D6 proliferates in response to murine IL-2, IL-4, IL-7 and IL-12 (Maruo et al., (1997) J. Leukocyte Biol. 61, 346-352). A significant proliferation was also detected in response to rat PBMC supernatants containing rat IL-12. The cells do not respond to dog, cynomolgus, baboon or human IL-12. Murine 2D6 cells were propagated in RPMI complete medium supplemented with 50 mM beta-mercaptoethanol (.beta.ME) and 30 ng/ml murine IL-12. One day prior to the assay, the murine IL-12 was washed out and the cells were incubated overnight in RPMI complete medium plus .beta.ME.

[0737] Serial dilutions of anti-IL-12 antibody were preincubated for 1 hour at 37.degree. C., 5% CO.sub.2 with 40 pg/ml murine IL-12 in 100 ml RPMI complete medium plus .beta.ME in a microtiter plate (U-bottom, 96-well, Costar). 2D6 cells were washed once and resuspended in RPMI complete medium containin.gamma. .beta.ME to a cell density of 1.times.10.sup.5 cells/ml. 2D6 cells (100 .mu.l, 1.times.10.sup.4 cells) were added to the antibody/hIL-12 mixture, incubated for 3 days at 37.degree. C., 5% CO.sub.2 and labeled for 4-6 hours with 0.5 mCi/well (3H)-Thymidine. The culture contents were harvested and counted by liquid scintillation counting. All samples were assayed in duplicate.

G. Species Cross-Reactivity of J695 with Non-Human IL-12

[0738] Species cross-reactivity of J695 with non-human IL-12 was analyzed using PBMC's isolated from several non-human species. The presence of non-human IL-12 activity in the rat, dog, cynomolgus and baboon PBMC supernatants was confirmed using several bioassays described above, such as the murine 2D6 cell proliferation assay, the human PHA blast proliferation assay and the interferon-gamma induction assay by blocking the non-human PBMC induced responses with rabbit and/or sheep polyclonal antibodies to murine and/or human IL-12. Cross-reactivity of the human anti-hIL-12 antibodies Y61 and J695 with non-human IL-12 in PBMC supernatants or purified murine and rhesus IL-12 was then assessed in the same bioassay(s) by determining the J695 antibody concentration at which 50% inhibition of the response was observed. The species cross-reactivity results are summarized in Table 5. The results demonstrate that Y61 and J695 are each able to recognize IL-12 from monkeys (e.g., cynomolgus and rhesus IL-12 for Y61, and cynomolgus, rhesus and baboon for J695) and that J695 is approximately 35 fold less active on dog IL-12; neither Y61 nor J695 cross reacts with mouse or rat IL-12.

H. Human Cytokine Specificity of J695

[0739] The specificity of J695 was tested in a competition ELISA in which a panel of human cytokines was tested for their ability to interfere with the binding of soluble J695 to immobilized human IL-12. The panel of human cytokines included IL-1.alpha. and IL-1.beta. (Genzyme, Boston, Mass.), IL-2 (Endogen), IL-4, IL-10, IL-17, IFN-gamma, and TGF-.beta.1 (R&D, Minneapolis, Minn.) IL-8 (Calbiochem), PDGF, IGF-I, and IGF-II (Boehringer Mannheim Corp., Indianapolis, Ind.), TNF.alpha. and lymphotoxin, IL-6, soluble IL-6 receptor, IL-11, IL-12 p70, IL-12 p40, M-CSF, and LIF. EBI-3, an IL-12 p40 related protein that is induced by Epstein-Barr virus infection in B lymphocytes (Devergne et

TABLE-US-00011 TABLE 5 Species Cross Reactivity Data IC.sub.50 (M) Antibody Mouse IL-12 Rat IL-12 Dog IL-12 Cyno IL-12 Rhesus IL-12 Baboon IL-12 Human IL-12 Name Specificity Purified PBMC sup PBMC sup PBMC sup Purified PBMC sup Purified C17.15 rat-.alpha.muIL12 3.0 .times. 10.sup.-11 R03B03 rabbit-.alpha.muIL12 1.5 .times. 10.sup.-10 6.0 .times. 10.sup.-10 C8.6.2 mouse-.alpha.huIL12 1.2 .times. 10.sup.-10 1.0 .times. 10.sup.-10 2.0 .times. 10.sup.-10 5.0 .times. 10.sup.-11 Y61 human-.alpha.huIL12 Non-neutralizing 2.2 .times. 10.sup.-10 1.0 .times. 10.sup.-10 1.7 .times. 10.sup.-10 J695 human-.alpha.huIL12 Non-neutralizing Non-neutralizing 3.5 .times. 10.sup.-10 1.0 .times. 10.sup.-11 1.0 .times. 10.sup.-11 1.5 .times. 10.sup.-11 5.0 .times. 10.sup.-12

al., (1996) J. Virol. 70, 1143-1153) was expressed as a human IgG-Fc chimera (EBI-3/Fc) Single-stranded salmon sperm DNA (Sigma) was also tested.

[0740] Flat-bottom ELISA immunoassay microtiter plates (96 well, high binding, Costar) were coated overnight at 4.degree. C. with 0.1 ml human IL-12 (2 .mu.g/ml in 0.1 M carbonate coating buffer (4 volumes 0.1 M NaHCO.sub.3 plus 8.5 volumes 0.1 M NaHCO.sub.3)). The plates were washed twice with PBS containing 0.05% Tween 20 (PBS-T), blocked with 200 .mu.l of 1 mg/ml bovine serum albumin (BSA, Sigma) in PBS-T for 1 hour at room temperature, and again washed twice with PBS-T. Samples (100 .mu.l) containing IL-12 antibody J695 (100 ng/ml) and each cytokine (2 nM) in PBS-T containing 50 .mu.g/ml BSA (PBS-T/BSA) were added and incubated for 2 h at room temperature. The plates were washed 4 times and incubated for 1 h at room temperature with 100 .mu.l mouse anti-human lambda-HRP (1:500 in PBS-T/BSA, Southern Biotech. Ass. Inc., Birmingham, Ala.). The plates were washed 4 times and developed with ABTS (Kirkegaard & Perry Lab., Gaithersburg, Md.) for 20-30 minutes in the dark. The OD.sub.450 nm was read using a microplate reader (Molecular Devices, Menlo Park, Calif.). Percent binding was determined relative to J695 binding to the IL-12 coated plate in the absence of any soluble cytokine.

[0741] The results demonstrated that J695 binding to immobilized human IL-12 was blocked only by human IL-12 p70 and to a lesser extent, by human IL-12 p40 and not by any of the other cytokines tested.

I. Binding to a Novel IL-12 Molecule

[0742] An alternative IL-12 heterodimer has been described, in which the p35 subunit is replaced by a novel p19 molecule. P19 was identified using 3D homology searching for IL-6/IL-12 family members, and is synthesized by activated dendritic cells. P19 binds to p40 to form a p19/p40 dimer, which has IL-12-like activity, but is not as potent as the p35/p40 heterodimer in IFN.gamma. induction. Antibodies which recognize p40 alone, but preferably in the context of a p70 molecule (e.g., J695 and Y61, see Example 3H) are expected to also neutralize both the p35/p40 molecules and the p19/p40 molecules.

Example 4

In Vivo Activity of Anti-hIL-12 Antibodies

[0743] The in vivo effects of IL-12 antibodies on IL-12 induced responses were examined in a model modified from one used by Bree et al. to study the effect of human IL-12 on peripheral hematology in cynomolgus monkey Bree et al., (1994) Biochem Biophys Res. Comm. 204: 1150-1157. In those previous studies, administration of human IL-12 at 1 .mu.g/kg/day for a period of 5 days resulted in a decrease in white blood cell count (WBC), especially in the lymphocyte and monocyte subsets after 24 hours. A decrease in the platelet count was observed at 72 hours. Levels of plasma neopterin, a marker of monocyte activation in response to IFN-.gamma., began to elevate at 24 hours and were the highest at 72 hours.

[0744] In the first study with human anti-hIL-12 antibodies, fifteen healthy cynomolgus monkeys with an average weight of 5 kg, were sedated and divided into 5 groups (n=3). Group 1 received an intravenous (IV) administration of 10 mg/kg human intravenous immunoglobulin (IVIG, Miles, Eckhart, IN, purified using protein A Sepharose). Group 2 received an intravenous administration of 1 mg/kg C8.6.2 (neutralizing mouse anti-human IL-12 monoclonal antibody). Group 3 received an intravenous administration of 10 mg/kg C8.6.2. Group 4 received an intravenous administration of 1 mg/kg Y61 (human anti-human IL-12 antibody, purified from CHO cell conditioned medium). Group 5 received an intravenous administration of 10 mg/kg Y61.

[0745] One hour after the antibody administration all animals received a single subcutaneous (SC) injection of human IL-12 (1 .mu.g/kg). Blood samples were taken at the following time points: baseline, 8, 24, 48, 96 and 216 hours, and analyzed for complete blood cell counts with differentials and serum chemistry. Serum human IL-12, C8.6.2 antibody, Y61 antibody, monkey IFN-gamma, monkey IL-10, monkey IL-6 and plasma neopterin levels were also measured.

[0746] Animals treated with IL-12 plus IVIG control antibody (Group 1) showed many of the expected hematological changes, including decreases in WBC, platelets, lymphocyte count and monocyte count. These decreases were not seen or were less pronounced in the animals treated with either the C8.6.2 or Y61 antibody at 1 or 10 mg/kg (Groups 2-5).

[0747] Serum or plasma samples were analyzed by ELISA specific for monkey IFN-gamma and monkey IL-10 (Biosource International, Camarillo, Calif.), monkey IL-6 (Endogen) and plasma neopterin (ICN Pharmaceuticals, Orangeburg, N.Y.). IFN-gamma, IL-10 or IL-6 were not detected in any of the IL-12 treated animals including the control animals treated with IL-12 plus IVIG. This was probably due to the low level exposure to IL-12 (only 1 dose of 1 .mu.g/kg). Nevertheless, plasma neopterin levels increased about three fold in the IL-12 plus IVIG treated animals but did not change in all C8.6.2 or Y61 treated animals, including the lower dose (1 mg/kg) Y61 treated animals, indicating that Y61 was effective in vivo in blocking this sensitive response to IL-12.

[0748] In a second study, in vivo activity and pharmacodynamics (PD) of J695 in cynomolgous monkeys were studied by administering exogenous rhIL-12 and determining if J695 could block or reduce the responses normally associated with rhIL-12 administration. Male cynomolgus monkeys (n=3 per group) were administered a single dose of 0.05, 0.2, or 1.0 mg/kg J695 or 1 mg/kg intravenous immunoglobulin (IVIG) as a bolus intravenous (IV) injection via a saphenous vein or subcutaneously (SC) in the dorsal skin. One hour following the administration of J695 or IVIG, all animals received a single SC dose of 1 .mu.g/kg rhIL-12 in the dorsal skin. Blood samples were collected via the femoral vein up to 28 days after J695 administration. Serum was acquired from each blood sample and assayed for IL-12, J695, IFN-.gamma., and anti-J695 antibodies by ELISA. Neopterin was assayed by reverse-phase high performance liquid chromatography.

[0749] The levels of neopterin, normalized with respect to the levels of neopterin that were measured before administration of J695 or rhIL-12, are shown in FIG. 3. To compare the suppression of neopterin between groups, the area under the curve (AUC) normalized for neopterin levels was calculated for each animal (Table 6). Neopterin exposure (AUC) was suppressed in a dose-dependent manner between approximately 71 and 93% in the IV groups and between 71 and 100% in SC groups, relative to the IVIG control groups. These results suggest that the dose of J695 necessary for 50% inhibition of the neopterin response (ED.sub.50) was less than 0.05 mg/kg when administered by either the IV or SC route.

TABLE-US-00012 TABLE 6 Dose-Dependent Suppression of IL-12 Induced Neopterin by J695 in Cynomolgus Monkeys Route of dosing IVIG J695 Dose IVIG Dose AUC of Normalized % Reduction of Neopterin or J695 and rhIL-12 (mg/kg) (mg/kg) Neopterin Levels AUC Compared with Control Single IV injection -- 1.0 1745 .+-. 845 0 followed 1 hr later by a 0.05 -- 502 .+-. 135 71.3 dose of 1 .mu.g/kg human 0.2 -- 199 .+-. 316 88.6 IL-12 given SC 1.0 -- 128 .+-. 292 92.7 Single SC injection -- 1.0 1480 .+-. 604 0 followed 1 hour later 0.05 -- 426 .+-. 108 71.2 by a dose of 1 .mu.g/kg 0.2 -- 395 .+-. 45.9 73.3 human IL-12 given SC 1.0 -- 0 .+-. 109 100

[0750] Treatment with J695 also prevented or reduced the changes in hematology normally associated with rhIL-12 administration (leukopenia and thrombocytopenia). At 24 hours after rhIL-12 administration lymphocyte counts were reduced by approximately 50% when compared to baseline values in the control IV and SC IVIG treated groups. Administration of J695 either SC or IV at all three dose levels prevented this reduction, resulting in lymphocyte counts at 24 hours approximately the same as baseline values. At 48 hours after IL-12 administration, platelet counts in the groups treated with IV and SC IVIG were reduced by approximately 25% when compared to baseline values.

[0751] An example dose schedule targeted to maintain serum levels above the 90% effect level would be 1 mg/kg IV and SC given approximately every other week, or 0.3 mg/kg given approximately every week, assuming slight accumulation during repeated dosing. This study demonstrates that antibody can be given safely to monkeys at such dosages. In independent toxicity studies, it was further found that up to 100 mg/kg of the antibody can be given safely to monkeys.

[0752] J695 was also effective in preventing IFN-.gamma. production in mice treated with a chimeric IL-12, a molecule which combines the murine p35 subunit with the human IL-12 p40 subunit. In contrast to human IL-12 which is biologically inactive in mice, this chimeric IL-12 retains biological function in mice, including induction of IFN-.gamma.. In addition, the human p40 subunit allows the molecule to be bound and neutralized by J695. Chimeric IL-12 at a dose of 0.05 mg/kg i.p. was administered to female C3H/HeJ mice (10/experimental group) in five daily doses on days 0, 1, 2, 3, and 4. J695 was given on days 0, 2 and 4 at doses of 0.05, 0.01, 0.002, 0.0004, 0.00008, and 0.000016 mg/kg i.p., 30' prior to the IL-12 injections. The control hulgG1.gamma. was given IP. at a dose of 0.05 mg/kg on days 0, 2, and 4. The mice were bled on day 5, and serum IFN-.gamma. levels were determined by ELISA. The results demonstrated that J695 caused dose-dependent inhibition of IFN-.gamma. production with an ED.sub.50 of approximately 0.001 mg/kg. Collectively, these results demonstrate that J695 is a potent inhibitor of Il -12 activity in vivo.

Example 5

Kinetic Analysis of Binding of Human Antibodies to Recombinant Human IL-12 (rhIL-12)

[0753] Real-time binding interactions between captured ligand (human anti-rhIL-12 antibody J695, captured on a biosensor matrix) and analyte (rhIL12 in solution) were measured by surface plasmon resonance (SPR) using the BIAcore system (Biacore AB, Uppsala, Sweden). The system utilizes the optical properties of SPR to detect alterations in protein concentration within a dextran biosensor matrix. Proteins are covalently bound to the dextran matrix at known concentrations. Antibodies are injected through the dextran matrix and specific binding between injected antibodies and immobilized ligand results in an increased matrix protein concentration and resultant change in the SPR signal. These changes in SPR signal are recorded as resonance units (RU) and are displayed with respect to time along the y-axis of a sensorgram.

[0754] To facilitate immobilization of goat anti-human IgG (Southern Biotechnology Associates, Cat. No. 2040-01, Birmingham, Ala.) on the biosensor matrix, goat anti-human IgG is covalently linked via free amine groups to the dextran matrix by first activating carboxyl groups on the matrix with 100 mM N-hydroxysuccinimide (NHS) and 400 mM N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). Next, goat anti-human IgG is injected across the activated matrix. Thirty-five microliters of goat anti-human IgG (25 .mu.g/ml), diluted in sodium acetate, pH 4.5, is injected across the activated biosensor and free amines on the protein are bound directly to the activated carboxyl groups. Unreacted matrix EDC-esters are deactivated by an injection of 1 M ethanolamine. Standard amine coupling kits were commercially available (Biacore AB, Cat. No. BR-1000-50, Uppsala, Sweden).

[0755] J695 was diluted in HBS running buffer (Biacore AB, Cat. No. BR-1001-88, Uppsala, Sweden) to be captured on the matrix via goat anti-human IgG. To determine the capacity of rhIL12-specific antibodies to bind immobilized goat anti-human IgG, a binding assay was conducted as follows. Aliquots of J695 (25 .mu.g/ml; 25 .mu.l aliquots) were injected through the goat anti-human IgG polyclonal antibody coupled dextran matrix at a flow rate of 5 .mu.l/min. Before injection of the protein and immediately afterward, HBS buffer alone flowed through each flow cell. The net difference in signal between the baseline and the point corresponding to approximately 30 seconds after completion of J695 injection was taken to represent the amount of IgG1 J695 bound (approximately 1200 RU's). Direct rhIL12 specific antibody binding to soluble rhIL12 was measured. Cytokines were diluted in HBS running buffer and 50 .mu.l aliquots were injected through the immobilized protein matrices at a flow rate of 5 .mu.l/min. The concentrations of rhIL-12 employed were 10, 20, 25, 40, 50, 80, 100, 150 and 200 nM. Prior to injection of rhIL-12, and immediately afterwards, HBS buffer alone flowed through each flow cell. The net difference in baseline signal and signal after completion of cytokine injection was taken to represent the binding value of the particular sample. Biosensor matrices were regenerated using 100 mM HCl before injection of the next sample. To determine the dissociation constant (off-rate), association constant (on-rate), BIAcore kinetic evaluation software (version 2.1) was used.

[0756] Representative results of CHO derived J695 binding to rhIL-12 as compared to the COS derived J695, are shown in Table 7.

TABLE-US-00013 TABLE 7 Binding of CHO or COS derived J695 to rhIL-12. rhIL12 bound, Ab, bound, Source rhIL12, nM RU's RU's rhIL12/AB CHO 200 1112 1613 1.48 CHO 150 1033 1525 1.45 CHO 100 994 1490 1.43 CHO 80 955 1457 1.40 CHO 50 912 1434 1.36 CHO 40 877 1413 1.33 CHO 25 818 1398 1.25 CHO 20 773 1382 1.20 CHO 10 627 1371 0.98 COS 200 1172 1690 1.49 COS 150 1084 1586 1.46 COS 100 1024 1524 1.44 COS 80 985 1489 1.42 COS 50 932 1457 1.37 COS 40 894 1431 1.34 COS 25 833 1409 1.27 COS 20 783 1394 1.20 COS 10 642 1377 1.00

[0757] Molecular kinetic interactions between captured J695 and soluble rhIL-12 were quantitatively analyzed using BIAcore technology. Several independent experiments were performed and the results were analyzed by the available BIAcore mathematical analysis software to derive kinetic rate constants, as shown in Table 8.

TABLE-US-00014 TABLE 8 Apparent kinetic rate and affinity constants of J695 for rhIL-12. On-rate (M-1s-1), Off-rate (s-1), Kd (M), Antibody Source Avg. Avg. Avg. J695 CHO 3.52E+05 4.72E-05 1.34E-10 J695 COS 3.40E+05 2.61E-05 9.74E-11

[0758] There was a small difference between the calculated apparent constant (Kd) for the interaction between CHO derived J695 (Kd=1.34.sup.-10M.sup.-1) and COS derived J695 (Kd=9.74.times.10.sup.-11M.sup.-1) antibodies. The apparent dissociation constant (Kd) between J695 and rhIL12 was estimated from the observed rate constants by the formula: Kd=off-rate/on-rate.

[0759] To determine the apparent association and dissociation rate constant for the interaction between J695 and rhIL-12, several binding reactions were performed using a fixed amount of J695 (2 .mu.g/ml) and varying concentrations of rhIL-12. Real-time binding interaction sensorgrams between captured J695 and soluble rhIL12 showed that both forms of antibody were very similar for both the association and dissociation phase.

[0760] To further evaluate the capacity of captured IgG1 J695 mAb to bind soluble recombinant cytokine, a direct BIAcore method was used. In this method, goat anti-human IgG (25 .mu.g/ml) coupled carboxymethyl dextran sensor surface was coated with IgG1 J695 (2 .mu.g/ml) and recombinant cytokine was then added. When soluble rhIL12 was injected across a biosensor surface captured with CHO or COS derived IgG1 J695, the amount of signal increased as the concentration of cytokine in the solution increased. No binding was observed with rmIL12 (R&D Systems, Cat. No. 419-ML, Minneapolis, Minn.) or rh IL12 any concentration tested up to 1000 nM. These results support the conclusion that IgG1 J695 antibodies recognize a distinct determinant on rhIL-12.

[0761] Table 9 shows the results of an experiment using BIAcore to demonstrate human IgG1 J695 mAb binding to only soluble rhIL12 and none of the other recombinant cytokines.

TABLE-US-00015 TABLE 9 Epitope mapping of J695 using BIAcore technology. Captured ligand Captured ligand Soluble analyte COS J695 CHO J695 rec. human IL12 Positive Positive rec. murine IL12 Negative Negative

Example 6

Further Studies of J695 Affinity for IL-12

[0762] Molecular kinetic interactions between J695 antibody and human IL-12 were quantitatively analyzed using BIAcore plasmon resonance technology, and apparent kinetic rate constants were derived.

[0763] BIAcore technology was used to measure the binding of soluble rhIL-12 to solid phase captured J695. A goat anti-human IgG antibody was immobilized on the biosensor chips, then a fixed amount of J695 was injected and captured on the surface. Varying concentrations of rhIL-12 were applied, and the binding of IL-12 at different concentrations to J695 was measured as a function of time. Apparent dissociation and association rate constants were calculated, assuming zero-order dissociation and first order association kinetics, as well as a simple one-to-one molecular interaction between J695 and IL-12. Three independent experiments were performed, and the values shown are averages for the three experiments. From these measurements, the apparent dissociation (k.sub.d) and association (k.sub.a) rate constants were derived and used to calculate a K.sub.d value for the interaction (see Table 10). The results indicated that J695 has a high affinity for rhIL-12.

TABLE-US-00016 TABLE 10 Kinetic Parameters for the Interaction Between J695 and Human IL-12 Kinetic Parameter Value k.sub.d 3.71 .+-. 0.40 .times. 10.sup.-5 s.sup.-1 k.sub.a 3.81 .+-. 0.48 .times. 10.sup.5 M.sup.-1s.sup.-1 K.sub.d 9.74 .times. 10.sup.-11 M (14 ng/mL)

Example 7

Characteristics and Neutralization Activity of C17.15, a Rat Monoclonal Antibody to Murine Interleukin-12

[0764] To assess the relevance of IL-12 treatment studies in mouse models of inflammation and autoimmunity using monoclonal antibodies specific for murine IL-12 to similar approaches in human disease, the interaction of C17.15, a rat anti-murine IL-12 monoclonal antibody with murine IL-12, was examined. The ability of C17.15 to neutralize murine IL-12 activity in a PHA blast proliferation assay, and to block murine IL-12 binding to cell surface receptors, was assessed, as were the kinetics of the C17.15-murine IL-12 binding interaction.

[0765] In a human PHA blast proliferation assay (See Example 3), serial dilutions of C17.15 or rat IgG2a (a control antibody) were preincubated with 230 pg/mL murine IL-12 for 1 hr at 37.degree. C. PHA-stimulated blast cells were added to the antibody-IL-12 mixtures and incubated for 3 days at 37.degree. C. The cells were subsequently labeled for 6 h with 1 .mu.Ci/well [.sup.3H]-thymidine. The cultures were harvested and [.sup.3H]-thymidine incorporation was measured. Background non-specific proliferation was measured in the absence of added murine IL-12. All samples were assayed in duplicate. The IC.sub.50 (M) of C17.15 for recombinant murine IL-12 in this assay was found to be 1.4.times.10.sup.-11, as compared to the IC.sub.50 value of 5.8.times.10.sup.-12 observed for J695 for recombinant human IL-12 under the same conditions (see Table 11).

TABLE-US-00017 TABLE 11 Comparison of the properties of anti-human IL-12 monoclonal antibody J695 and the rat anti-mouse IL-12 monoclonal antibody C17.15 Biomolecular Interaction Assay Receptor PHA blast k.sub.a, on-rate k.sub.d, off-rate Binding Assay Assay Antibody Epitope (M.sup.-1s.sup.-1) (s.sup.-1) K.sub.d (M) IC.sub.50 (M) IC.sub.50 (M) J695 Hu p40 3.81 .times. 10.sup.5 3.71 .times. 10.sup.-5 9.74 .times. 10.sup.-11 1.1 .times. 10.sup.-11 5.8 .times. 10.sup.-12 C17.15 Mu p40 3.80 .times. 10.sup.5 1.84 .times. 10.sup.-4 4.80 .times. 10.sup.-10 1.5 .times. 10.sup.-10 1.4 .times. 10.sup.-11

[0766] The ability of C17.15 to inhibit the binding of murine IL-12 to cellular receptors was also measured. Serial dilutions of C17.15 were pre-incubated for 1 hr at 37.degree. C. with 100 pM [.sup.125I]-murine IL-12 in binding buffer. The 2D6 cells (2.times.10.sup.6) were added to the antibody/[.sup.125I]-murine IL-12 mixture and incubated for 2 hours at room temperature. Cell-bound radioactivity was separated from free [.sup.125I]-IL-12, and the remaining cell-bound radioactivity was determined. Total binding of the labeled murine IL-12 to receptors on 2D6 cells was determined in the absence of antibody, and non-specific binding was determined by the inclusion of 25 nM unlabelled murine IL-12 in the assay. Specific binding was calculated as the total binding minus the non-specific binding. Incubations were carried out in duplicate. The results showed that C17.15 has an IC.sub.50 (M) of 1.5.times.10.sup.-10 for inhibition of binding of murine IL-12 to cellular receptors.

[0767] The affinity of C17.15 for recombinant murine IL-12 was assessed by biomolecular interaction analysis. A goat anti-rat IgG antibody was immobilized on the biosensor chips, followed by an injection of a fixed amount of the C17.15 antibody, resulting in capture of C17.15 on the surface of the chip. Varying concentrations of recombinant murine IL-12 were applied to the C17.15 surface, and the binding of murine IL-12 to the immobilized C17.15 was measured as a function of time. Apparent dissociation and association rate constants were calculated, assuming a zero order dissociation and first order association kinetics as well as a simple one to one molecular interaction between the immobilized C17.15 and murine IL-12. From these measurements, the apparent dissociation (k.sub.d, off-rate) and association (k.sub.a, on-rate) rate constants were calculated. These results were used to calculate a K.sub.d value for the interaction. An on-rate of 3.8.times.10.sup.5 M.sup.-1s.sup.-1, an off-rate of 1.84.times.10.sup.-4 s.sup.-1, and a K.sub.d of 4.8.times.10.sup.-10 was observed for the recombinant murine IL-12-C17.15 interaction.

[0768] The observed activities of C17.15 in neutralizing murine IL-12 activity and binding to cell surface receptors, as well as the kinetics of binding of C17.15 to murine IL-12 correlate with similar measurements for the J695-rhIL-12 interaction. This indicates that the modes of action of the rat anti-mouse IL-12 antibody C17.15 and anti-human IL-12 antibody J695 are nearly identical based upon on-rate, off-rate, K.sub.d, IC.sub.50, and the PHA blast assay. Therefore, C17.15 was used as a homologous antibody to J695 in murine models of inflammation and autoimmune disease to study the effects of IL-12 blockade on the initiation or progression of disease in these model animals (see Example 8).

Example 8

Treatment of Autoimmune or Inflammation-Based Diseases in Mice by .alpha.-Murine IL-12 Antibody Administration

A. Suppression of Collagen-Induced Arthritis in Mice by the .alpha.-Il-12 Antibody C17.15

[0769] A correlation between IL-12 levels and rheumatoid arthritis (RA) has been demonstrated. For example, elevated levels of IL-12 p70 have been detected in the synovia of RA patients compared with healthy controls (Morita et al (1998) Arthritis and Rheumatism. 41: 306-314). Therefore, the ability of C17.15, a rat anti-mouse IL-12 antibody, to suppress collagen-induced arthritis in mice was assessed.

[0770] Male DBA/1 mice (10/group) were immunized with type II collagen on Day 0 and treated with C17.15, or control rat IgG, at 10 mg/kg intraperitoneally on alternate days from Day-1 (1 day prior to collagen immunization) to Day 12. The animals were monitored clinically for the development of arthritis in the paws until Day 90. The arthritis was graded as: 0--normal; 1--arthritis localized to one joint; 2--more than one joint involved but not whole paw; 3--whole paw involved; 4--deformity of paw; 5--ankylosis of involved joints. The arthritis score of a mouse was the sum of the arthritic grades in each individual paw of the mouse (max=20). The results are expressed as mean.+-.SEM in each group.

[0771] The results, as shown in FIG. 4, indicate that an arthritic score was measurable in the C17.15-treated mice only after day 50 post-treatment, and that the peak mean arthritic score obtained with the C17.15-treated mice was at least 5-fold lower than that measured in the IgG-treated mice. This demonstrated that the rat anti-mouse IL-12 antibody C17.15 prevented the development of collagen-induced arthritis in mice.

B. Suppression of Colitis in Mice by the Rat .alpha.-Murine IL-12 Antibody C17.15

[0772] IL-12 has also been demonstrated to play a role in the development/pathology of colitis. For example, anti-IL-12 antibodies have been shown to suppress disease in mouse models of colitis, e.g., TNBS induced colitis IL-2 knockout mice (Simpson et al. (1998) J. Exp. Med. 187(8): 1225-34). Similarly, anti-IL-2 antibodies have been demonstrated to suppress colitis formation in IL-10 knock-out mice. The ability of the rat anti-mouse IL-12 antibody, C17.15, to suppress TNBS colitis in mice was assessed in two studies (Davidson et al. (1998) J. Immunol. 161(6): 3143-9).

[0773] In the first study, colitis was induced in pathogen free SJL mice by the administration of a 150 .mu.L 50% ethanol solution containing 2.0 mg TNBS delivered via a pediatric umbilical artery catheter into the rectum. Control animals were treated with a 150 .mu.L 50% ethanol solution only. A single dose of 0.75, 0.5, 0.25, or 0.1 mg C17.15 or 0.75 mg control rat IgG2a was given intravenously via the tail vein at day 11, and the therapeutic effect of the treatment was assessed by weighing the animals on days 11 and 17, and histological scoring at day 17. The weight of the mice treated with C17.15 increased within 48 hours of antibody treatment and normalized on day 6 after treatment. The effect of treatment with C17.15 was confirmed histologically. Further, assessments of IFN-.gamma. secretion by CD4.sup.+ T-cells from spleen and colon of the treated mice, as well as IL-12 levels from spleen or colon-derived macrophages from the treated mice were also made (see Table 12).

[0774] In the second study, the dosing was optimized and the mice were treated with a total dose of 0.1 mg or 0.5 mg C17.15 or 0.1 mg control IgG2a, respectively, split between days 12 and 14. It was found that the administration of C17.15 in a single dose at the dosage of 0.1 mg/mouse or 0.25 mg/mouse led to only partial improvement in TNBS-induced colitis and did not result in a significant reduction in the CD4.sup.+ T cell production of IFN-.gamma. in vitro, but did result in a significant decrease in secretion of IL-12, compared to untreated controls. At a single dose of 0.5 mg/mouse or greater a response was observed. Taking the lowest dose of antibody tested and administering it in two divided injections (at days 12 and 14) improved the dosing regimen, indicating that multiple low doses can be more effective than a single bolus dose. The data obtained are shown in Table 12.

TABLE-US-00018 TABLE 12 Anti-mouse Il-12 mAb C17.15 Suppresses Established Colitis in Mice Disease Treatment Weight (g) IFN-.gamma. spleen IL-12 spleen Induction Day 0 Day 11 Day 11 Day 17 CD4.sup.+ cells (U/mL) macrophages (pg/ml) TNBS + Ethanol Control IgG2a 16.0 15.26 3326 300 0.75 mg TNBS + Ethanol C17.15 0.75 mg 16.0 20.21 1732 0 TNBS + Ethanol C17.15 0.5 mg 16.36 19.94 1723 0 TNBS + Ethanol C17.15 0.25 mg 16.28 17.7 3618 7 TNBS + Ethanol C17.15 0.1 mg 16.2 17.98 3489 22 Ethanol control -- 20.76 21.16 1135 0

[0775] Administration of C17.15 monoclonal anti-IL-12 in two divided doses spaced one day apart totaling 0.1 mg/mouse or 0.05 mg/mouse led to complete reversal of colitis as assessed by wasting and macroscopic appearance of the colon. In addition, this dose schedule led to significant down-regulation of lamina propria T-cell production of IFN-.gamma. and macrophage production of IL-12, so that the latter were comparable to levels seen in control ethanol-treated mice without TNBS-colitis. Thus, C17.15 administration to mouse models for TNBS colitis reversed the progression of the disease in a dose-dependent manner.

C. Suppression of Experimental Autoimmune Encephalomyelitis (EAE) in Mice by .alpha.-IL-12 Antibodies

[0776] It is commonly believed that IL-12 plays a role in the pathogenesis of multiple sclerosis (MS). The inducible IL-12 p40 message has been shown to be expressed in acute plaques of MS patients but not in inflammatory brain infarct lesions (Windhagen, A. et al. (1995) J. Exp. Med. 182: 1985-1996). T cells from MS patients (but not control T cells) stimulate IL-12 production from antigen-presenting cells through unregulated CD40L expression (Balashov, K. E. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 599-603). MS patients have enhanced IFN-.gamma. secretion that can be blocked with .alpha.-IL-12 antibodies in vitro (Balashov, K. E. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 599-603). Elevated levels of serum IL-12 are detected in MS patients, but not in other neurological diseases (Nicoletti, F. et al. (1996) J. Neuroimmunol. 70: 87-90). Increased IL-12 production has been shown to correlate with disease activity in MS patients (Cormabella, M. et al. (1998) J. Clin. Invest. 102: 671-678). The role of IL-12 in the pathogenesis of a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), has been studied (Leonard, J. P. et al. (1995) J. Exp. Med. 181: 281-386; Banerjee, S. et al. (1998) Arthritis Rheum. (1998) 41: S33; and Segal, B. M. et al. (1998) J. Exp. Med. 187: 537-546). The disease in this model is known to be induced by T cells of the TH.sub.1 subset. Therefore, the ability of .alpha.-IL-12 antibodies to prevent the onset of acute EAE was assessed.

[0777] An .alpha.-IL-12 antibody was found to be able to inhibit the onset of acute EAE, to suppress the disease after onset, and to decrease the severity of relapses in mice immunized with the autoantigen, myelin basic protein (Banerjee, S. et al. (1998) Arthritis Rheum. (1998) 41: S33). The beneficial effects of .alpha.-IL-12 antibody treatment in the mice persisted for over two months after stopping treatment. It has also been demonstrated that anti-IL-12 antibodies suppress the disease in mice that are recipients of encephalitogenic T cells by adoptive transfer (Leonard, J. P. et al. (1995) J. Exp. Med. 181: 281-386).

Example 9

Clinical Pharmacology of J695

[0778] In a double blind, crossover study, 64 healthy, human male subjects were administered ascending doses of J695 or placebo. Measurement of complement fragment C3a prior to and 0.25 h after dosing did not demonstrate activation of the complement system. CRP and fibrinogen levels were only increased in subjects in whom symptoms of concurrent infections were observed.

[0779] All subjects survived and the overall tolerability of J695 was very good. In no case did treatment have to be stopped because of adverse events (AEs). The most commonly observed AEs were headache and common cold/bronchitis, neither of which were categorized as severe.

[0780] One of the study subjects, a 33-year-old single male, was suffering from psoriasis guttata at the start of the study. According to the randomized study design, this subject by chance received 5 mg/kg J695 by SC administration. Ten days prior to administration of the antibody, the subject showed only small discrete papular lesions on the arms and legs. At the time of the antibody administration, the subject displayed increased reddening, thickness of the erythematous plaques, and increased hyperkaratosis. One week after J695 administration, the subject reported an improvement in skin condition, including flattening of the lesions and a decrease in scaling. Shortly after the second administration of J695 (5 mg/kg IV), the subject's skin was totally cleared of psoriatic lesions, in the absence of any local treatment. Erythematous plaques covered with white scales reappeared concomitant with the expected clearance of J695 after the second administration of antibody.

Example 10

Comparison of J695 Produced by Two CHO Cell Lines

[0781] For recombinant expression of J695, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells (Urlaub, G. and Chasin, L. A. (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220) by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.

[0782] One hundred and fifty micrograms of an expression vector encoding the peptide sequences of the human antibody J695 were dissolved in 2.7 ml water in a 50 ml conical tube. Three hundred .mu.L of 2.5 M CaCl.sub.2 were added and this DNA mixture was added dropwise to 3 ml of 2.times.HEPES buffered saline in a 50 ml conical tube. After vortexing for 5 sec and incubating at room temperature for 20 min, 1 mL was distributed evenly over each plate (still in F12 medium), and the plates were incubated at 37.degree. C. for 4 h. Liquid was removed by aspiration and 2 ml of 10% DMSO in F12 were added to each plate. The DMSO shock continued for 1 min, after which the DMSO was diluted by the addition of 5 ml PBS to each plate. Plates were washed twice in PBS, followed by the addition of 10 ml of alpha MEM, supplemented with H/T and 5% FBS (selective for cells expressing DHFR) and overnight incubation at 37.degree. C. Cells were seeded into 96-well plates at a density of 100 cells per well, and plates were incubated at 37.degree. C., 5% CO.sub.2 for two weeks, with one change of medium per week.

[0783] Five days after the final medium change, culture supernatants were diluted 1:50 and tested using an ELISA specific for human IgG gamma chain. The clones yielding the highest ELISA signal were transferred from the 96-well plates to 12-well plates in 1.5 ml/well of Alpha MEM+5% dialyzed serum. After 3 days, another ELISA specific for human IgG gamma chain was performed, and the 12 clones with the greatest activity were split into the alpha MEM+5% dialyzed serum and 20 nM MTX. Cell line 031898 218 grew in the presence of 20 nM MTX without any apparent cell death or reduction in growth rate, produced 1.8 .mu.g/ml hIgG in a three-day assay. T-25 cultures of 031898 218, growing in medium containing MTX, produced an average of 11.9 .mu.g/ml of J695. The line, designated ALP903, was adapted to growth in suspension under serum-free conditions, where it produced 7.5 pg J695/cell/24 h.

[0784] ALP903 cells, after initial selection in alpha MEM/5% FBS/20 nM MTX medium, were passed again in 20 nM MTX. The cells were cultured under 100 nM MTX selection, followed by passaging in 500 nM MTX twice in the next 30 days. At that time the culture was producing 32 .mu.g J695/mL/24 h. The culture was subcloned by limiting dilution. Subclone 218-22 produced 16.5 .mu.g/mL in a 96-well plate in 2 days and 50.3 .mu.g/mL of J695 in a 12-well dish in 2 days. Clone 218-22 was cultured in alpha MEM/5% dialyzed FBS/500 nM MTX for 38 days, followed by adaptation to serum-free spinner culture, as above. The average cell-specific productivity of the serum-free suspension culture, designated ALP 905, was 58 pg/cell/24 h.

[0785] The first cell line used to produce J695 (ALP 903) resulted in lower yields of the antibody from culture than a second cell line, ALP 905. To assure that the ALP 905-produced J695 was functionally identical to that produced from ALP 903, both batches of antibodies were assessed for IL-12 affinity, for the ability to block IL-12 binding to cellular receptors, for the ability to inhibit IFN-.gamma. induction by IL-12, and for the ability to inhibit IL-12-mediated PHA blast proliferation.

[0786] The affinities of J695 batches ALP 903 and ALP 905 for IL-12 were determined by measuring the kinetic rate constants of binding to IL-12 by surface plasmon resonance studies (BIAcore analyses). The off-rate constant (k.sub.d) and the on-rate constant (k.sub.a) of antibody batches ALP903 and ALP905 for binding to rhIL-12 were determined in three experiments (as described in Example 3). The affinity, K.sub.d, of binding to IL-12 was calculated by dividing the off-rate constant by the on-rate constant. K.sub.d was calculated for each separate experiment and then averaged. The results showed that the determined kinetic parameters and affinity of binding to rhIL-12 were very similar for J695 batches ALP 903 and ALP 905: the calculated K.sub.d was 1.19.+-.0.22.times.10.sup.-10 M for batch ALP 903 and 1.49.+-.0.47.times.10.sup.-10 M for batch ALP 905 (see Table 13).

[0787] The ability of J695 derived from both ALP 903 and ALP 905 to block binding of rhIL-12 to IL-12 receptors on human PHA-activated T-lymphoblasts was assessed (see Example 3). Each sample of J695 was tested at a starting concentration of 1.times.10.sup.-8 with 10-fold serial dilutions. The antibody was preincubated for 1 hour at 37.degree. C. with 50 pM [.sup.125I]-human IL-12 in binding buffer. PHA blast cells were added to the antibody/[.sup.125I]-human IL-12 mixture and incubated for 2 h at room temperature. Cell bound radioactivity was separated from free [.sup.125I]-IL-12 by centrifugation and washing steps, and % inhibition was calculated. The IC.sub.50 values for J695 were determined from the inhibition curves using 4-parameter curve fitting and were confirmed by two independent experiments. Incubations were carried out in duplicate. The results for the two batches of J695 were very similar (see Table 13).

[0788] The ability of J695 from both ALP 903 and ALP 905 cells to inhibit rhIL-12-induced IFN-.gamma. production by human PHA-activated lymphoblasts in vitro was assessed. Serial dilutions of J695 were preincubated with 200 pg/mL rhIL-12 for 1 h at 37.degree. C. PHA lymphoblast cells were added and incubated for 18 hours at 37.degree. C. After incubation, cell free supernatant was withdrawn and the level of human IFN-.gamma. determined by ELISA. The IC.sub.50 values from the inhibition curves were plotted against the antibody concentration using 4-parameter curve fitting. The results demonstrate that the ability of the two batches to inhibit IFN-.gamma. production is very similar.

[0789] The in vitro PHA blast cell proliferation assay was used to measure the neutralization capacity of ALP 903 and ALP 905 J695 for rhIL-12. Serial dilutions of J695 of each type were preincubated with 230 pg/mL human IL-12 for 1 h at 37.degree. C. Next PHA blast cells were added and incubated for 3 days at 37.degree. C. The cells were then labeled for 6 hours with 1 .gamma.Ci/well [.sup.3H]-thymidine. The cultures were harvested and [.sup.3H]-thymidine incorporation measured. Non-specific proliferation (background) was measured in the absence of rhIL-12. The IC.sub.50 values for ALP 903 and ALP 905 J695 were found to be very similar and are set forth in Table 13.

[0790] The activity of the J695 antibodies in neutralizing rhIL-12 activity, in blocking IL-12 binding to cell surface receptors, and in binding to rhIL-12 did not significantly differ from batch ALP 903 to batch ALP 905, and thus the antibodies produced from these two different cell types were equivalent.

TABLE-US-00019 TABLE 13 Comparison of the Properties of J695 lots ALP 903 and ALP 905 k.sub.a, On-rate k.sub.d, Off-rate RB assay PHA blast IFN-.gamma. Antibody (M.sup.-1, s.sup.-1) (s.sup.-1) K.sub.d(M) IC.sub.50 (M) Assay IC.sub.50 (M) Assay IC.sub.50 (M) J695 3.75 .times. 10.sup.5 4.46 .times. 10.sup.-5 1.19 .times. 10.sup.-10 3.4 .times. 10.sup.-11 5.5 .times. 10.sup.-12 5.8 .times. 10.sup.-12 ALP 903 J695 3.91 .times. 10.sup.5 5.59 .times. 10.sup.5 1.49 .times. 10.sup.-10 3.0 .times. 10.sup.-11 4.4 .times. 10.sup.-12 4.3 .times. 10.sup.-12 ALP 905

Example 11

Anti-rhIL12 Monoclonal Antibody Epitope Map Determination by Biacore

[0791] Real-time biospecific interaction analysis (BIA) based on surface plasmon resonance technology was used to map the epitope specificity patterns of J695 and 4 other monoclonal antibodies against soluble recombinant human IL12. The technique does not require labeling of either antibodies or antigen. Antibodies directed against separate and distinct epitopes will bind simultaneously to the antigen, whereas antibodies directed against closely related epitopes will interfere with each other's binding. Furthermore, if the second antibody fails to bind, the epitopes defined by the two antibodies may be identical or overlapping, or binding of the first antibody may prevent binding of the second antibody through allosteric inhibition caused by a conformational alteration in the target molecule.

[0792] An epitope mapping assay using Biacore was performed. First, carboxyl groups on the dextran matrix were activated with 100 mM N-hydroxysuccinimide (NHS) and 400 mM N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), across four different flow cells. Next, Antibody 1 was injected across the activated matrix. Approximately fifty microliters of anti-rhIL12 antibody (25 .mu.g/ml), diluted in sodium acetate, pH 4.5, was injected across the activated biosensor and free amines on the protein are bound directly to the activated carboxyl groups. Typically, 5000 resonance units were immobilized. Unreacted matrix EDC-esters were deactivated by an injection of 1 M ethanolamine. Standard amine coupling kits were commercially available (Biacore AB, Cat. No. BR-1000-50, Uppsala, Sweden). SPR measurements were performed using CM biosensor chip (Biacore AB, Cat No BR-1000-14, Uppsala, Sweden). All antibodies and antigens to be analyzed on the biosensor surface were diluted in HBS-EP running buffer (Biacore AB, Cat No BR-1001-88, Uppsala, Sweden).

[0793] Next, rhuIL12 (100 nM) was injected across covalently immobilized antibody on the CM5 biosensor surface at a flow rate of 25 .mu.l/min. Before injection of the antigen and immediately afterward, HBS-EP buffer alone flowed through each flow cell. Excess soluble Antibody 2 (25 .mu.g/ml) was then injected across captured rhuIL12 (5 minute contact time). Before injection of Antibody 2 and immediately afterward, HBS-EP buffer alone flowed through each flow cell. The net difference in the signals between the baseline and the point corresponding to approximately 30 seconds after completion of Mab injection was taken to represent the final binding value. Again, the response was measured in Resonance Units. Biosensor matrices were regenerated using 10 mM HCl (5 minute contact time) before injection of the next sample.

[0794] Antibodies and antigens supplied by Abbott Bioresearch Center and/or commercial vendors were as follows: Human J695; Mouse C8.6.2 Mab; Human 1D4.7 Mab; Human C340 Mab; Mouse 7G3 Mab; Human IgG1 control (1.0 mg/ml, Sigma Catalog No. 1-3889). Each of the antibodies used in this study binds to an epitope of the p40 subunit of human IL-12. The antibodies ID4.7 and 7G3 are described in U.S. Provisional Patent Application No. 60/695,679, filed Jun. 29, 2005, and U.S. patent application Ser. No. 11/478,096, filed Jun. 29, 2006. The antibody C340 is described in U.S. Pat. No. 7,063,964 and U.S. Pat. No. 6,902,734. The entire contents of each of the foregoing patents and patent applications are hereby incorporated herein by reference. The antibody C8.6.2 is a subclone of, and thus has the same characteristics as, the antibody C8.6, which is described in A. D'Andrea et al., 1992 J. Exp. Med. 176:1387-1398, the entire contents of which are hereby incorporated herein by reference. Recombinant human interleukin 12 (rhIL12, commercially available by Wyeth).

[0795] The reactivity patterns for the monoclonal antibodies tested are displayed in Table 14. In these experiments, J695 and 7G3 monoclonal antibodies could bind recombinant human IL-12 concurrently. In this case, simultaneous binding with recombinant human IL-12 confirms that the antibodies occupy different sites. Sensorgrams obtained from mapping experiments revealed that 1D4.7 and 7G3 monoclonal antibodies recognize independent epitopes. Another pair of monoclonal antibodies showed simultaneous binding in the order [C340]-[rhIL12]-[7G3]. Again, a positive result in this sequence indicated distinct and independent epitopes for these two antibodies. Test antibodies gave negative results when used in the order [C8.6.2]-[rhIL12]-[C8.6.2], [C340]-[rhIL12]-[C8.6.2] and [C340]-[rhIL12]-[1D4.7]. The lack of binding of the second antibody is presumably due to occupation of the epitope. In a negative control, omission of recombinant human IL-12 from the normal Biacore assay sequence reduced the response from the second antibody to background levels. Further, murine and human IgG matched isotype controls gave negative results, regardless of orientation.

TABLE-US-00020 TABLE 14 Summary of pair-wise epitope mapping of IL-12 antibodies by Biacore Directly immobilized ant-human IL-12 Mab (approximately 5000 RU's) Sample FC 1 FC 2 FC 3 FC 4 1 2.degree. Soluble Mouse IgG1 J695 Human IgG 1 Human IgG1 anti-human IL-12 Mab C8.6.2 C340 1D4.7 2 Mouse IgG C8.6.2 - slow rhIL-12 - - displacement 3 J695 rapid rhIL-12 - rapid rIL-12 rapid rhIL-12 displacement displacement displacement 4 Human IgG1 C340 - slow rhIL-12 - - displacement 5 Human IgG1 1D4.7 - slow rhIL-12 - - displacement 6 Mouse IgG1 7G3 + + + + If both anti-human IL-12 antibodies can bind simultaneously to the target molecule (rhIL-12), they define separate and independent epitopes. If the second antibody fails to bind, then the epitopes defined by the two antibodies may be identical or overlapping, or binding of the first antibody may prevent binding of the second through a conformational alteration in the target molecule. Soluble antigen = recombinant IL-12 (p40/p35).

[0796] The results described in this Example demonstrate that Biacore can be used to characterize monoclonal antibody epitopes. Each antibody and antigen was injected over a long time period, until a plateau was reached in the SPR signal. Examination of the sensorgrams showed that all human anti-rhIL12 monoclonal antibodies bound specifically to rhIL12 when they were directly immobilized across carboxymethyl dextran surface. The results presented in this Example demonstrate that the antibodies that can bind simultaneously to IL-12 are binding to non-overlapping epitopes. In particular, 7G3 binds to a different epitope that is not shared by J695, C8.6.2, C340 or 1D4.7. The results further demonstrate that the antibodies tested that cannot bind simultaneously to IL-12 are binding to overlapping epitopes (i.e., no binding of antibody 2 observed when IL-12 is bound to antibody 1). In particular, C8.6.2, C340 and 1D4.7 bind to overlapping epitopes as binding of one of these antibodies to IL-12 does not allow binding of the second antibody in all cases. Finally, the results demonstrate that the antibodies that cannot bind simultaneously to IL-12 are prevented from binding due to an allosteric interaction that prevents the antibodies from binding simultaneously (i.e., antibody 2 releases IL-12 from antibody 1). In particular, J695 cannot bind rhuIL-12 simultaneously with either C8.6.2, C340 or 1D4.7. The inability of the antibodies to bind IL-12 simultaneously does not indicate a simple competition between the antibodies for the same binding site, but rather that a conformational alteration in the target molecule IL-12 occurs upon binding. This unusual allosteric mechanism is evident in the observation that soluble J695 rapidly displaced IL-12 bound to immobilized C8.6.2, C340 or 1D4.7. Conversely, C8.6.2, C340 and 1D4.7 slowly displaced IL-12 bound to immobilized J695.

Example 12

Identification of Antibodies Capable of Altering the Conformational Structure of an Interleukin Containing a p40 Subunit

[0797] Antibodies are identified that bind to the p40 subunit of an interleukin, e.g., IL-12 or IL-23, and are capable of altering its conformational structure by using real-time biospecific interaction analysis (BIA). The experimental protocol used is essentially as described above in Example 11.

[0798] Specifically, carboxyl groups on a dextran matrix are activated, typically with 100 mM N-hydroxysuccinimide (NHS) and 400 mM N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). Next, a first antibody that specifically binds to the p40 subunit of an interleukin, e.g., IL-12 or IL-23, is then injected across the activated matrix. In particular, the first antibody injected across the activated matrix is an antibody that binds to an epitope of the p40 subunit of IL-12 to which the antibodies C8.6.2, C340 or 1D4.7 bind. Preferably, the first antibody is either C8.6.2, C340 or 1D4.7. Approximately fifty microliters of the first antibody (e.g., at 25 .mu.g/ml, diluted in sodium acetate, pH 4.5) is then injected across the activated biosensor and free amines on the protein are bound directly to the activated carboxyl groups. Typically, 5000 resonance units are immobilized. Unreacted matrix EDC-esters are deactivated by an injection of 1 M ethanolamine. SPR measurements are performed using CM biosensor chip (Biacore AB, Cat No BR-1000-14, Uppsala, Sweden) as described above in Example 11. All antibodies and antigens to be analyzed on the biosensor surface are diluted in HBS-EP running buffer (Biacore AB, Cat No BR-1001-88, Uppsala, Sweden).

[0799] Next, the p40 subunit of the interleukin, e.g., IL-12 or IL-23 (e.g., 100 nM) is injected across the covalently immobilized antibody on the CM5 biosensor surface, typically at a flow rate of approximately 25 .mu.l/min. The p40 subunit of the interleukin, e.g., IL-12 or IL-23, can be injected, for example, either as an isolated single subunit (or fragment thereof) or alternatively can be injected in a heterodimeric form, wherein the heterodimer comprises the p40 subunit and a second subunit, e.g., the heterodimer p40/p35 (IL-12), or an alternate heterodimer comprising the p40 subunit of IL-12 and a p19 subunit (p40/p19; IL-23). Before injection of the antigen and immediately afterward, HBS-EP buffer alone is flowed through each flow cell. Following this, an excess of a soluble test antibody (e.g., at a concentration of 25 .mu.g/ml) is injected across the captured p40 subunit of the interleukin (e.g., IL-12 or IL-23), typically for a contact time of approximately 5 minutes. Before injection of the test antibody and immediately afterward, HBS-EP buffer alone is again flowed through each flow cell. The net difference in the signals between the baseline and the point corresponding to approximately 30 seconds after completion of antibody injection is taken to represent the final binding value and is measured in Resonance Units. Antibodies which rapidly displace the p40 subunit of the interleukin (e.g., IL-12 or IL-23) from the first antibody immobilized on the CM5 biosensor surface are identified as antibodies that can alter the conformational structure of the interleukin, e.g., the p40 subunit of the interleukin.

[0800] This conformational alteration in the structure of the interleukin ((e.g., IL-12 or IL-23) may be confirmed by any of the well known techniques in the art for monitoring 3-dimensional structures of proteins. For example X-ray crystallography or Circular Dichroism may be used.

EQUIVALENTS

[0801] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence CWU 1

67516PRTHomo sapiensXaa at position 1 could be either His or Ser 1Xaa Gly Ser Xaa Asp Xaa1 5212PRTHomo sapiensXaa at position 2 could be either Ser or Thr 2Gln Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10317PRTHomo sapiens 3Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly47PRTHomo sapiensXaa at position 1 could be either Gly or Tyr 4Xaa Asn Xaa Xaa Arg Pro Ser1 559PRTHomo sapiensXaa represents either Ser or Glu 5Phe Thr Phe Ser Xaa Tyr Gly Met His1 5613PRTHomo sapiensXaa at position 1 could be either Ser or Thr 6Xaa Gly Xaa Xaa Ser Asn Ile Xaa Xaa Xaa Xaa Val Xaa1 5 107115PRTHomo sapiensXaa at position 6 could be either Gln or Glu 7Gln Val Gln Leu Val Xaa Ser Gly Gly Gly Val Val Gln Pro Gly Xaa1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Asx 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Xaa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Xaa Xaa Xaa Gly Ser Xaa Asp Xaa Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 1158112PRTHomo sapiensXaa at position 1 could be either Ser or Gln 8Xaa Xaa Val Leu Thr Gln Pro Pro Ser Val Ser Gly Xaa Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Xaa Gly Xaa Xaa Ser Asn Ile Xaa Xaa Xaa 20 25 30Xaa Val Xaa Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Xaa Asn Xaa Xaa Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Xaa Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Xaa Tyr Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11096PRTHomo sapiensXaa at position 2 could be either Gly, Val, Cys or His 9His Xaa Xaa Xaa Xaa Xaa1 51012PRTHomo sapiensXaa at position 4 could be either Asp or Ser 10Gln Ser Tyr Xaa Xaa Xaa Thr His Pro Ala Leu Leu1 5 101117PRTHomo sapiensXaa at position 1 could be either Phe, Thr or Tyr 11Xaa Ile Xaa Tyr Xaa Xaa Ser Xaa Lys Xaa Tyr Ala Asp Ser Val Lys1 5 10 15Gly127PRTHomo sapiensXaa at position 1 could be either Gly, Tyr, Ser, Thr, Asn or Gln 12Xaa Asn Asp Gln Arg Pro Ser1 5139PRTHomo sapiensXaa at position 4 and 5 represents any amino acid 13Phe Thr Phe Xaa Xaa Xaa Xaa Met His1 51413PRTHomo sapiensXaa at position 9 could be either Ser, Cys, Arg, Asn, Asp or Thr 14Ser Gly Gly Arg Ser Asn Ile Gly Xaa Xaa Xaa Val Lys1 5 1015114PRTHomo sapiensXaa at position 30 could be Ser or Glu 15Gln Val Gln Val Xaa Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser1 5 10 15Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Tyr Gly 20 25 30Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Xaa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys 85 90 95Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr Val 100 105 110Ser Ser16112PRTHomo sapiensXaa at position 1 could be either Ser or Gln 16Xaa Xaa Val Leu Thr Gln Pro Pro Ser Val Ser Gly Xaa Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Xaa Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Xaa Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Xaa Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Xaa Thr 85 90 95His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110176PRTHomo sapiens 17His Gly Ser His Asp Asn1 51812PRTHomo sapiens 18Gln Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu1 5 101917PRTHomo sapiens 19Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly207PRTHomo sapiens 20Gly Asn Asp Gln Arg Pro Ser1 5219PRTHomo sapiens 21Phe Thr Phe Ser Ser Tyr Gly Met His1 52213PRTHomo sapiens 22Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Thr Val Lys1 5 1023115PRTHomo sapiens 23Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11524112PRTHomo sapiens 24Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Trp Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110256PRTHomo sapiens 25His Gly Ser His Asp Asn1 52612PRTHomo sapiens 26Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala Leu Leu1 5 102717PRTHomo sapiens 27Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly287PRTHomo sapiens 28Tyr Asn Asp Gln Arg Pro Ser1 5299PRTHomo sapiens 29Phe Thr Phe Ser Ser Tyr Gly Met His1 53013PRTHomo sapiens 30Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn Thr Val Lys1 5 1031115PRTHomo sapiens 31Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11532112PRTHomo sapiens 32Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Tyr Thr 85 90 95His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11033115PRTHomo sapiens 33Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr Ser Gly Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11534112PRTHomo sapiens 34Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95Arg Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11035115PRTHomo sapiens 35Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Gly Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11536112PRTHomo sapiensXaa at position 32 represents either Gly or Tyr 36Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Xaa 20 25 30Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95Ser Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11037115PRTHomo sapiens 37Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11538112PRTHomo sapiens 38Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95Arg Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11039115PRTHomo sapiens 39Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr Ser Gly Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11540112PRTHomo sapiens 40Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Phe 85 90 95Thr Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11041115PRTHomo sapiens 41Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr Ser Gly Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11542112PRTHomo sapiens 42Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75

80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95Trp Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11043115PRTHomo sapiens 43Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11544112PRTHomo sapiens 44Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Phe 85 90 95Thr Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11045115PRTHomo sapiens 45Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11546112PRTHomo sapiens 46Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95Trp Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11047115PRTHomo sapiens 47Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11548112PRTHomo sapiens 48Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Lys Gly Phe 85 90 95Thr Gly Ser Ser Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11049115PRTHomo sapiens 49Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11550112PRTHomo sapiens 50Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Lys Gly Phe 85 90 95Thr Gly Ser Ser Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11051115PRTHomo sapiens 51Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr His Gly Ser His Asp Thr Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11552112PRTHomo sapiens 52Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95Trp Gly Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11053115PRTHomo sapiens 53Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11554112PRTHomo sapiens 54Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Val Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Phe 85 90 95Thr Gly Ser Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11055115PRTHomo sapiens 55Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11556112PRTHomo sapiens 56Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Val Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Phe 85 90 95Thr Gly Ala Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11057115PRTHomo sapiens 57Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11558112PRTHomo sapiens 58Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Tyr Asp Lys Gly Phe 85 90 95Thr Gly Ser Ser Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11059115PRTHomo sapiens 59Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11560112PRTHomo sapiens 60Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Glu Arg Gly Phe 85 90 95Thr Gly Ser Met Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11061115PRTHomo sapiens 61Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11562112PRTHomo sapiens 62Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95His Pro Leu Thr Ile Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11063115PRTHomo sapiens 63Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr

100 105 110Val Ser Ser 11564112PRTHomo sapiens 64Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Ser 85 90 95His Pro Ala Leu Thr Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11065115PRTHomo sapiens 65Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11566112PRTHomo sapiens 66Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95His Pro Leu Thr Met Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11067115PRTHomo sapiens 67Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11568112PRTHomo sapiens 68Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95His Pro Leu Thr Met Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11069115PRTHomo sapiens 69Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11570112PRTHomo sapiens 70Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11071115PRTHomo sapiens 71Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11572112PRTHomo sapiens 72Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11073115PRTHomo sapiens 73Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11574112PRTHomo sapiens 74Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Tyr Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Gly Thr 85 90 95His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 11075115PRTHomo sapiens 75Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly Thr Met Val Thr 100 105 110Val Ser Ser 11576112PRTHomo sapiens 76Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Tyr Thr 85 90 95His Pro Ala Leu Leu Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110776PRTHomo sapiens 77Ser Gly Ser Tyr Asp Tyr1 5786PRTHomo sapiens 78His Gly Ser His Asp Asn1 5796PRTHomo sapiens 79His Gly Ser Tyr Asp Tyr1 5806PRTHomo sapiens 80Arg Arg Arg Ser Asn Tyr1 5816PRTHomo sapiens 81Ser Gly Ser Ile Asp Tyr1 5826PRTHomo sapiens 82His Gly Ser His Asp Asp1 5836PRTHomo sapiens 83His Gly Ser His Asp Asn1 58412PRTHomo sapiens 84Thr Thr His Gly Ser His Asp Asn Trp Gly Gln Gly1 5 108512PRTHomo sapiens 85Ala Lys His Gly Ser His Asp Asn Trp Gly Gln Gly1 5 108612PRTHomo sapiens 86Thr Thr His Gly Ser His Asp Asn Trp Ser Gln Gly1 5 108712PRTHomo sapiens 87Thr Thr His Gly Ser His Asp Thr Trp Gly Gln Gly1 5 108812PRTHomo sapiens 88Lys Thr His Gly Ser His Asp Asn Trp Gly Gln Gly1 5 108912PRTHomo sapiens 89Lys Thr His Gly Ser His Asp Asn Trp Gly His Gly1 5 109012PRTHomo sapiens 90Thr Thr His Gly Ser His Asp Asn Trp Ser Gln Gly1 5 109112PRTHomo sapiens 91Thr Thr His Arg Ser His Asn Asn Trp Gly Gln Gly1 5 10928PRTHomo sapiens 92Thr Thr His Gly Ser His Asp Asn1 5938PRTHomo sapiens 93Thr Thr His Gly Ser His Asp Thr1 5948PRTHomo sapiens 94Thr Lys His Gly Ser His Asp Asn1 5958PRTHomo sapiens 95Thr Thr Gln Gly Arg His Asp Asn1 5968PRTHomo sapiens 96Lys Thr Arg Gly Arg His Asp Asn1 5978PRTHomo sapiens 97Thr Thr His Gly Ser His Asp Lys1 5988PRTHomo sapiens 98Thr Thr His Gly Ser His Asp Asp1 5998PRTHomo sapiens 99Lys Thr His Gly Ser His Asp Asn1 51008PRTHomo sapiens 100Lys Thr His Gly Ser His Asp Asn1 51018PRTHomo sapiens 101Thr Thr His Gly Ser His Asp Asn1 51028PRTHomo sapiens 102Thr Thr Ser Gly Ser Tyr Asp Tyr1 51038PRTHomo sapiens 103Thr Thr His Gly Ser His Asp Asn1 51048PRTHomo sapiens 104Thr Thr His Gly Ser Gln Asp Asn1 51058PRTHomo sapiens 105Ala Thr His Gly Ser Gln Asp Asn1 51066PRTHomo sapiens 106His Gly Ser Gln Asp Thr1 51076PRTHomo sapiens 107Ser Gly Ser Tyr Asp Tyr1 51086PRTHomo sapiens 108His Gly Ser Gln Asp Asn1 51099PRTHomo sapiens 109Cys Lys Thr His Gly Ser His Asp Asn1 511012PRTHomo sapiens 110Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser Arg Val1 5 1011112PRTHomo sapiens 111Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Arg Val1 5 1011212PRTHomo sapiens 112Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser Arg Val1 5 1011312PRTHomo sapiens 113Gln Ser Tyr Asp Ser Ser Leu Thr Gly Ser Arg Val1 5 1011412PRTHomo sapiens 114Gln Ser Tyr Asp Ser Ser Leu Trp Gly Ser Arg Val1 5 1011512PRTHomo sapiens 115Gln Thr Tyr Asp Ile Ser Glu Ser Gly Ser Arg Val1 5 1011612PRTHomo sapiens 116Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Arg Val1 5 1011712PRTHomo sapiens 117Gln Thr Tyr Asp Arg Gly Phe Thr Gly Ser Arg Val1 5 1011812PRTHomo sapiens 118Gln Thr Tyr Asp Lys Gly Phe Thr Gly Ser Ser Val1 5 1011912PRTHomo sapiens 119Gln Ser Tyr Asp Arg Arg Phe Thr Gly Ser Arg Val1 5 1012012PRTHomo sapiens 120Gln Ser Tyr Asp Trp Asn Phe Thr Gly Ser Arg Val1 5 1012112PRTHomo sapiens 121Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Arg Val1 5 1012212PRTHomo sapiens 122Gln Ser Tyr Asp Asn Gly Phe Thr Gly Ser Arg Val1 5 1012312PRTHomo sapiens 123Gln Ser Tyr Asp Asn Ala Val Thr Ala Ser Lys Val1 5 1012412PRTHomo sapiens 124Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Arg Val1 5 1012512PRTHomo sapiens 125Gln Ser Tyr Asp Ser Ser Leu Trp Gly Thr Arg Val1 5 1012612PRTHomo sapiens 126Gln Ser Tyr Asp Arg Asp Phe Thr Gly Ser Arg Val1 5 1012712PRTHomo sapiens 127Gln Ser Tyr Glu Arg Gly Phe Thr Gly Ser Met Val1 5 1012812PRTHomo sapiens 128Gln Ser Tyr Asp Asn Gly Phe Thr Gly Ala Arg Val1 5 1012912PRTHomo sapiens 129Gln Ser Tyr Asp Arg Arg Phe Thr Gly Ser Arg Val1 5 1013012PRTHomo sapiens 130Gln Thr Tyr Asp Lys Gly Phe Thr Gly Ser Ser Val1 5 1013112PRTHomo sapiens 131Gln Ser Tyr Asp Arg Asp Phe Thr Gly Thr Arg Val1 5 1013212PRTHomo sapiens 132Gln Ser Tyr Asp Arg Gly Phe Tyr Gly Ser Met Val1 5 1013312PRTHomo sapiens 133Gln Thr Tyr Asp Lys Gly Phe Thr Gly Ser Ser Val1 5 1013412PRTHomo sapiens 134Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ala Arg Val1 5 1013512PRTHomo sapiens 135Gln Ser Tyr Glu Arg Gly Phe Thr Gly Ala Arg Val1 5 1013613PRTHomo sapiens 136Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Arg Val Phe1 5 1013713PRTHomo sapiens 137Gln Ser Tyr Asp Arg Gly Phe Thr Gly Phe Lys Val Phe1 5 1013813PRTHomo sapiens 138Gln Ser Tyr Asp Arg Gly Phe Val Ser Ala Tyr Val Phe1 5 1013913PRTHomo sapiens 139Gln Ser Tyr Asp Arg Gly Leu Thr Val Thr Lys Val Phe1 5 1014013PRTHomo sapiens 140Gln Ser Tyr Asp Arg Gly Tyr Thr Ala Ser Arg Val Phe1 5 1014113PRTHomo sapiens 141Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Lys Val Phe1 5 1014213PRTHomo sapiens 142Gln Ser Tyr Asp Arg Gly Leu Thr Gly Phe Arg Val Phe1 5 1014313PRTHomo sapiens 143Gln Ser Tyr Asp Arg Gly Phe Thr Gly Tyr Lys Val Phe1 5 1014413PRTHomo sapiens 144Gln Ser Tyr Asp Arg Gly Leu Thr Gly Tyr Arg Leu Phe1 5 1014513PRTHomo sapiens 145Gln Ser Tyr Asp Arg Gly Phe Thr Asp Tyr Lys Val Phe1 5 1014613PRTHomo sapiens 146Gln Ser Tyr Asp Arg Gly Phe Thr Gly Pro Arg Leu Phe1 5 1014713PRTHomo sapiens 147Gln Ser Tyr Asp Arg Gly Leu Thr Gly Ser Arg Val Phe1 5 1014813PRTHomo sapiens 148Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ala Arg Val Trp1 5 1014913PRTHomo sapiens 149Gln Ser Tyr Asp Arg Gly Phe Thr Gly Tyr Arg Val Phe1 5

1015013PRTHomo sapiens 150Gln Ser Tyr Asp Arg Gly Phe Thr Gly Pro Arg Val Phe1 5 1015113PRTHomo sapiens 151Gln Ser Tyr Asp Arg Gly Met Thr Ser Ser Arg Val Phe1 5 1015213PRTHomo sapiens 152Gln Ser Tyr Asp Arg Asp Ser Thr Gly Ser Arg Val Phe1 5 1015313PRTHomo sapiens 153Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser Arg Val Phe1 5 1015413PRTHomo sapiens 154His Ser Tyr Asp Ser Asp Phe Thr Gly Ser Arg Val Phe1 5 1015513PRTHomo sapiens 155His Ser Ser Glu Ser Gly Phe Thr Gly Ser Arg Val Phe1 5 1015613PRTHomo sapiens 156His Ser Tyr Asp Asn Arg Phe Thr Gly Ser Arg Val Phe1 5 1015713PRTHomo sapiens 157His Ser Tyr Asp Ser Arg Phe Thr Gly Ser Arg Val Phe1 5 1015813PRTHomo sapiens 158Gln Ser Tyr Asp Ser Glu Phe Thr Gly Ser Arg Val Phe1 5 1015913PRTHomo sapiens 159Gln Ser Tyr Asp Thr Gly Phe Thr Gly Ser Arg Val Phe1 5 1016013PRTHomo sapiens 160His Ser Tyr Asp Ser Gly Phe Thr Gly Ser Arg Val Phe1 5 1016113PRTHomo sapiens 161Gln Ser Tyr Asp Thr Gly Phe Thr Gly Ser Arg Val Phe1 5 1016213PRTHomo sapiens 162His Ser Tyr Asp Thr Lys Phe Thr Gly Ser Arg Val Phe1 5 1016313PRTHomo sapiens 163His Ser Ser Asp Ser Gly Phe Thr Gly Ser Arg Val Phe1 5 1016413PRTHomo sapiens 164Gln Ser Tyr Asp Ser Asp Phe Thr Gly Ser Arg Val Phe1 5 1016513PRTHomo sapiens 165His Ser Tyr Glu Ser Gly Phe Thr Gly Ser Arg Val Phe1 5 1016613PRTHomo sapiens 166Gln Ser Tyr Asp Ala Pro Trp Ser Gly Ser Arg Val Phe1 5 1016713PRTHomo sapiens 167Gln Ser Tyr Asp Ser Asp Phe Thr Gly Ser Lys Val Phe1 5 1016813PRTHomo sapiens 168His Thr Asn Asp Ser Gly Phe Thr Gly Ser Arg Val Phe1 5 1016913PRTHomo sapiens 169His Ser Tyr Asp Thr Arg Phe Thr Gly Ser Arg Val Phe1 5 1017013PRTHomo sapiens 170Gln Ser Tyr Asp Met Arg Phe Thr Gly Ser Arg Val Phe1 5 1017113PRTHomo sapiens 171His Ser Ser Asp Ser Asp Ser Thr Gly Ser Arg Val Phe1 5 1017213PRTHomo sapiens 172Gln Ser Tyr Asn Thr Asp Phe Thr Gly Ser Arg Val Phe1 5 1017313PRTHomo sapiens 173Gln Ser Tyr Asp Ser Gly Phe Thr Gly Ser Arg Val Phe1 5 1017413PRTHomo sapiens 174His Ser Tyr Asp Met Gly Phe Thr Gly Ser Arg Val Phe1 5 1017513PRTHomo sapiens 175His Ser Tyr Asp Asn Gly Phe Thr Gly Ser Arg Val Phe1 5 1017613PRTHomo sapiens 176His Ser His Asp Arg Asp Phe Thr Gly Ser Arg Val Phe1 5 1017712PRTHomo sapiens 177Gln Ser Tyr Asp Ser Ser Leu Arg Gly Ser Arg Val1 5 1017813PRTHomo sapiens 178Gln Ser Tyr Asp Arg Gly Ile His Gly Ser Arg Val Phe1 5 1017913PRTHomo sapiens 179Gln Ser Tyr Asp Ser Gly Phe Pro Gly Ser Arg Val Phe1 5 1018013PRTHomo sapiens 180Gln Ser Tyr Asp Ile Gly Ser Thr Gly Ser Arg Val Phe1 5 1018113PRTHomo sapiens 181Gln Ser Tyr Asp Ser Gly Leu Thr Gly Ser Arg Val Phe1 5 1018213PRTHomo sapiens 182Gln Ser Tyr Asp Ile Gly Met Thr Gly Ser Arg Val Phe1 5 1018313PRTHomo sapiens 183Gln Ser Tyr Asp Ile Gly Leu Thr Gly Ser Arg Val Phe1 5 1018413PRTHomo sapiens 184Gln Ser Tyr Asp Ser Gly Val Thr Gly Ser Arg Val Phe1 5 1018513PRTHomo sapiens 185Gln Ser Tyr Asp Arg Gly Leu Thr Ala Ser Arg Val Phe1 5 1018613PRTHomo sapiens 186Gln Ser Tyr Asp Thr Gly Leu Thr Gly Ser Arg Val Phe1 5 1018713PRTHomo sapiens 187Gln Ser Tyr Asp Thr Ala Leu Thr Gly Ser Arg Val Phe1 5 1018813PRTHomo sapiens 188Gln Ser Tyr Asp Ile Arg Phe Thr Gly Ser Arg Val Phe1 5 1018913PRTHomo sapiens 189Gln Ser Tyr Asp Ile Arg Ser Thr Gly Ser Arg Val Phe1 5 1019013PRTHomo sapiens 190Gln Ser Tyr Asp Asn Arg Leu Thr Gly Ser Arg Val Phe1 5 1019113PRTHomo sapiens 191Gln Ser Tyr Glu Thr Ser Phe Thr Gly Ser Arg Val Phe1 5 1019213PRTHomo sapiens 192Gln Ser Tyr Asp Ser Ser Ser Thr Gly Ser Arg Val Phe1 5 1019313PRTHomo sapiens 193Gln Ser Tyr Asp Ser Gly Phe Thr Ala Ser Arg Val Phe1 5 1019413PRTHomo sapiens 194Gln Thr Tyr Asp Lys Gly Phe Thr Gly Ser Ser Val Phe1 5 1019513PRTHomo sapiens 195Gln Ser Tyr Asp Asn Gly Phe Thr Gly Ser Arg Val Phe1 5 1019613PRTHomo sapiens 196Gln Ser Tyr Asp Thr Gly Phe Thr Lys Ser Arg Val Phe1 5 1019713PRTHomo sapiens 197Gln Ser Tyr Asp Ser Asp Val Thr Gly Ser Arg Val Phe1 5 1019813PRTHomo sapiens 198Gln Ser Tyr Asp Ala Gly Phe Thr Gly Ser Arg Val Phe1 5 1019912PRTHomo sapiens 199Gln Ser Tyr Asp Arg Gly Thr His Pro Ser Met Leu1 5 1020012PRTHomo sapiens 200Gln Ser Tyr Asp Arg Gly Thr Thr Pro Arg Pro Met1 5 1020112PRTHomo sapiens 201Gln Ser Tyr Asp Arg Gly Arg Asn Pro Ala Leu Thr1 5 1020212PRTHomo sapiens 202Gln Ser Tyr Asp Arg Gly Thr His Pro Trp Leu His1 5 1020312PRTHomo sapiens 203Gln Ser Tyr Asp Arg Gly Asn Ser Pro Ala Thr Val1 5 1020412PRTHomo sapiens 204Gln Ser Tyr Asp Arg Gly Thr Phe Pro Ser Pro Gln1 5 1020512PRTHomo sapiens 205Gln Ser Tyr Asp Arg Gly Leu Asn Pro Ser Ala Thr1 5 1020612PRTHomo sapiens 206Gln Ser Tyr Asp Arg Gly Lys Ser Asn Lys Met Leu1 5 1020712PRTHomo sapiens 207Gln Ser Tyr Asp Arg Gly His Thr Ala His Leu Tyr1 5 1020812PRTHomo sapiens 208Gln Ser Tyr Asp Arg Gly Gln Thr Pro Ser Ile Thr1 5 1020912PRTHomo sapiens 209Gln Ser Tyr Asp Arg Gly Tyr Pro Arg Asn Ile Leu1 5 1021012PRTHomo sapiens 210Gln Ser Tyr Asp Arg Gly Ile Thr Pro Gly Leu Ala1 5 1021112PRTHomo sapiens 211Gln Ser Tyr Asp Arg Gly Gln Pro His Ala Val Leu1 5 1021212PRTHomo sapiens 212Gln Ser Tyr Asp Arg Gly Asn Ser Pro Ile Pro Thr1 5 1021312PRTHomo sapiens 213Gln Ser Tyr Asp Arg Gly Thr Pro Asn Asn Ser Phe1 5 1021412PRTHomo sapiens 214Gln Ser Tyr Asp Ser Gly Val Asp Pro Gly Pro Tyr1 5 1021512PRTHomo sapiens 215Gln Ser Tyr Asp Arg Gly Arg Pro Arg His Ala Leu1 5 1021612PRTHomo sapiens 216Gln Ser Tyr Asp Arg Gly Pro Tyr His Pro Ile Arg1 5 1021712PRTHomo sapiens 217Gln Ser Tyr Asp Arg Gly Pro His Thr Gln Pro Thr1 5 1021812PRTHomo sapiens 218Gln Ser Tyr Asp Arg Gly His Asn Asn Phe Ser Pro1 5 1021912PRTHomo sapiens 219Gln Ser Tyr Asp Arg Gly Pro Thr His Leu Pro His1 5 1022012PRTHomo sapiens 220Gln Ser Tyr Asp Arg Gly Thr Pro Ser Tyr Pro Thr1 5 1022112PRTHomo sapiens 221Gln Ser Tyr Asp Ser Gly Thr Ser Asn Leu Leu Pro1 5 1022212PRTHomo sapiens 222Gln Ser Tyr Asp Arg Gly Asp Ser Asn His Asp Leu1 5 1022312PRTHomo sapiens 223Gln Ser Tyr Asp Arg Gly Leu Pro Arg Leu Thr His1 5 1022412PRTHomo sapiens 224Gln Ser Tyr Asp Arg Gly Ile Pro Thr Ser Tyr Leu1 5 1022512PRTHomo sapiens 225Gln Ser Tyr Asp Arg Gly Leu Arg Val Gln Ala Pro1 5 1022612PRTHomo sapiens 226Gln Ser Tyr Asp Arg Gly Leu Ser Asp Ser Pro Leu1 5 1022712PRTHomo sapiens 227Gln Ser Tyr Asp Ser Gly Ser Leu Arg Arg Ile Leu1 5 1022812PRTHomo sapiens 228Gln Ser Tyr Asp Arg Gly Pro Ala Arg Thr Ser Pro1 5 1022912PRTHomo sapiens 229Gln Ser Tyr Asp Arg Gly Arg Ala Ala His Pro Gln1 5 1023012PRTHomo sapiens 230Gln Ser Tyr Asp Arg Gly Thr Gln Pro Ala Asx Ile1 5 1023112PRTHomo sapiens 231Gln Ser Tyr Asp Arg Gly Thr His Pro Thr Met Ile1 5 1023212PRTHomo sapiens 232Gln Ser Tyr Asp Arg Gly Arg Ile Pro Ala Asx Thr1 5 1023312PRTHomo sapiens 233Gln Ser Tyr Asp Arg Gly Thr His Pro Val Pro Ala1 5 1023412PRTHomo sapiens 234Gln Ser Tyr Asp Arg Gly Ser Asx Pro Ile Pro Ala1 5 1023512PRTHomo sapiens 235Gln Ser Tyr Asp Arg Gly Thr His Pro Val Pro Ala1 5 1023612PRTHomo sapiens 236Gln Ser Tyr Asp Arg Gly Thr His Pro Thr Met Tyr1 5 1023712PRTHomo sapiens 237Gln Ser Tyr Asp Arg Gly His His Tyr Thr Thr Phe1 5 1023812PRTHomo sapiens 238Gln Ser Tyr Asp Arg Gly Ser His Pro Ala Ala Glu1 5 1023912PRTHomo sapiens 239Gln Ser Tyr Asp Arg Gly Thr Ile Pro Ser Ile Glu1 5 1024012PRTHomo sapiens 240Gln Ser Tyr Asp Arg Gly Ser Ser Pro Ala Ile Met1 5 1024112PRTHomo sapiens 241Gln Ser Tyr Asp Arg Gly Ile Trp Pro Asn Leu Asn1 5 1024212PRTHomo sapiens 242Gln Ser Tyr Asp Arg Gly Thr His Pro Asn Leu Asn1 5 1024312PRTHomo sapiens 243Gln Ser Tyr Asp Arg Gly Thr His Pro Ser Ile Ser1 5 1024412PRTHomo sapiens 244Gln Ser Tyr Asp Arg Gly Ser Ala Pro Met Ile Asn1 5 1024512PRTHomo sapiens 245Gln Ser Tyr Asp Arg Gly His His Pro Ala Met Ser1 5 1024612PRTHomo sapiens 246Gln Ser Tyr Asp Arg Gly Thr His Pro Ser Ile Thr1 5 1024712PRTHomo sapiens 247Gln Ser Tyr Asp Arg Gly Thr Asp Pro Ala Ile Val1 5 1024812PRTHomo sapiens 248Gln Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu1 5 1024912PRTHomo sapiens 249Gln Ser Tyr Asp Arg Gly Ser His Pro Ala Leu Thr1 5 1025012PRTHomo sapiens 250Gln Ser Tyr Asp Arg Gly Thr Thr Pro Ala Pro Glu1 5 1025112PRTHomo sapiens 251Gln Ser Tyr Asp Arg Gly Ser His Pro Thr Leu Ile1 5 1025212PRTHomo sapiens 252Gln Ser Tyr Asp Arg Gly Thr His Pro Ser Met Leu1 5 1025312PRTHomo sapiens 253Gln Ser Tyr Asp Arg Gly Thr Thr Pro Arg Pro Met1 5 1025412PRTHomo sapiens 254Gln Ser Tyr Asp Arg Gly Arg Leu Pro Ala Gln Thr1 5 1025512PRTHomo sapiens 255Gln Ser Tyr Asp Arg Gly Thr His Pro Leu Thr Ile1 5 1025612PRTHomo sapiens 256Gln Ser Tyr Asp Arg Gly Gln Thr Pro Ser Ile Thr1 5 1025712PRTHomo sapiens 257Gln Ser Tyr Asp Arg Gly Thr His Phe Gln Met Tyr1 5 1025812PRTHomo sapiens 258Gln Ser Tyr Asp Arg Gly Arg Asn Pro Ala Leu Thr1 5 1025912PRTHomo sapiens 259Gln Ser Tyr Asp Arg Gly Thr His Pro Leu Thr Met1 5 1026012PRTHomo sapiens 260Gln Ser Tyr Asp Arg Gly Thr His Pro Leu Thr Met1 5 1026112PRTHomo sapiens 261Gln Ser Tyr Asp Ser Gly Tyr Thr Gly Ser Arg Val1 5 1026212PRTHomo sapiens 262Gln Ser Tyr Asp Ser Gly Phe Thr Gly Ser Arg Val1 5 1026312PRTHomo sapiens 263Gln Ser Tyr Asp Ser Arg Phe Thr Gly Ser Arg Val1 5 1026412PRTHomo sapiens 264Gln Ser Tyr Pro Asp Gly Thr Pro Ala Ser Arg Val1 5 1026512PRTHomo sapiens 265Gln Ser Tyr Ser Thr His Met Pro Ile Ser Arg Val1 5 1026612PRTHomo sapiens 266Gln Ser Tyr Asp Ser Gly Ser Thr Gly Ser Arg Val1 5 1026712PRTHomo sapiens 267Gln Ser Tyr Pro Asn Ser Tyr Pro Ile Ser Arg Val1 5 1026810PRTHomo sapiens 268Gln Ser Tyr Ile Arg Ala Pro Gln Gln Val1 5 1026912PRTHomo sapiens 269Gln Ser Tyr Leu Lys Ser Arg Ala Phe Ser Arg Val1 5 1027012PRTHomo sapiens 270Gln Ser Tyr Asp Ser Arg Phe Thr Gly Ser Arg Val1 5 1027112PRTHomo sapiens 271Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val1 5 1027212PRTHomo sapiens 272Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val1 5 1027312PRTHomo sapiens 273Gln Ser Tyr Asp Arg Gly Phe Thr Gly Phe Asp Gly1 5 1027412PRTHomo sapiens 274Gln Ser Tyr Asp Arg Gly Thr Ala Pro Ala Leu Ser1 5 1027512PRTHomo sapiens 275Gln Ser Tyr Asp Arg Gly Ser Tyr Pro Ala Leu Arg1 5 1027612PRTHomo sapiens 276Gln Ser Tyr Asp Arg Gly Asn Trp Pro Asn Ser Asn1 5 1027712PRTHomo sapiens 277Gln Ser Tyr Asp Arg Gly Thr Ala Pro Ser Leu Leu1 5 1027812PRTHomo sapiens 278Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val1 5 1027912PRTHomo sapiens 279Gln Ser Tyr Asp Arg Gly Thr Thr Pro Arg Ile Arg1 5 1028012PRTHomo sapiens 280Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val1 5 1028112PRTHomo sapiens 281Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val1 5 1028212PRTHomo sapiens 282Gln Ser Tyr Asp Arg Gly Met Ile Pro Ala Leu Thr1 5 1028312PRTHomo sapiens 283Gln Ser Tyr Asp Arg Asn Thr His Pro Ala Leu Leu1 5 1028412PRTHomo sapiens 284Gln Ser Tyr Asp Arg Phe Thr His Pro Ala Leu Leu1 5 1028512PRTHomo sapiens 285Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala Leu Leu1 5 1028612PRTHomo sapiens 286Gln Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu1 5 1028712PRTHomo sapiens 287Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala Leu Leu1 5 102889PRTHomo sapiens 288Phe Thr Phe Glu Ser Tyr Gly Met His1 5 2899PRTHomo sapiens 289Phe Thr Phe Ser Ser Tyr Gly Met His1 52909PRTHomo sapiens 290Phe Thr Phe Tyr Ser Tyr Gly Met His1 52919PRTHomo sapiens 291Phe Thr Phe His Ser Tyr Gly Met His1 52929PRTHomo sapiens 292Phe Thr Phe Lys Ser Tyr Gly Met His1 52939PRTHomo sapiens 293Phe Thr Phe Arg Ser Tyr Gly Met His1 52949PRTHomo sapiens 294Phe Thr Phe Asn Ser Tyr Gly Met His1 52959PRTHomo sapiens 295Phe Thr Phe Thr Ser Tyr Gly Met His1 52969PRTHomo sapiens 296Phe Thr Phe Gly Ser Tyr Gly Met His1 52979PRTHomo sapiens 297Phe Thr Phe Val Ser Tyr Gly Met His1 52989PRTHomo sapiens 298Phe Thr Phe Ile Ser Tyr Gly Met His1 52999PRTHomo sapiens 299Phe Thr Phe Trp Ser Tyr Gly Met His1 53009PRTHomo sapiens 300Phe Thr Phe Ser Glu Tyr Gly Met His1 53019PRTHomo sapiens 301Phe Thr Phe Ser Cys Tyr Gly Met His1 53029PRTHomo sapiens 302Phe Thr Phe Ser Ser Tyr Gly Met His1 53039PRTHomo sapiens 303Phe Thr Phe Ser Tyr Tyr Gly Met His1 53049PRTHomo sapiens 304Phe Thr Phe Ser His Tyr Gly Met His1 53059PRTHomo sapiens 305Phe Thr Phe Ser Arg Tyr Gly Met His1 53069PRTHomo sapiens 306Phe Thr Phe Ser Asn Tyr Gly Met His1 53079PRTHomo sapiens 307Phe Thr Phe Ser Gln Tyr Gly Met His1 53089PRTHomo sapiens 308Phe Thr Phe Ser Thr Tyr Gly Met His1 53099PRTHomo sapiens 309Phe Thr Phe Ser Ala Tyr Gly Met His1 53109PRTHomo sapiens 310Phe Thr Phe Ser Ile Tyr Gly Met His1 53119PRTHomo sapiens 311Phe Thr Phe Ser Ser Glu Gly Met His1 53129PRTHomo sapiens 312Phe Thr Phe Ser Ser Cys Gly Met His1 53139PRTHomo sapiens 313Phe Thr Phe Ser Ser Ser Gly Met His1 53149PRTHomo sapiens 314Phe Thr Phe Ser Ser Tyr Gly Met His1 53159PRTHomo sapiens 315Phe Thr Phe Ser Ser His Gly Met His1 53169PRTHomo sapiens 316Phe Thr Phe Ser Ser Arg Gly Met His1 53179PRTHomo sapiens 317Phe Thr Phe Ser Ser Asn Gly Met His1 53189PRTHomo sapiens 318Phe Thr Phe Ser Ser Thr Gly Met His1 53199PRTHomo sapiens 319Phe Thr Phe Ser Ser Ala Gly Met His1 53209PRTHomo sapiens 320Phe Thr Phe Ser Ser Val Gly Met His1 53219PRTHomo sapiens 321Phe Thr Phe Ser Ser Leu Gly Met His1 53229PRTHomo sapiens 322Phe Thr Phe Ser Ser Ile Gly Met His1 53239PRTHomo sapiens 323Phe Thr Phe Ser Ser Tyr Asp Met His1 53249PRTHomo sapiens 324Phe Thr Phe Ser Ser Tyr Glu Met His1 53259PRTHomo sapiens 325Phe Thr Phe Ser Ser Tyr Cys Met His1 53269PRTHomo sapiens 326Phe Thr Phe Ser Ser Tyr Ser Met His1

53279PRTHomo sapiens 327Phe Thr Phe Ser Ser Tyr Tyr Met His1 53289PRTHomo sapiens 328Phe Thr Phe Ser Ser Tyr Asn Met His1 53299PRTHomo sapiens 329Phe Thr Phe Ser Ser Tyr Gly Met His1 53309PRTHomo sapiens 330Phe Thr Phe Ser Ser Tyr Ala Met His1 53319PRTHomo sapiens 331Phe Thr Phe Ser Ser Tyr Val Met His1 53329PRTHomo sapiens 332Phe Thr Phe Ser Ser Tyr Met Met His1 53339PRTHomo sapiens 333Phe Thr Phe Ser Ser Tyr Ile Met His1 53349PRTHomo sapiens 334Phe Thr Phe Ser Ser Tyr Pro Met His1 533517PRTHomo sapiens 335Glu Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly33617PRTHomo sapiens 336Cys Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly33717PRTHomo sapiens 337Tyr Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly33817PRTHomo sapiens 338His Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly33917PRTHomo sapiens 339Lys Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34017PRTHomo sapiens 340Asn Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34117PRTHomo sapiens 341Gln Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34217PRTHomo sapiens 342Thr Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34317PRTHomo sapiens 343Leu Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34417PRTHomo sapiens 344Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34517PRTHomo sapiens 345Phe Ile Glu Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34617PRTHomo sapiens 346Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34717PRTHomo sapiens 347Phe Ile Tyr Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34817PRTHomo sapiens 348Phe Ile His Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly34917PRTHomo sapiens 349Phe Ile Lys Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35017PRTHomo sapiens 350Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35117PRTHomo sapiens 351Phe Ile Gln Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35217PRTHomo sapiens 352Phe Ile Thr Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35317PRTHomo sapiens 353Phe Ile Gly Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35417PRTHomo sapiens 354Phe Ile Ala Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35517PRTHomo sapiens 355Phe Ile Val Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35617PRTHomo sapiens 356Phe Ile Leu Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35717PRTHomo sapiens 357Phe Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35817PRTHomo sapiens 358Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly35917PRTHomo sapiens 359Phe Ile Arg Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36017PRTHomo sapiens 360Phe Ile Arg Tyr Ser Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36117PRTHomo sapiens 361Phe Ile Arg Tyr Tyr Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36217PRTHomo sapiens 362Phe Ile Arg Tyr Lys Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36317PRTHomo sapiens 363Phe Ile Arg Tyr Arg Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36417PRTHomo sapiens 364Phe Ile Arg Tyr Asn Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36517PRTHomo sapiens 365Phe Ile Arg Tyr Gln Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36617PRTHomo sapiens 366Phe Ile Arg Tyr Thr Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36717PRTHomo sapiens 367Phe Ile Arg Tyr Ala Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36817PRTHomo sapiens 368Phe Ile Arg Tyr Val Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly36917PRTHomo sapiens 369Phe Ile Arg Tyr Leu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37017PRTHomo sapiens 370Phe Ile Arg Tyr Ile Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37117PRTHomo sapiens 371Phe Ile Arg Tyr Phe Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37217PRTHomo sapiens 372Phe Ile Arg Tyr Asp Asp Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37317PRTHomo sapiens 373Phe Ile Arg Tyr Asp Glu Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37417PRTHomo sapiens 374Phe Ile Arg Tyr Asp Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37517PRTHomo sapiens 375Phe Ile Arg Tyr Asp Tyr Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37617PRTHomo sapiens 376Phe Ile Arg Tyr Asp Lys Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37717PRTHomo sapiens 377Phe Ile Arg Tyr Asp Arg Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37817PRTHomo sapiens 378Phe Ile Arg Tyr Asp Asn Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly37917PRTHomo sapiens 379Phe Ile Arg Tyr Asp Gln Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38017PRTHomo sapiens 380Phe Ile Arg Tyr Asp Thr Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38117PRTHomo sapiens 381Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38217PRTHomo sapiens 382Phe Ile Arg Tyr Asp Val Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38317PRTHomo sapiens 383Phe Ile Arg Tyr Asp Phe Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38417PRTHomo sapiens 384Phe Ile Arg Tyr Asp Gly Ser Ser Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38517PRTHomo sapiens 385Phe Ile Arg Tyr Asp Gly Ser Tyr Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38617PRTHomo sapiens 386Phe Ile Arg Tyr Asp Gly Ser His Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38717PRTHomo sapiens 387Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38817PRTHomo sapiens 388Phe Ile Arg Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly38917PRTHomo sapiens 389Phe Ile Arg Tyr Asp Gly Ser Gly Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly39017PRTHomo sapiens 390Phe Ile Arg Tyr Asp Gly Ser Met Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly39117PRTHomo sapiens 391Phe Ile Arg Tyr Asp Gly Ser Leu Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly39217PRTHomo sapiens 392Phe Ile Arg Tyr Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly39317PRTHomo sapiens 393Phe Ile Arg Tyr Asp Gly Ser Pro Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly39417PRTHomo sapiens 394Phe Ile Arg Tyr Asp Gly Ser Phe Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly39517PRTHomo sapiens 395Phe Ile Arg Tyr Asp Gly Ser Asn Lys Glu Tyr Ala Asp Ser Val Lys1 5 10 15Gly39617PRTHomo sapiens 396Phe Ile Arg Tyr Asp Gly Ser Asn Lys Ser Tyr Ala Asp Ser Val Lys1 5 10 15Gly39717PRTHomo sapiens 397Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly39817PRTHomo sapiens 398Phe Ile Arg Tyr Asp Gly Ser Asn Lys Asn Tyr Ala Asp Ser Val Lys1 5 10 15Gly39917PRTHomo sapiens 399Phe Ile Arg Tyr Asp Gly Ser Asn Lys Val Tyr Ala Asp Ser Val Lys1 5 10 15Gly40017PRTHomo sapiens 400Phe Ile Arg Tyr Asp Gly Ser Asn Lys Leu Tyr Ala Asp Ser Val Lys1 5 10 15Gly40117PRTHomo sapiens 401Phe Ile Arg Tyr Asp Gly Ser Asn Lys Ile Tyr Ala Asp Ser Val Lys1 5 10 15Gly40217PRTHomo sapiens 402Phe Ile Arg Tyr Asp Gly Ser Asn Lys Pro Tyr Ala Asp Ser Val Lys1 5 10 15Gly40317PRTHomo sapiens 403Phe Ile Arg Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val Lys1 5 10 15Gly4046PRTHomo sapiens 404Glu Gly Ser His Asp Asn1 54056PRTHomo sapiens 405Ser Gly Ser His Asp Asn1 54066PRTHomo sapiens 406His Gly Ser His Asp Asn1 54076PRTHomo sapiens 407Lys Gly Ser His Asp Asn1 54086PRTHomo sapiens 408Gln Gly Ser His Asp Asn1 54096PRTHomo sapiens 409Thr Gly Ser His Asp Asn1 54106PRTHomo sapiens 410Ala Gly Ser His Asp Asn1 54116PRTHomo sapiens 411Leu Gly Ser His Asp Asn1 54126PRTHomo sapiens 412Pro Gly Ser His Asp Asn1 54136PRTHomo sapiens 413Phe Gly Ser His Asp Asn1 54146PRTHomo sapiens 414His Asp Ser His Asp Asn1 54156PRTHomo sapiens 415His Cys Ser His Asp Asn1 54166PRTHomo sapiens 416His His Ser His Asp Asn1 54176PRTHomo sapiens 417His Arg Ser His Asp Asn1 54186PRTHomo sapiens 418His Thr Ser His Asp Asn1 54196PRTHomo sapiens 419His Gly Ser His Asp Asn1 54206PRTHomo sapiens 420His Val Ser His Asp Asn1 54216PRTHomo sapiens 421His Met Ser His Asp Asn1 54226PRTHomo sapiens 422His Leu Ser His Asp Asn1 54236PRTHomo sapiens 423His Ile Ser His Asp Asn1 54246PRTHomo sapiens 424His Pro Ser His Asp Asn1 54256PRTHomo sapiens 425His Trp Ser His Asp Asn1 54266PRTHomo sapiens 426His Gly Asp His Asp Asn1 54276PRTHomo sapiens 427His Gly Ser His Asp Asn1 54286PRTHomo sapiens 428His Gly Tyr His Asp Asn1 54296PRTHomo sapiens 429His Gly His His Asp Asn1 54306PRTHomo sapiens 430His Gly Arg His Asp Asn1 54316PRTHomo sapiens 431His Gly Asn His Asp Asn1 54326PRTHomo sapiens 432His Gly Thr His Asp Asn1 54336PRTHomo sapiens 433His Gly Gly His Asp Asn1 54346PRTHomo sapiens 434His Gly Ala His Asp Asn1 54356PRTHomo sapiens 435His Gly Ile His Asp Asn1 54366PRTHomo sapiens 436His Gly Pro His Asp Asn1 54376PRTHomo sapiens 437His Gly Trp His Asp Asn1 54386PRTHomo sapiens 438His Gly Phe His Asp Asn1 54396PRTHomo sapiens 439His Gly Ser His Asp Asn1 54406PRTHomo sapiens 440His Gly Ser Arg Asp Asn1 54416PRTHomo sapiens 441His Gly Ser Thr Asp Asn1 54426PRTHomo sapiens 442His Gly Ser Ala Asp Asn1 54436PRTHomo sapiens 443His Gly Ser Val Asp Asn1 54446PRTHomo sapiens 444His Gly Ser Leu Asp Asn1 54456PRTHomo sapiens 445His Gly Ser Ile Asp Asn1 54466PRTHomo sapiens 446His Gly Ser Phe Asp Asn1 54476PRTHomo sapiens 447His Gly Ser His Asp Asn1 54486PRTHomo sapiens 448His Gly Ser His Ser Asn1 54496PRTHomo sapiens 449His Gly Ser His Tyr Asn1 54506PRTHomo sapiens 450His Gly Ser His His Asn1 54516PRTHomo sapiens 451His Gly Ser His Arg Asn1 54526PRTHomo sapiens 452His Gly Ser His Asn Asn1 54536PRTHomo sapiens 453His Gly Ser His Gly Asn1 54546PRTHomo sapiens 454His Gly Ser His Ala Asn1 54556PRTHomo sapiens 455His Gly Ser His Val Asn1 54566PRTHomo sapiens 456His Gly Ser His Ile Asn1 54576PRTHomo sapiens 457His Gly Ser His Asp Ser1 54586PRTHomo sapiens 458His Gly Ser His Asp His1 54596PRTHomo sapiens 459His Gly Ser His Asp Lys1 54606PRTHomo sapiens 460His Gly Ser His Asp Arg1 54616PRTHomo sapiens 461His Gly Ser His Asp Asn1 54626PRTHomo sapiens 462His Gly Ser His Asp Thr1 54636PRTHomo sapiens 463His Gly Ser His Asp Gly1 54646PRTHomo sapiens 464His Gly Ser His Asp Ala1 54656PRTHomo sapiens 465His Gly Ser His Asp Leu1 54666PRTHomo sapiens 466His Gly Ser His Asp Ile1 54676PRTHomo sapiens 467His Gly Ser His Asp Pro1 54686PRTHomo sapiens 468His Gly Ser His Asp Trp1 54696PRTHomo sapiens 469His Gly Ser His Asp Phe1 547013PRTHomo sapiens 470Ser Gly Gly Arg Ser Asn Ile Gly Asp Asn Thr Val Lys1 5 1047113PRTHomo sapiens 471Ser Gly Gly Arg Ser Asn Ile Gly Cys Asn Thr Val Lys1 5 1047213PRTHomo sapiens 472Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Thr Val Lys1 5 1047313PRTHomo sapiens 473Ser Gly Gly Arg Ser Asn Ile Gly Tyr Asn Thr Val Lys1 5 1047413PRTHomo sapiens 474Ser Gly Gly Arg Ser Asn Ile Gly Lys Asn Thr Val Lys1 5 1047513PRTHomo sapiens 475Ser Gly Gly Arg Ser Asn Ile Gly Arg Asn Thr Val Lys1 5 1047613PRTHomo sapiens 476Ser Gly Gly Arg Ser Asn Ile Gly Asn Asn Thr Val Lys1 5 1047713PRTHomo sapiens 477Ser Gly Gly Arg Ser Asn Ile Gly Thr Asn Thr Val Lys1 5 1047813PRTHomo sapiens 478Ser Gly Gly Arg Ser Asn Ile Gly Pro Asn Thr Val Lys1 5 1047913PRTHomo sapiens 479Ser Gly Gly Arg Ser Asn Ile Gly Ser Asp Thr Val Lys1 5 1048013PRTHomo sapiens 480Ser Gly Gly Arg Ser Asn Ile Gly Ser Glu Thr Val Lys1 5 1048113PRTHomo sapiens 481Ser Gly Gly Arg Ser Asn Ile Gly Ser Ser Thr Val Lys1 5 1048213PRTHomo sapiens 482Ser Gly Gly Arg Ser Asn Ile Gly Ser Tyr Thr Val Lys1 5 1048313PRTHomo sapiens 483Ser Gly Gly Arg Ser Asn Ile Gly Ser His Thr Val Lys1 5 1048413PRTHomo sapiens 484Ser Gly Gly Arg Ser Asn Ile Gly Ser Lys Thr Val Lys1 5 1048513PRTHomo sapiens 485Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Thr Val Lys1 5 1048613PRTHomo sapiens 486Ser Gly Gly Arg Ser Asn Ile Gly Ser Gln Thr Val Lys1 5 1048713PRTHomo sapiens 487Ser Gly Gly Arg Ser Asn Ile Gly Ser Thr Thr Val Lys1 5 1048813PRTHomo sapiens 488Ser Gly Gly Arg Ser Asn Ile Gly Ser Gly Thr Val Lys1 5 1048913PRTHomo sapiens 489Ser Gly Gly Arg Ser Asn Ile Gly Ser Met Thr Val Lys1 5 1049013PRTHomo sapiens 490Ser Gly Gly Arg Ser Asn Ile Gly Ser Ile Thr Val Lys1 5 1049113PRTHomo sapiens 491Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Asp Val Lys1 5 1049213PRTHomo sapiens 492Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Cys Val Lys1 5 1049313PRTHomo sapiens 493Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Ser Val Lys1 5 1049413PRTHomo sapiens 494Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Tyr Val Lys1 5

1049513PRTHomo sapiens 495Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn His Val Lys1 5 1049613PRTHomo sapiens 496Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Lys Val Lys1 5 1049713PRTHomo sapiens 497Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Arg Val Lys1 5 1049813PRTHomo sapiens 498Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Asn Val Lys1 5 1049913PRTHomo sapiens 499Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Gln Val Lys1 5 1050013PRTHomo sapiens 500Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Thr Val Lys1 5 1050113PRTHomo sapiens 501Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Ala Val Lys1 5 1050213PRTHomo sapiens 502Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Val Val Lys1 5 1050313PRTHomo sapiens 503Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Leu Val Lys1 5 1050413PRTHomo sapiens 504Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Ile Val Lys1 5 1050513PRTHomo sapiens 505Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn Pro Val Lys1 5 105067PRTHomo sapiens 506Asp Asn Asp Gln Arg Pro Ser1 55077PRTHomo sapiens 507Glu Asn Asp Gln Arg Pro Ser1 55087PRTHomo sapiens 508Cys Asn Asp Gln Arg Pro Ser1 55097PRTHomo sapiens 509Ser Asn Asp Gln Arg Pro Ser1 55107PRTHomo sapiens 510Tyr Asn Asp Gln Arg Pro Ser1 55117PRTHomo sapiens 511His Asn Asp Gln Arg Pro Ser1 55127PRTHomo sapiens 512Lys Asn Asp Gln Arg Pro Ser1 55137PRTHomo sapiens 513Arg Asn Asp Gln Arg Pro Ser1 55147PRTHomo sapiens 514Asn Asn Asp Gln Arg Pro Ser1 55157PRTHomo sapiens 515Gln Asn Asp Gln Arg Pro Ser1 55167PRTHomo sapiens 516Thr Asn Asp Gln Arg Pro Ser1 55177PRTHomo sapiens 517Gly Asn Asp Gln Arg Pro Ser1 55187PRTHomo sapiens 518Ala Asn Asp Gln Arg Pro Ser1 55197PRTHomo sapiens 519Val Asn Asp Gln Arg Pro Ser1 55207PRTHomo sapiens 520Met Asn Asp Gln Arg Pro Ser1 55217PRTHomo sapiens 521Leu Asn Asp Gln Arg Pro Ser1 55227PRTHomo sapiens 522Ile Asn Asp Gln Arg Pro Ser1 55237PRTHomo sapiens 523Pro Asn Asp Gln Arg Pro Ser1 55247PRTHomo sapiens 524Trp Asn Asp Gln Arg Pro Ser1 55257PRTHomo sapiens 525Phe Asn Asp Gln Arg Pro Ser1 55267PRTHomo sapiens 526Gly Asn Asp Ser Arg Pro Ser1 55277PRTHomo sapiens 527Gly Asn Asp Tyr Arg Pro Ser1 55287PRTHomo sapiens 528Gly Asn Asp Arg Arg Pro Ser1 55297PRTHomo sapiens 529Gly Asn Asp Gln Arg Pro Ser1 55307PRTHomo sapiens 530Gly Asn Asp Thr Arg Pro Ser1 55317PRTHomo sapiens 531Gly Asn Asp Ala Arg Pro Ser1 55327PRTHomo sapiens 532Gly Asn Asp Ile Arg Pro Ser1 55337PRTHomo sapiens 533Gly Asn Asp Pro Arg Pro Ser1 553412PRTHomo sapiens 534Gln Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu1 5 1053512PRTHomo sapiens 535Gln Ser Tyr Cys Arg Gly Thr His Pro Ala Leu Leu1 5 1053612PRTHomo sapiens 536Gln Ser Tyr Ser Arg Gly Thr His Pro Ala Leu Leu1 5 1053712PRTHomo sapiens 537Gln Ser Tyr Tyr Arg Gly Thr His Pro Ala Leu Leu1 5 1053812PRTHomo sapiens 538Gln Ser Tyr Asn Arg Gly Thr His Pro Ala Leu Leu1 5 1053912PRTHomo sapiens 539Gln Ser Tyr Gln Arg Gly Thr His Pro Ala Leu Leu1 5 1054012PRTHomo sapiens 540Gln Ser Tyr Thr Arg Gly Thr His Pro Ala Leu Leu1 5 1054112PRTHomo sapiens 541Gln Ser Tyr Gly Arg Gly Thr His Pro Ala Leu Leu1 5 1054212PRTHomo sapiens 542Gln Ser Tyr Ala Arg Gly Thr His Pro Ala Leu Leu1 5 1054312PRTHomo sapiens 543Gln Ser Tyr Leu Arg Gly Thr His Pro Ala Leu Leu1 5 1054412PRTHomo sapiens 544Gln Ser Tyr Ile Arg Gly Thr His Pro Ala Leu Leu1 5 1054512PRTHomo sapiens 545Gln Ser Tyr Trp Arg Gly Thr His Pro Ala Leu Leu1 5 1054612PRTHomo sapiens 546Gln Ser Tyr Phe Arg Gly Thr His Pro Ala Leu Leu1 5 1054712PRTHomo sapiens 547Gln Ser Tyr Asp Asp Gly Thr His Pro Ala Leu Leu1 5 1054812PRTHomo sapiens 548Gln Ser Tyr Asp Cys Gly Thr His Pro Ala Leu Leu1 5 1054912PRTHomo sapiens 549Gln Ser Tyr Asp Ser Gly Thr His Pro Ala Leu Leu1 5 1055012PRTHomo sapiens 550Gln Ser Tyr Asp Tyr Gly Thr His Pro Ala Leu Leu1 5 1055112PRTHomo sapiens 551Gln Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu1 5 1055212PRTHomo sapiens 552Gln Ser Tyr Asp Asn Gly Thr His Pro Ala Leu Leu1 5 1055312PRTHomo sapiens 553Gln Ser Tyr Asp Gln Gly Thr His Pro Ala Leu Leu1 5 1055412PRTHomo sapiens 554Gln Ser Tyr Asp Thr Gly Thr His Pro Ala Leu Leu1 5 1055512PRTHomo sapiens 555Gln Ser Tyr Asp Gly Gly Thr His Pro Ala Leu Leu1 5 1055612PRTHomo sapiens 556Gln Ser Tyr Asp Ala Gly Thr His Pro Ala Leu Leu1 5 1055712PRTHomo sapiens 557Gln Ser Tyr Asp Val Gly Thr His Pro Ala Leu Leu1 5 1055812PRTHomo sapiens 558Gln Ser Tyr Asp Met Gly Thr His Pro Ala Leu Leu1 5 1055912PRTHomo sapiens 559Gln Ser Tyr Asp Leu Gly Thr His Pro Ala Leu Leu1 5 1056012PRTHomo sapiens 560Gln Ser Tyr Asp Ile Gly Thr His Pro Ala Leu Leu1 5 1056112PRTHomo sapiens 561Gln Ser Tyr Asp Pro Gly Thr His Pro Ala Leu Leu1 5 1056212PRTHomo sapiens 562Gln Ser Tyr Asp Trp Gly Thr His Pro Ala Leu Leu1 5 1056312PRTHomo sapiens 563Gln Ser Tyr Asp Arg Asp Thr His Pro Ala Leu Leu1 5 1056412PRTHomo sapiens 564Gln Ser Tyr Asp Arg Cys Thr His Pro Ala Leu Leu1 5 1056512PRTHomo sapiens 565Gln Ser Tyr Asp Arg Ser Thr His Pro Ala Leu Leu1 5 1056612PRTHomo sapiens 566Gln Ser Tyr Asp Arg Tyr Thr His Pro Ala Leu Leu1 5 1056712PRTHomo sapiens 567Gln Ser Tyr Asp Arg His Thr His Pro Ala Leu Leu1 5 1056812PRTHomo sapiens 568Gln Ser Tyr Asp Arg Arg Thr His Pro Ala Leu Leu1 5 1056912PRTHomo sapiens 569Gln Ser Tyr Asp Arg Asn Thr His Pro Ala Leu Leu1 5 1057012PRTHomo sapiens 570Gln Ser Tyr Asp Arg Gln Thr His Pro Ala Leu Leu1 5 1057112PRTHomo sapiens 571Gln Ser Tyr Asp Arg Thr Thr His Pro Ala Leu Leu1 5 1057212PRTHomo sapiens 572Gln Ser Tyr Asp Arg Gly Thr His Pro Ala Leu Leu1 5 1057312PRTHomo sapiens 573Gln Ser Tyr Asp Arg Ala Thr His Pro Ala Leu Leu1 5 1057412PRTHomo sapiens 574Gln Ser Tyr Asp Arg Val Thr His Pro Ala Leu Leu1 5 1057512PRTHomo sapiens 575Gln Ser Tyr Asp Arg Leu Thr His Pro Ala Leu Leu1 5 1057612PRTHomo sapiens 576Gln Ser Tyr Asp Arg Ile Thr His Pro Ala Leu Leu1 5 1057712PRTHomo sapiens 577Gln Ser Tyr Asp Arg Pro Thr His Pro Ala Leu Leu1 5 1057812PRTHomo sapiens 578Gln Ser Tyr Asp Arg Trp Thr His Pro Ala Leu Leu1 5 1057912PRTHomo sapiens 579Gln Ser Tyr Asp Arg Phe Thr His Pro Ala Leu Leu1 5 1058048DNAsynthetic constructnucleotides at positions 16 to 34 can be substituted with any nucleotide such that the randomized nucleotides represent 12% of the sequence 580tgtcccttgg ccccagtagt catagctccc actggtcgta cagtaata 4858135DNAsynthetic construct 581gacacctcga tcagcggata acaatttcac acagg 3558215DNAsynthetic construct 582tggggccaag ggaca 1558345DNAsynthetic construct 583attcgtccta taccgttcta ctttgtcgtc tttccagacg ttagt 4558418DNAsynthetic construct 584attcgtccta taccgttc 1858566DNAsynthetic constructnucleotides from position 28 to 42 can be substituted with any nucleotide such that the randomized nucleotides represent 12% of the sequence 585ggtcccagtt ccgaagaccc tcgaacccct caggctgctg tcatatgact ggcagtaata 60gtcagc 6658615DNAsynthetic construct 586tggggccaag ggaca 1558724DNAsynthetic construct 587tgaagagacg gtgaccattg tccc 2458816DNAsynthetic construct 588gacacctcga tcagcg 1658948DNAsynthetic construct 589gagtcattct cgacttgcgg ccgcacctag gacggtcagc ttggtccc 4859012PRTHomo sapiens 590Gln Ser Tyr Asp Arg Gly Phe Thr Gly Ser Met Val1 5 1059112PRTHomo sapiensXaa is encoded by a randomized codon of sequence NNS with N being any nucleotide and S being either deoxycytosine or deoxyguanidine 591Xaa Xaa Xaa Xaa Xaa Xaa Phe Thr Gly Ser Met Val1 5 1059212PRTHomo sapiensXaa is encoded by a randomized codon of sequence NNS with N being any nucleotide and S being either deoxycytosine or deoxyguanidine 592Gln Ser Tyr Xaa Xaa Xaa Xaa Xaa Xaa Ser Met Val1 5 1059312PRTHomo sapiensXaa is encoded by a randomized codon of sequence NNS with N being any nucleotide and S being either deoxycytosine or deoxyguanidine 593Gln Ser Tyr Asp Arg Gly Xaa Xaa Xaa Xaa Xaa Xaa1 5 10594100PRTHomo sapiens 594Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Thr Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg 100595100PRTHomo sapiens 595Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Leu Val 35 40 45Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55 60Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr 85 90 95Tyr Cys Ala Arg 100596100PRTHomo sapiens 596Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Leu Val 35 40 45Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55 60Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr65 70 75 80Met Tyr Leu Gln Met Ser Asn Leu Lys Thr Glu Asp Leu Ala Val Tyr 85 90 95Tyr Cys Ala Arg 100597100PRTHomo sapiens 597Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Leu Val 35 40 45Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55 60Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr 85 90 95Tyr Cys Ala Arg 10059898PRTHomo sapiens 598Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Lys 59998PRTHomo sapiens 599Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys 85 90 95Ala Arg 60098PRTHomo sapiens 600Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Lys 60198PRTHomo sapiens 601Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 60298PRTHomo sapiens 602Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 603100PRTHomo sapiens 603Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Ala Ala 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Arg 100604100PRTHomo sapiens 604Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10

15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr 100605100PRTHomo sapiens 605Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Glu Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr 100606100PRTHomo sapiens 606Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr 100607100PRTHomo sapiens 607Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asn Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr 100608100PRTHomo sapiens 608Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr 100609100PRTHomo sapiens 609Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr 10061098PRTHomo sapiens 610Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Pro Ala Ser Gly Phe Thr Phe Ser Asn His 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Gly Asp Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser Pro Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys 61198PRTHomo sapiens 611Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn His 20 25 30Tyr Thr Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ser Ser Gly Asn Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys 61298PRTHomo sapiens 612Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30Asp Met Asn Trp Val His Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Val Ser Trp Asn Gly Ser Arg Thr His Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr65 70 75 80Leu Gln Thr Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg 61398PRTHomo sapiens 613Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30Asp Met Asn Trp Ala Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Val Ser Trp Asn Gly Ser Arg Thr His Tyr Val Asp Ser Val 50 55 60Lys Arg Arg Phe Ile Ile Ser Arg Asp Asn Ser Arg Asn Ser Leu Tyr65 70 75 80Leu Gln Lys Asn Arg Arg Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys 85 90 95Val Arg 61498PRTHomo sapiens 614Thr Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Glu Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Val Ser Trp Asn Gly Ser Arg Thr His Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Arg Asn Phe Leu Tyr65 70 75 80Gln Gln Met Asn Ser Leu Arg Pro Glu Asp Met Ala Val Tyr Tyr Cys 85 90 95Val Arg 61597PRTHomo sapiens 615Glu Val His Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Asn Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg 61697PRTHomo sapiens 616Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg 61797PRTHomo sapiens 617Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95Arg 61897PRTHomo sapiens 618Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95Arg 61998PRTHomo sapiens 619Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys 62098PRTHomo sapiens 620Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Val Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys 62198PRTHomo sapiens 621Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Val Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys 62298PRTHomo sapiens 622Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asn Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys 85 90 95Ala Arg 62398PRTHomo sapiens 623Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys 62498PRTHomo sapiens 624Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 62598PRTHomo sapiens 625Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 62698PRTHomo sapiens 626Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 62798PRTHomo sapiens 627Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 62898PRTHomo sapiens 628Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65

70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 62998PRTHomo sapiens 629Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 63098PRTHomo sapiens 630Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 63198PRTHomo sapiens 631Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 63298PRTHomo sapiens 632Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 63398PRTHomo sapiens 633Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Val Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys 63498PRTHomo sapiens 634Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 63598PRTHomo sapiens 635Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 63698PRTHomo sapiens 636Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 63798PRTHomo sapiens 637Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys 63897PRTHomo sapiens 638Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg 63998PRTHomo sapiens 639Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64098PRTHomo sapiens 640Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Leu Arg Ala Arg Leu Cys Ile Thr Val 85 90 95Arg Glu 64198PRTHomo sapiens 641Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64298PRTHomo sapiens 642Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64398PRTHomo sapiens 643Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64498PRTHomo sapiens 644Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64598PRTHomo sapiens 645Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64698PRTHomo sapiens 646Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64798PRTHomo sapiens 647Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64898PRTHomo sapiens 648Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Gly Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 64998PRTHomo sapiens 649Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys 65098PRTHomo sapiens 650Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys 65198PRTHomo sapiens 651Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 65298PRTHomo sapiens 652Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys 65395PRTHomo sapiens 653Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40

45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Arg Lys 85 90 9565498PRTHomo sapiens 654Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 65598PRTHomo sapiens 655Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Ala 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Thr Asn Thr Leu Phe65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 65698PRTHomo sapiens 656Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 65798PRTHomo sapiens 657Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 65897PRTHomo sapiens 658Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg 65998PRTHomo sapiens 659Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 66098PRTHomo sapiens 660Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 66197PRTHomo sapiens 661Glu Asp Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Pro Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30Val Leu His Trp Val Arg Arg Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45Ser Ala Ile Gly Thr Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Met 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Ile Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95Arg 66298PRTHomo sapiens 662Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 66398PRTHomo sapiens 663Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 66498PRTHomo sapiens 664Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 66598PRTHomo sapiens 665Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val 35 40 45Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Met 50 55 60Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys 85 90 95Thr Arg 66698PRTHomo sapiens 666Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg 66798PRTHomo sapiens 667Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr 66898PRTHomo sapiens 668Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln65 70 75 80Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95Ser Ala 66998PRTHomo sapiens 669Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asp Met Gly Asn Tyr 20 25 30Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Glu Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Trp65 70 75 80Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Thr Ser Pro 85 90 95Arg Ala 67098PRTHomo sapiens 670Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly 67198PRTHomo sapiens 671Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Ser Gly 67298PRTHomo sapiens 672Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ala Pro Arg Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly 67399PRTHomo sapiens 673Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Gln Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Lys Ala Trp Asp Asn Ser 85 90 95Leu Asn Ala 67499PRTHomo sapiens 674Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95Leu Ser Gly 67598PRTHomo sapiens 675Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Gly Arg Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Lys Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Gly Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Val Gln65 70 75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95Arg Gly

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