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United States Patent Application 20070178082
Kind Code A1
Silence; Karen ;   et al. August 2, 2007

Stabilized single domain antibodies

Abstract

The present invention relates to heterospecific polypeptide constructs comprising at least one single domain antibody directed against a therapeutic and/or diagnostic target and at least one single domain antibody directed against a serum protein, said construct having a prolonged lifetime in biological circulatory systems. The invention further relates to methods for stabilising VHHs in biological circulatory systems.


Inventors: Silence; Karen; (Overijse, BE) ; Lauwereys; Marc; (Haaltert, BE) ; Dreier; Torsten; (Munich, DE)
Correspondence Address:
    WOLF GREENFIELD & SACKS, P.C.
    600 ATLANTIC AVENUE
    BOSTON
    MA
    02210-2206
    US
Assignee: ABLYNX N.V.
TECHNOLOGIEPARK 4
GHENT-ZWIJNAARDE
BE
B-9052

Serial No.: 534349
Series Code: 10
Filed: November 7, 2003
PCT Filed: November 7, 2003
PCT NO: PCT/BE03/00193
371 Date: May 9, 2005

Current U.S. Class: 424/131.1; 435/320.1; 435/327; 435/69.1; 514/44R; 530/387.2; 536/23.53
Class at Publication: 424/131.1; 514/044; 435/069.1; 435/320.1; 435/327; 530/387.2; 536/023.53
International Class: A61K 48/00 20060101 A61K048/00; A61K 39/395 20060101 A61K039/395; C07K 16/42 20060101 C07K016/42; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101 C12P021/06


Foreign Application Data

DateCodeApplication Number
Jan 10, 2003EP03447005.4
Jun 23, 2003EPPCT/EP03/06581
Jul 8, 2003EPPCT/EP03/07313

Claims



1. A polypeptide construct comprising: at least one single domain antibody directed against a therapeutic and/or diagnostic target, and at least one single domain antibody directed against a serum protein.

2. A polypeptide construct according to claim 1 wherein: the number of anti-target single domain antibodies is at least two, and at least two anti-target single domain antibodies do not share the same sequence, or all the anti-target single domain antibodies share the same sequence.

3. A polypeptide construct according to claim 1 wherein: the number of anti-serum protein single domain antibodies is at least two, and at least two anti-serum-protein single domain antibodies do not share the same sequence, or all the anti-serum-protein single domain antibodies share the same sequence.

4. A polypeptide construct according to claim 1 wherein the at least one single domain antibody is a Camelidae VHHs antibody.

5. A polypeptide construct according to claim 1 wherein the at least one single domain antibody is a humanised Camelidae VHHs antibody.

6. A polypeptide construct according to claim 1 wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen or a fragment thereof.

7. A polypeptide construct according to claim 1 wherein the at least one single domain anti-serum protein antibody corresponds to a sequence represented by any of SEQ ID NOS: 1 to 4, and 28 to 40.

8. A polypeptide construct according to claim 1 wherein the target is TNF-alpha.

9. A polypeptide construct according to claim 7 corresponding to the sequence represented by any of SEQ ID NO: 5 to 18.

10. (canceled)

11. A nucleic acid encoding a polypeptide construct according to claim 9.

12. (canceled)

13. A method for treatment, prevention and/or alleviation of disorders relating to inflammatory processes comprising administering to a subject in need of such treatment a polypeptide construct according to claim 8.

14. (canceled)

15. The method according to claim 13 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

16. A polypeptide construct according to claim 1 wherein a the target is vWF

17. A polypeptide construct according to claim 1 wherein the target is collagen.

18. A polypeptide construct according to claim 16 wherein at least one anti-target single domain antibody is anti-vWF VHHs.

19. A polypeptide construct according to claim 18 corresponding to the sequence represented by any of SEQ ID NOS: 19 to 21.

20. (canceled)

21. A nucleic acid encoding a polypeptide construct according to claim 19.

22. A method for treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof comprising administering to a subject in need of such treatment A polypeptide construct according to claim 16.

23. (canceled)

24. A method according to claim 22 wherein said disorders are any of cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis, and said conditions are those arising from coronary by-pass graft, or coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.

25. A method according to claim 22 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

26. A polypeptide construct according to claim 1 wherein the target is IgE.

27. A polypeptide construct according to claim 26 wherein the at least one anti-target single domain antibody is anti-IgE VHHs.

28. A polypeptide construct according to claim 26 corresponding to the sequence represented by any of SEQ ID NOS: 22 to 24.

29. (canceled)

30. A nucleic acid encoding a polypeptide construct according to claim 28.

31. (canceled)

32. A method for the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions comprising administering to a subject in need of such treatment a polypeptide construct according to claim 26.

33. A method according to claim 32 wherein said disorders or conditions are any of hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.

34. A method according to claim 32 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

35. A polypeptide construct according to claim 1 wherein the target is IFN-gamma.

36. A polypeptide construct according to claim 35 wherein at least one anti-target single domain antibody is anti-IFN-gamma VHHs.

37. A polypeptide construct according to claim 35 corresponding to a sequence represented by SEQ ID NOs: 25 to 27.

38. (canceled)

39. A nucleic acid encoding a polypeptide construct according to claim 37.

40. (canceled)

41. A method for the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active comprising administering to a subject in need of such treatment a polypeptide construct according to claim 35.

42. A method according to claim 41 wherein said disorders or conditions are any of Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.

43. A method according to claim 41 wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

44. A composition comprising a polypeptide construct according to claim 8 or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.

45. A composition comprising a polypeptide construct according to claim 16 or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.

46. A composition comprising a polypeptide construct according to claim 26 or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.

47. A polypeptide construct according to claim 1 directed against a single target wherein said target is involved in a disease process.

48. (canceled)

49. A nucleic acid encoding a polypeptide construct according to claim 47.

50.-52. (canceled)

53. A method for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation comprising administering to a subject in need of such treatment the polypeptide construct according to claim 47.

54. (canceled)

55. A method for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time comprising administering to a subject in need of such treatment the polypeptide construct according to claim 47.

56. A according to any of claims 53 and 55, wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

57. (canceled)

58. A method of producing a polypeptide according to claim 1 comprising (a) culturing host cells comprising nucleic acid capable of encoding a polypeptide according to claim 1, under conditions allowing the expression of the polypeptide, and, (b) recovering the produced polypeptide from the culture.

59. A method according to claim 58, wherein said host cells are bacterial or yeast.

60. A method for prolonging the half-life of a single domain antibody in the blood stream of a subject, said antibody directed against a therapeutic and/or diagnostic target, comprising joining thereto one or more single domain antibodies directed against a serum protein.

61. A method according to claim 60 wherein said anti-target single domain antibodies do not share the same sequence.

62. A method according to claim 60 wherein said anti-serum protein single domain antibodies do not share the same sequence.

63. A method according to claim 60 wherein said single domain antibodies are Camelidae VHH antibodies.

64. A method according to claim 60 wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen or a fragment thereof.

65. A method according to claim 60 wherein said serum protein comprises a sequence corresponding to any of SEQ ID NOs: 1 to 4, a homologous sequence, a functional portion thereof, or a homologous sequence of a functional portion thereof.

66. A composition comprising a polypeptide according to claim 1 or a nucleic acid capable of encoding said polypeptide and a pharmaceutically acceptable vehicle.
Description



FIELD OF THE INVENTION

[0001] The present invention provides heterospecific polypeptide constructs comprising one or more single domain antibodies, said constructs having improved stability in vivo and their use in diagnosis and therapy.

BACKGROUND OF THE INVENTION

[0002] Polypeptide therapeutics and in particular antibody-based therapeutics have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. However, in order to be effective as therapeutic agent, their pharmacokinetic profile should be optimized. The majority of current antibody applications are for acute disorders. There are however significant opportunities to develop antibody therapeutics for chronic conditions. This will require large doses of protein over a long period of time. Since the cost of antibody production in mammalian cells is high, the development of traditional antibody therapeutics for these applications has been discouraged. An alternative approach has been to express fragments of antibodies such as Fab's or single-chain Fv's in microbial expression systems such as yeast and bacteria. These fragments however have very short circulation times in vivo.

[0003] Some of the initial approaches to increase the circulation in the bloodstream of proteins and peptides were based on chemical modification, such as pegylation (U.S. Pat. No. 4,179,337). Examples of such products are PEG-Intron, i.e. pegylated interferon alpha-2b for the treatment of HCV, and treatment of chronic disorder with PEG-modified antibodies (A. P Chapman, Adv.Drug Delivery Reviews (2002), 54, 531-545). Such chemical methods, however, suffer from a number of disadvantages, such as inactivation of the target protein or peptide due to the chemical modification of certain amino acid side chains, instability of the target protein/peptide during the chemical reaction.

[0004] To overcome these limitations, alternative approaches have been developed, first of all by using non-conventional or modified proteins, secondly by using alternative methods to increase half-life in vivo. Stabilisation of the protein drug can therefore be carried out by choosing an inherently stable protein scaffold and providing methods to bind such scaffold to plasma proteins which occur in high concentrations, such as immunoglobulins or albumin. Binding to plasma protein can be an effective means to improving the pharmacokinetic properties of molecules in general. More precisely, binding to albumin to improve the half-life of proteins has been described: M. S. Dennis et al. (J. Biol. Chem. 33, 238390, 2002) isolated peptides having affinity for serum albumin. When bound to a Fab molecule, half-lives comparable to pegylated Fab's were obtained. Peptide ligands having affinity for IgG or serum albumin have been disclosed (WO 01/45746). Cemu Bioteknik (Nygren, Wigzell, Uhlen, EP 486525 B1; U.S. Pat. No. 6,267,964) described fusions of active proteins or peptides to polypeptides from bacterial origin that bind to serum albumin (e.g. Staph A). The drawback of these peptide-based approaches is that the peptides have to fold properly and be accessible to binding to serum albumin when fused to the therapeutic protein. Therefore, these peptides are inherently unstable and have affinities in the submicromolar range rather than subnanomolar or low nanomolar range, as is the case with conventional antibodies. As part of a larger protein, such as a conventional antibody molecule, binding of these peptides to albumin may be sterically hindered.

[0005] An alternative hybrid molecule with two functional units is based on a heterospecific antibody. Such a hybrid would consist of a bifunctional or heterospecific antibody construct with one entity having specificity and affinity for the target, the second entity having specificity and affinity for a serum protein, such as albumin. However, such heterospecific constructs based on conventional antibodies or Fab fragments have several important drawbacks: these are complex, large molecules composed of two polypeptide chains (VH and VL) and therefore difficult and expensive to produce in high amounts in mammalian expression systems. Furthermore, producing bifunctional antibodies composed of 4 chains (2 VH's and 2 VL's) have the inherent risk of resulting in molecules with the unproductive VH-VL combinations and consequent loss of activity. Several alternatives have been tried with mixed results based on peptide derivatives of conventional antibodies, such as diabodies and bifunctional scFv's (WO0220615; WO 9413804; WO 9119739; WO 9409131). Holliger et al (Nature Biotech. 15, 632-636, 1979) suggests that binding one of the antibody fragments of a diabody (bispecific construct derived from a conventional antibody) to serum immunoglobulin (IgG) may prolong serum residence time of such diabodies but no suggestion is made that bispecific diabodies may be stabilised using antibodies against a serum protein other than serum IgG. Diabodies are known to be inherently difficult to produce due to stickiness of their exposed surface and due to non-productive associations between the four different V-regions (2 VH+2 VL).

[0006] Covalent binding to serum proteins as disdosed in, for example, EP0793506B1, U.S. Pat. Nos. 5,612,034, 6,103,233, and US20020009441 using reactive groups forming stable covalent bonds to a serum protein or a cell have the inherent disadvantage of unwanted target modification through the reactive groups.

[0007] Fusions to large, long lived proteins such as albumin (Syed et al, Blood 89, 3243-3252 (1997), Yeh et al, PNAS 89, 1904-1908 (1992); Celltech (WO0027435)) or N-terminal fusions of albumin polypeptides (Delta Biotech/HGS, U.S. Pat. Nos. 5,380,712, 5,766,883) or the Fc portion of IgG (Capon et al, Nature 337, 525-531(1989); Ashkenazi et al, Curr. Op.Immunol. 9, 195-200 (1997)) have been described. Such fusions have the disadvantage of inefficient production and causing unwanted immunological reactions.

[0008] A complex of interferon with a monoclonal antibody to increase the serum half-life of interferon has been described in U.S. Pat. No. 5,055,289. Such approach has the inherent risk of impairing the biological activity of the interferon since the size of the construct raises the problem of steric hindrance.

THE AIMS OF THE PRESENT INVENTION

[0009] It is an aim of the present invention to provide therapeutic heterospecific antibody polypeptide constructs which overcome the problems of therapeutic antibodies of the art namely, low half-life in vivo, poor folding, low expression, and poor stability. It is a further aim of the present invention to provide methods for providing said heterospecific antibodies.

SUMMARY OF THE INVENTION

[0010] One-embodiment of the present invention is a polypeptide construct comprising: [0011] at least one single domain antibody directed against a therapeutic and/or diagnostic target, and [0012] at least one single domain antibody directed against a serum protein.

[0013] Another embodiment of the present invention is a polypeptide construct as described above wherein: [0014] the number of anti-target single domain antibodies is at least two, and [0015] at least two anti-target single domain antibodies do not share the same sequence, or all the anti-target single domain antibodies share the same sequence.

[0016] One embodiment of the present invention is a polypeptide construct as described above wherein: [0017] the number of anti-serum protein single domain antibodies is at least two, and [0018] at least two anti-serum-protein single domain antibodies do not share the same sequence, or all the anti-serum-protein single domain antibodies share the same sequence.

[0019] One embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody is a Camelidae VHHs antibody.

[0020] One embodiment of the present invention is a polypeptide construct as described above wherein at least one single domain antibody is a humanised Camelidae VHHs antibody.

[0021] One embodiment of the present invention is a polypeptide construct as described above wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.

[0022] One embodiment of the present invention is a polypeptide construct as described above wherein a single domain anti-serum protein antibody correspond to a sequence represented by any of SEQ ID NOS: 1 to 4, and 28 to 40.

[0023] One embodiment of the present invention is a polypeptide construct as described above wherein a target is TNF-alpha-alpha.

[0024] One embodiment of the present invention is a polypeptide construct as described above corresponding to the sequence represented by any of SEQ ID NO: 5 to 18.

[0025] One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.

[0026] One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.

[0027] One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.

[0028] One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.

[0029] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said disorders are any of rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.

[0030] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

[0031] One embodiment of the present invention is a polypeptide construct as described above wherein a target is vWF

[0032] One embodiment of the present invention is a polypeptide construct as described above wherein a target is collagen.

[0033] One embodiment of the present invention is a polypeptide construct as described above wherein at least one anti-target single domain antibody is anti-vWF VHHs.

[0034] One embodiment of the present invention is a polypeptide construct as described above corresponding to the sequence represented by any of SEQ ID NOS: 19 to 21.

[0035] One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.

[0036] One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.

[0037] One embodiment of the present invention is a polypeptide construct as described above or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.

[0038] One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.

[0039] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis, and said conditions are those arising from coronary by-pass graft, or coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.

[0040] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

[0041] One embodiment of the present invention is a polypeptide construct as described above wherein a target is IgE.

[0042] One embodiment of the present invention is a polypeptide construct as described above wherein at least anti-target single domain antibody is anti-IgE VHHs.

[0043] One embodiment of the present invention is a polypeptide construct as described above corresponding to the sequence represented by any of SEQ ID NOS: 22 to 24.

[0044] One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.

[0045] One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.

[0046] One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions.

[0047] One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to allergic reactions.

[0048] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.

[0049] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

[0050] One embodiment of the present invention is a polypeptide construct as described above wherein a target is IFN-gamma.

[0051] One embodiment of the present invention is a polypeptide construct as described above wherein at least one anti-target single domain antibody is anti-IFN-gamma VHHs.

[0052] One embodiment of the present invention is a polypeptide construct as described above corresponding to a sequence represented by SEQ ID NOS: 25 to 27.

[0053] One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion of said polypeptide construct, or an homologous sequence of a functional portion of said polypeptide construct.

[0054] One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.

[0055] One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active.

[0056] One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions wherein the immune system is over-active.

[0057] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct or nucleic acid as described above wherein said disorders are any of Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.

[0058] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above or a use of a polypeptide construct as described above wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

[0059] One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.

[0060] One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.

[0061] One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid encoding said polypeptide construct and a pharmaceutically acceptable vehicle.

[0062] One embodiment of the present invention is a polypeptide construct as described above directed against a single target wherein said target is involved in a disease process.

[0063] One embodiment of the present invention is a polypeptide construct as described above, wherein said polypeptide construct is a homologous sequence of said polypeptide construct, a functional portion thereof, of an homologous sequence of a functional portion thereof.

[0064] One embodiment of the present invention is a nucleic acid encoding a polypeptide construct as described above.

[0065] One embodiment of the present invention is a polypeptide construct as described above, or a nucleic add as described above for use in the treatment, prevention and/or alleviation of disorders or conditions in which the target is involved.

[0066] One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions in which the target is involved.

[0067] One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation.

[0068] One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation.

[0069] One embodiment of the present invention is a polypeptide construct as described above, or a nucleic acid as described above for use in treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time.

[0070] One embodiment of the present invention is a use of a polypeptide construct as described above, or a nucleic acid as described above for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disease requiring a therapeutic or diagnostic compound which is remains active in the circulation for extended periods of time.

[0071] One embodiment of the present invention is a polypeptide construct or nucleic acid as described above, or use of a polypeptide construct or nucleic acid as described above, wherein said polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally, subcutaneously or by inhalation.

[0072] One embodiment of the present invention is a composition comprising a polypeptide construct as described above, or a nucleic acid as described above and a pharmaceutically acceptable vehicle.

[0073] One embodiment of the present invention is a method of producing a as described above comprising

[0074] (a) culturing host cells comprising nucleic acid capable of encoding a polypeptide as described above, under conditions allowing the expression of the polypeptide, and,

[0075] (b) recovering the produced polypeptide from the culture.

[0076] One embodiment of the present invention is a method as described above, wherein said host cells are bacterial or yeast.

[0077] One embodiment of the present invention is a method for prolonging the half-life of a single domain antibody in the blood stream of a subject, said antibody directed against a therapeutic and/or diagnostic target by joining thereto one or more single domain antibodies directed against a serum protein.

[0078] One embodiment of the present invention is a method as described above wherein said anti-target single domain antibodies do not share the same sequence.

[0079] One embodiment of the present invention is a method as described above wherein said anti-serum protein single domain antibodies do not share the same sequence.

[0080] One embodiment of the present invention is a method as described above wherein said single domain antibodies are Camelidae VHH antibodies.

[0081] One embodiment of the present invention is a method as described above wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.

[0082] One embodiment of the present invention is a method as described above wherein said serum protein comprises a sequence corresponding to any of SEQ ID NOS: 1 to 4, a homologous sequence, a functional portion thereof, or a homologous sequence of a functional portion thereof.

[0083] One embodiment of the present invention is a composition comprising a polypeptide as described above or a nucleic acid capable of encoding said polypeptide and a pharmaceutically acceptable vehicle.

BRIEF DESCRIPTION OF FIGURES AND TABLES

[0084] FIG. 1 phage ELISA to show that HSA-specific nanobodies are present in the library as described in Example 4.

[0085] FIG. 2 Binding of phages expressing the albumin binders, to plasma blotted on nitrocellulose as described in Example 8.

[0086] FIG. 3 Coomassie staining of plasma samples on SDS-PAGE as described in example 8.

[0087] FIG. 4 Binding of purified nanobodies to mouse albumin as determined by ELISA as described in Example 10.

[0088] FIG. 5 Multiple cloning site of PAX011 for construction of bispecific nanobodies as described in Example 11.

[0089] FIG. 6 Sandwich ELISA to show the functionality of both nanobodies in the bispecific construct as described in Example 12.

[0090] FIG. 7 Optimization of ELISA to determine nanobody concentration in 10% plasma or in 10% blood as described in Example 14.

[0091] FIG. 8 Pharmacokinetics for the monovalent anti-TNF-.alpha. nanobody in mice as determined by ELISA as described in Example 16.

[0092] FIG. 9 Pharmacokinetics for the bispecific nanobody MSA21/TNF3E in mice as determined by ELISA as described in Example 16.

[0093] FIG. 10 Pharmacokinetics for the bispecific nanobody MSA21/TNF3E in mice as determined by ELISA with K208 as compared to URL49 as described in Example 16.

[0094] FIG. 11 Pharmacokinetics for the bispecific nanobody MSA24/TNF3E in mice as determined by ELISA as described in Example 16.

[0095] FIG. 12 Binding to vWF as determined by ELISA, by purified VHH as described in Example 23.

[0096] FIG. 13 ELISA to test inhibition by VHH of binding of vWF to collagen as described in Example 24.

[0097] FIG. 14 Sandwich ELISA showing the functionality of both VHHs in a bispecific construct as described in example 27.

[0098] Table 1 Immunization scheme according to Example 1

[0099] Table 2 Results after one and two rounds of panning on mouse serum albumin as described in example 5.

[0100] Table 3 Clones were selected after one and two rounds of selection and periplasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 6.

[0101] Table 4 Sequence listing

[0102] Table 5 Affinities (koff, kon and KD) for albumin binders as determined by BIACORE as described in Example 13.

[0103] Table 6 Results for the LAL-assay for monovalent and bispecific nanobodies after purification on polymyxin as described in Example 15.

[0104] Table 7 Immunization scheme used for llama 002 according to Example 17.

[0105] Table 8 Plaque forming units (pfu) after one or two round(s) of panning on vWF as compared to PBS-casein as described in example 19. Pfu vWF (antigen) divided by pfu casein (a specific binding)=enrichment.

[0106] Table 9 Number of inhibitors versus the number of clones tested after the first and the second round of panning as described in Example 20.

[0107] Table 10 Concentration of VHH (nM) needed to inhibit binding of vWF to collagen by 50% (IC50) as described in Example 23.

[0108] Table 11 IC50 values for bispecific nanobodies against albumin and against vWF as described in Example 28.

[0109] Table 12 Fractional homologies between the amino acid sequences of anti-mouse serum albumin VHHs of the invention.

[0110] Table 13 Fractional homologies between anti-TNF-alpha VHHs of the invention.

[0111] Table 14 Percentage homologies between anti-IFN-gamma VHHs of the invention.

[0112] Table 15 Fractional homologies between anti-vWF VHHs of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0113] The present invention relates to a heterospecific polypeptide construct comprising one or more single domain antibodies each directed against a serum protein(s) of a subject, and one or more single domain antibodies each directed against a target molecule(s) and the finding that the construct has a significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-target single domain antibody when not part of such a construct.

[0114] Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. According to one aspect of the invention, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678 for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.

[0115] The one or more single domain antibodies of the polypeptide construct which are directed against a target may be of the same sequence. Alternatively they may not all have the same sequence. It is within the scope of the invention that a heterospecific polypeptide construct comprises anti-target single domain antibodies which do not all share the same sequence, but which are directed against the same target, or fragment thereof, one or more antigens thereof.

[0116] In accordance with the present invention there are provided methods for the utilization of a plurality of anti-target and/or anti-serum protein single domain antibodies to increase the avidity and/or affinity of the heterospecific molecule. In this manner, serum half-lives of molecules modified in accordance with the invention can be extended. Such modification will modify and/or extend the therapeutic window of a specific therapeutic molecule. This flexibility cannot be achieved with alternative methods in the art, such as when using peptides with specificity to serum proteins, diabodies which are difficult to produce in a multivalent form, chemical modifications (such as pegylation, acylation).

[0117] The one or more single domain antibodies of the polypeptide construct which are directed against a serum protein may be of the same sequence. Alternatively they may not all have the same sequence. It is within the scope of the invention that a heterospecific polypeptide construct comprises anti-serum protein single domain antibodies which do not all share the same sequence, but which are directed against serum protein, or fragment thereof, one or more antigens thereof.

[0118] In another embodiment, one or more anti-target single domain antibodies of the polypeptide construct may be directed to more than one target (e.g. vWF and collagen). Similarly, the anti-serum protein single domain antibodies of the polypeptide construct may be directed against more than one serum protein (e.g. serum albumin and fibrinogen).

[0119] VHHs, according to the present invention, and as known to the skilled addressee are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains such as those derived from Camelids as described in WO9404678 (and referred to hereinafter as VHH domains or nanobodies). VHH molecules are about 10.times. smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs. In addition, antibodies generated in Camelids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunisation of mammals other than Camelids (WO 9749805). As such, anti-albumin VHH's may interact in a more efficient way with serum albumin which is known to be a carrier protein. As a carrier protein some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds. Since VHH's are known to bind into `unusual` or non-conventional epitopes such as cavities (WO9749805), the affinity of such VHH's to circulating albumin may be increased.

[0120] The present invention also relates to the finding that a heterospecific polypeptide construct comprising one or more VHHs directed against one or more serum proteins of a subject, and one or more VHHs directed against one or more target molecule of said subject surprisingly has significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-target VHH when not part of said construct. Furthermore, such prolonged half-life is in the range of several days due to the high affinity anti-serum albumin VHH's compared to several hours when using low affinity peptides specific for albumin (Dennis et al, JBC, 277, 35035). The extension of the half-life is demonstrated by the inventors herein, for example, in Example 16, and by the polypeptide represented by SEQ ID NO: 5. Furthermore, the said construct was found to exhibit the same favourable properties of VHHs such as high stability remaining intact in mice for at least 19 days (Example 16), extreme pH resistance, high temperature stability and high target affinity.

[0121] A target according to the invention is any biological substance capable of binding to a heterospecific polypeptide construct of the invention. Targets may be, for example, proteins, peptides, nucleic acids, oligonucleic acids, saccharides, polysaccharides, glycoproteins. Examples include, but are not limited to therapeutic targets, diagnostic targets, receptors, receptor ligands, viral coat proteins, immune system proteins, hormones, enzymes, antigens, cell signaling proteins, or a fragment thereof. Targets may be native protein or a fragment thereof, a homologous sequence thereof, a functional portion thereof, or a functional portion of an homologous sequence.

[0122] The properties of single domain antibodies, in particular VHHs, compare favourably with those of antibodies derived from sources such as mouse, sheep, goat, rabbit etc. (i.e. traditional antibodies), and humanised derivatives thereof. Traditional antibodies are not stable at room temperature, and have to be refrigerated for preparation and storage, requiring necessary refrigerated laboratory equipment, storage and transport, which contribute towards time and expense. Refrigeration is sometimes not feasible in developing countries. Furthermore, the manufacture or small-scale production of said antibodies is expensive because the mammalian cellular systems necessary for the expression of intact and active antibodies require high levels of support in terms of time and equipment, and yields are very low. Furthermore, traditional antibodies have a binding activity which depends upon pH, and hence are unsuitable for use in environments outside the usual physiological pH range such as, for example, in treating gastric bleeding, gastric surgery. Furthermore, traditional antibodies are unstable at low or high pH and hence are not suitable for oral administration.

[0123] However, it has been demonstrated that VHHs resist harsh conditions, such as extreme pH, denaturing reagents and high temperatures (Ewert S et al, Biochemistry 2002 Mar 19;41 (11):3628-36), so making them suitable for delivery by oral administration. Furthermore, traditional antibodies have a binding activity which depends upon temperature, and hence are unsuitable for use in assays or kits performed at temperatures outside biologically active-temperature ranges (e.g. 37.+-.20.degree. C.).

[0124] Furthermore VHHs are more soluble, meaning they may be stored and/or administered in higher concentrations compared with conventional antibodies. The polypeptides of the present invention also retain binding activity at a pH and temperature outside those of usual physiological ranges, which means they may be useful in situations of extreme pH and temperature which require a modulation of platelet-mediated aggregation, such as in gastric surgery, control of gastric bleeding, assays performed at room temperature etc. The polypeptides of the present invention also exhibit a prolonged stability at extremes of pH, meaning they would be suitable for delivery by oral administration. The polypeptides of the present invention may be cost-effectively produced through fermentation in convenient recombinant host organisms such as Escherichia coli and yeast; unlike conventional antibodies which also require expensive mammalian cell culture facilities, achievable levels of expression are high. Examples of yields of the polypeptides of the present invention are 1 to 10 mg/ml (E. coli) and up to 1 g/l (yeast). The polypeptides of the present invention also exhibit high binding affinity for a broad range of different antigen types, and ability to bind to epitopes not recognised by conventional antibodies; for example they display long CDR-based loop structures with the potential to penetrate into cavities and exhibit enzyme function inhibition. Furthermore, since binding often occurs through the CDR3 loop only, it is envisaged that peptides derived from CDR3 could be used therapeutically (Desmyter et al, J Biol Chem, 2001, 276: 26285-90). The polypeptides of the invention are also able to retain full binding capacity as fusion protein with an enzyme or toxin.

[0125] The present invention also relates to a heterospecific polypeptide construct comprising one or more VHHs each directed against one or more serum proteins of a subject, and one or more VHH each directed against one or more target molecules wherein the VHHs belong to the traditional class of Camelidae single domain heavy chain antibodies. The present invention also relates to a heterospecific polypeptide construct comprising one or more VHH each directed against one or more serums protein of a subject, and one or more VHH each directed against one or more target molecules wherein the VHHs belong to a class of Camelidae single domain heavy chain antibodies that have human-like sequences. A VHH sequence represented by SEQ ID NO: 12 which binds to TNF-alpha and a second VHH which binds to mouse albumin, belongs to this class of VHH peptides. As such, peptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to patients directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization.

[0126] A human-like class of Camelidae single domain antibodies represented by SEQ ID No. 1, 3 and 4 have been described in WO03035694 and contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation.

[0127] Therefore, one aspect of the present invention allows for the direct administration of an anti-serum albumin polypeptide, wherein the single domain antibodies belong to the humanized class of VHH, and comprise a sequence represented by any of SEQ ID NO: 1, 3 or 4 to a patient in need of the same.

[0128] A subject as used herein is any mammal having a circulatory system in which the fluid therein comprises serum proteins. Examples of circulatory system include blood and lymphatic systems. Examples of animals include, but are not limited to, rabbits, humans, goats, mice, rats, cows, calves, camels, llamas, monkeys, donkeys, guinea pigs, chickens, sheep, dogs, cats, horses etc.

[0129] One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a therapeutic and/or diagnostic target, and at least one single domain antibodies each directed against one or more serum proteins or polypeptides. As already mentioned, the anti-target single domain antibodies may have the same sequence. Alternatively, at least two anti-target single domain antibodies may have the different sequences, but are directed against the same epitope or different epitopes on the same target, fragments thereof, or antigen thereof. Similarly, the anti-serum protein single domain antibodies may have the same sequence. Alternatively, at least two anti-serum protein single domain antibodies may have the different sequences, but are directed against the same epitope or different epitopes on the same serum protein, fragments thereof, or antigen thereof.

[0130] In another embodiment of the present invention, where more than one anti-target single domain antibodies is present in the heterospecific polypeptide construct, each anti-target single domain antibody may be directed to a different target (e.g. one to vWF and one to collagen). Similarly, where more than one anti-serum protein single domain antibody is present, each anti-serum single domain antibody may be directed to a different serum protein (e.g. one to serum albumin and one to fibrinogen).

[0131] One embodiment of the invention, is a heterospecific polypeptide, wherein an anti-serum protein single domain antibody corresponds to a sequence represented by any of SEQ ID NOS:1 to 4 and 28 to 40.

[0132] The constructs disclosed herein retain the advantageous properties of single domain antibodies (e.g. VHHs) and have a prolonged lifetime in the circulation of an individual. Thus, such constructs are able to circulate in the subject's serum for several days, reducing the frequency of treatment, the inconvenience to the subject and resulting in a decreased cost of treatment. Furthermore, it is an aspect of the invention that the half-life of the heterospecific polypeptide constructs may be controlled by the number of anti-serum protein single domain antibodies present in the construct. A controllable half-life is desirable in several circumstances, for example, in the application of a timed dose of a therapeutic heterospecific polypeptide construct, or to obtain a desired therapeutic effect.

[0133] According to an aspect of the Invention a heterospecific polypeptide construct may be a homologous sequence of a full-length heterospecific polypeptide construct. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion of a full-length heterospecific polypeptide construct. According to another aspect of the invention, a heterospecific polypeptide construct may be a homologous sequence of a full-length heterospecific polypeptide construct. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion of a homologous sequence of a full-length heterospecific polypeptide construct. According to an aspect of the invention a heterospecific polypeptide construct may comprise a sequence of a heterospecific polypeptide construct.

[0134] According to an aspect of the invention a single domain antibody used to form a heterospecific polypeptide construct may be a complete single domain antibody (e.g. a VHH) or a homologous sequence thereof. According to another aspect of the invention, a single domain antibody used to form the heterospecific polypeptide construct may be a functional portion of a complete single domain antibody. According to another aspect of the invention, a single domain antibody used to form the heterospecific polypeptide construct may be a homologous sequence of a complete single domain antibody. According to another aspect of the invention, a single domain antibody used to form the heterospecific polypeptide construct may be a functional portion of a homologous sequence of a complete single domain antibody.

[0135] According to another aspect of the invention a heterospecific polypeptide construct may be an homologous sequence of the parent sequence. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion parent sequence. According to another aspect of the invention, a heterospecific polypeptide construct may be a functional portion of a homologous sequence of the parent sequence.

[0136] As used herein, an homologous sequence of the present invention may comprise additions, deletions or substitutions of one or more amino acids, which do not substantially alter the functional characteristics of the polypeptides of the invention. The number of amino acid deletions or substitutions is preferably up to 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

[0137] A homologous sequence of the present invention may include a single domain antibody of the invention which has been humanised.

[0138] By humanised is meant mutated so that immunogenicity upon administration in human patients is minor or nonexistent. Humanising a single domain antibody, according to the present invention, comprises a step of replacing one or more of amino acids by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e. the humanisation does not significantly affect the antigen binding capacity of the resulting polypeptide. Such methods are known by the skilled addressee. A humanisation technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of any of the following residues either alone or in combination: some VHH contain typical Camelidae hallmark residues at position 37, 44, 45 and 47 with hydrophilic characteristics. Replacement of the hydrophilic residues by human hydrophobic residues at positions 44 and 45 (E44G and R45L) did not have an effect on binding and/or inhibition. Further humanization may be required by substitution of residues in FR 1, such as position 1, 5, 28 and 30; FR3, such as positions 74, 75, 76, 83, 84, 93 and 94; and FR4, such as position 103, 104, 108 and 111 (all numbering according to the Kabat).

[0139] One embodiment of the present invention is a method for humanizing a VHH comprising the steps of replacing of any of the following residues either alone or in combination: [0140] FR1 position 1, 5, 28 and 30, [0141] the hallmark amino acid at position 44 and 45 in FR2, [0142] FR3 residues 74, 75, 76, 83, 84, 93 and 94, [0143] and positions 103, 104, 108 and 111 in FR4; (numbering according to the Kabat numbering).

[0144] Some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanisation. Therefore, one aspect of the present invention allows for the formation of a heterospecific polypeptide construct without humanisation of the VHH, when said VHH exhibit high homology to human VH framework regions.

[0145] A homologous sequence of the present invention may be a sequence of the invention derived from another species such as, for example, camel, llama, dromedary, alpaca, guanaco etc.

[0146] Where homologous sequence indicates sequence identity, it means a sequence which presents a high sequence identity (more than 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with a single domain antibody of the invention, and is preferably characterised by similar properties of the parent sequence, namely affinity, said identity calculated using known methods.

[0147] A homologous sequence according to the present invention may refer to nucleotide sequences of more than 50, 100, 200, 300, 400, 500, 600, 800 or 1000 nucleotides able to hybridise to the reverse-complement of the nucleotide sequence capable of encoding a native sequence under stringent hybridisation conditions (such as the ones described by SAMBROOK et al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratory press, New York).

[0148] As used herein, a functional portion refers to a single domain antibody of sufficient length such that the interaction of interest is maintained with affinity of 1.times.10.sup.-6 M or better.

[0149] Alternatively a functional portion of a single domain antibody of the invention comprises a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and protein domain(s) necessary for the binding of and interaction with the target or serum protein.

[0150] As used herein, a functional portion of a single domain antibody of the invention refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but comprising 5 or more amino acids or 15 or more nucleotides.

[0151] A portion of a single domain antibody of the invention refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.), but comprising 5 or more amino acids or 15 or more nucleotides.

[0152] Targets as mentioned herein such as TNF-alpha, IFN-gamma receptor, serum proteins (e.g. serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, fibrinogen) and IFN-gamma may be fragments of said targets. Thus a target is also a fragment of said target, capable of eliciting an immune response. A target is also a fragment of said target, capable of binding to a single domain antibody raised against the full length target.

[0153] A fragment as used herein refers to less than 100% of the sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. A fragment is of sufficient length such that the interaction of interest is maintained with affinity of 1.times.10.sup.-6 M or better.

[0154] A fragment as used herein also refers to optional insertions, deletions and substitutions of one or more amino acids which do not substantially alter the ability of the target to bind to a single domain antibody raised against the wild-type target. The number of amino acid insertions deletions or substitutions is preferably up to 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

[0155] The serum protein may be any suitable protein found in the serum of subject, or fragment thereof. In one aspect of the invention, the serum protein is serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen. Depending on the intended use such as the required half-life for effective treatment and/or compartimentalisation of the target antigen, the VHH-partner can be directed to one of the above serum proteins.

[0156] A single domain antibody directed against a target means single domain antibody that it is capable of binding to its target with an affinity of better than 10.sup.-6 M.

[0157] The heterospecific polypeptide constructs disclosed herein may be made by the skilled artisan according to methods known in the art or any future method. For example, VHHs may be obtained using methods known in the art such as by immunising a camel and obtaining hybridomas therefrom, or by cloning a library of single domain antibodies using molecular biology techniques known in the art and subsequent selection by using phage display.

[0158] The anti-serum protein single domain antibody may be directed against a polypeptide of a serum protein or a whole protein. The anti-target single domain antibody may be directed against a polypeptide of said target of the whole target. Methods for scanning a protein for immunogenic polypeptides are well known in the art.

[0159] The single domain antibodies may be joined using methods known in the art or any future method. For example, they may be fused by chemical cross-linking by reacting amino acid residues with an organic derivatising agent such as described by Blattler et al, Biochemistry 24,1517-1524; EP294703. Alternatively, the single domain antibody may be fused genetically at the DNA level i.e. a polynucleotide construct formed which encodes the complete polypeptide construct comprising one or more anti-target single domain antibodies and one or more anti-serum protein single domain antibodies. A method for producing bivalent or multivalent VHH polypeptide constructs is disclosed in PCT patent application WO 96/34103. One way of joining multiple single domain antibodies is via the genetic route by linking single domain antibody coding sequences either directly or via a peptide linker. For example, the C-terminal end of the first single domain antibody may be linked to the N-terminal end of the next single domain antibody. This linking mode can be extended in order to link additional single domain antibodies for the construction and production of tri-, tetra-, etc. functional constructs.

[0160] An aspect of the present invention is the administration of heterospecific polypeptide constructs according to the invention which avoids the need for injection. Conventional antibody-based therapeutics have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity, however, they have one important drawback: these are complex, large molecules and therefore relatively unstable, and they are sensitive to breakdown by proteases. This means that conventional antibody drugs cannot be administered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation because they are not resistant to the low pH at these sites, the action of proteases at these sites and in the blood and/or because of their large size. They have to be administered by injection (intravenously, subcutaneously, etc.) to overcome some of these problems. Administration by injection requires specialist training in order to use a hypodermic syringe or needle correctly and safely. It further requires sterile equipment, a liquid formulation of the therapeutic polypeptide, vial packing of said polypeptide in a sterile and stable form and, of the subject, a suitable site for entry of the needle. Furthermore, subjects commonly experience physical and psychological stress prior to and upon receiving an injection. An aspect of the present invention overcomes these problems of the prior art, by providing the heterospecific polypeptides constructs of the present invention. Said constructs are sufficiently small, resistant and stable to be delivered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation substantial without loss of activity. The heterospecific polypeptides constructs of the present invention avoid the need for injections, are not only cost/time savings, but are also more convenient and more comfortable for the subject.

[0161] One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the gastric environment without being inactivated.

[0162] As known by persons skilled in the art, once in possession of said polypeptide construct, formulation technology may be applied to release a maximum amount of VHHs in the right location (in the stomach, in the colon, etc.). This method of delivery is important for treating, prevent and/or alleviate the symptoms of disorder whose targets that are located in the gut system.

[0163] An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder susceptible to modulation by a therapeutic compound that is able pass through the gastric environment without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies specific for antigen related to the disorder.

[0164] Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the gastric environment without being inactivated.

[0165] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the gut system without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0166] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by orally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0167] Another embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the vaginal and/or rectal tract.

[0168] In a non-limiting example, a formulation according to the invention comprises a heterospecific polypeptide construct as disclosed herein comprising one or more VHHs directed against one or more targets in the form of a gel, cream, suppository, film, or in the form of a sponge or as a vaginal ring that slowly releases the active ingredient over time (such formulations are described in EP 707473, EP 684814, U.S. Pat. No. 5,629,001).

[0169] An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound to the vaginal and/or rectal tract, by vaginally and/or rectally administering to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies specific for antigen related to the disorder.

[0170] Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the vaginal and/or rectal tract without being inactivated.

[0171] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the vaginal and/or rectal tract without being inactivated, by administering to the vaginal and/or rectal tract of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0172] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering to the vaginal and/or rectal tract of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0173] Another embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target comprising at least one single domain antibody directed against a target, for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the nose, upper respiratory tract and/or lung.

[0174] In a non-limiting example, a formulation according to the invention, comprises a heterospecific polypeptide construct as disclosed herein directed against one or more targets in the form of a nasal spray (e.g. an aerosol) or inhaler. Since the construct is small, it can reach its target much more effectively than therapeutic IgG molecules.

[0175] An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound delivered to the upper respiratory tract and lung, by administering to a subject a heterospecific polypeptide construct as disclosed herein wherein one or more single domain antibodies are specific for an antigen related to the disorder, by inhalation through the mouth or nose.

[0176] Another aspect of the invention is a dispersible VHH composition, in particular dry powder dispersible VHH compositions, such as those described in U.S. Pat. No. 6,514,496. These dry powder compositions comprise a plurality of discrete dry particles with an average particle size in the range of 0.4-10 mm. Such powders are capable of being readily dispersed in an inhalation device. VHH's are particularly suited for such composition as lyophilized material can be readily dissolved (in the lung subsequent to being inhaled) due to its high solubilisation capacity (Muyldermans, S., Reviews in Molecular Biotechnology, 74, 277-303, (2001)). Alternatively, such lyophilized VHH formulations can be reconstituted with a diluent to generate a stable reconstituted formulation suitable for subcutaneous administration. For example, anti-IgE antibody formulations (Example 1; U.S. Pat. No. 6,267,958, EP 841946) have been prepared which are useful for treating allergic asthma.

[0177] Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the nose, upper respiratory tract and/or lung without being inactivated.

[0178] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the nose, upper respiratory tract and lung, by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0179] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the nose, upper respiratory tract and/or lung without being inactivated, by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0180] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated by administering to the nose, upper respiratory tract and/or lung of a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0181] One embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa. Because of their small size, a heterospecific polypeptide construct as disclosed herein can pass through the intestinal mucosa and reach the bloodstream more efficiently in subjects suffering from disorders which cause an increase in the permeability of the intestinal mucosa.

[0182] An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa, by orally administering to a subject a heterospecific polypeptide construct as disclosed herein.

[0183] This process can be even further enhanced by an additional aspect of the present invention--the use of active transport carriers. In this aspect of the invention, VHH is fused to a carrier that enhances the transfer through the intestinal wall into the bloodstream. In a non-limiting example, this "carrier" is a second VHH which is fused to the therapeutic VHH. Such fusion constructs are made using methods known in the art. The "carrier" VHH binds specifically to a receptor on the intestinal wall which induces an active transfer through the wall.

[0184] Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa.

[0185] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the intestinal mucosa without being inactivated, by administering orally to a subject a heterospecific polypeptide construct of the invention.

[0186] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering orally to a subject a heterospecific polypeptide construct of the invention.

[0187] This process can be even further enhanced by an additional aspect of the present invention--the use of active transport carriers. In this aspect of the invention, a heterospecific polypeptide construct as described herein is fused to a carrier that enhances the transfer through the intestinal wall into the bloodstream. In a non-limiting example, this "carrier" is a VHH which is fused to said polypeptide. Such fusion constructs made using methods known in the art. The "carriers" VHH binds specifically to a receptor on the intestinal wall which induces an active transfer through the wall.

[0188] One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody directed against a target for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the tissues beneath the tongue effectively. A formulation of said polypeptide construct as disclosed herein, for example, a tablet, spray, drop is placed under the tongue and adsorbed through the mucus membranes into the capillary network under the tongue.

[0189] An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound that is able pass through the tissues beneath the tongue effectively, by sublingually administering to a subject a VHH specific for an antigen related to the disorder.

[0190] Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able to pass through the tissues beneath the tongue.

[0191] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the tissues beneath the tongue without being inactivated, by administering orally to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0192] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject without being inactivated, by administering orally to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0193] One embodiment of the present invention is a heterospecific polypeptide construct comprising at least one single domain antibody for use in treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the skin effectively. A formulation of said polypeptide construct, for example, a cream, film, spray, drop, patch, is placed on the skin and passes through.

[0194] An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a therapeutic compound that is able pass through the skin effectively, by topically administering to a subject a heterospecific polypeptide construct as disclosed herein comprising one or more single domain antibodies specific for an antigen related to the disorder.

[0195] Another aspect of the invention is the use of a heterospecific polypeptide construct as disclosed herein as a topical ophthalmic composition for the treatment of ocular disorder, such as allergic disorders, which method comprises the topical administration of an ophthalmic composition comprising polypeptide construct as disclosed herein, said construct comprising one or more anti-IgE VHH (Example 1, Example 2).

[0196] Another embodiment of the present invention is a use of a heterospecific polypeptide construct as disclosed herein for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by an anti-target therapeutic compound that is able pass through the skin effectively.

[0197] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the skin without being inactivated, by administering topically to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0198] An aspect of the invention is a method for delivering an anti-target therapeutic compound to the bloodstream of a subject, by administering topically to a subject a heterospecific polypeptide construct comprising one or more single domain antibodies directed against said target.

[0199] In another embodiment of the present invention, a heterospecific polypeptide construct further comprises a carrier single domain antibody (e.g. VHH) which acts as an active transport carrier for transport said heterospecific polypeptide construct, the lung lumen to the blood.

[0200] A polypeptide construct further comprising a carrier binds specifically to a receptor present on the mucosal surface (bronchial epithelial cells) resulting in the active transport of the polypeptide from the lung lumen to the blood. The carrier single domain antibody may be fused to the polypeptide construct. Such fusion constructs made using methods known in the art and are describe herein. The "carrier" single domain antibody binds specifically to a receptor on the mucosal surface which induces an active transfer through the surface.

[0201] Another aspect of the present invention is a method to determine which single domain antibodies (e.g. VHHs) are actively transported into the bloodstream upon nasal administration. Similarly, a naive or immune VHH phage library can be administered nasally, and after different time points after administration, blood or organs can be isolated to rescue phages that have been actively transported to the bloodstream. A non-limiting example of a receptor for active transport from the lung lumen to the bloodstream is the Fc receptor N (FcRn). One aspect of the invention includes the VHH molecules identified by the method. Such VHH can then be used as a carrier VHH for the delivery of a therapeutic VHH to the corresponding target in the bloodstream upon nasal administration.

[0202] One embodiment of the present invention is a heterospecific polypeptide construct for use in treating, preventing and/or alleviating the symptoms of disorders requiring the delivery of a therapeutic compound intraveneously. An aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of disorders requiring the delivery of a therapeutic compound via the bloodstream.

[0203] Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation. An aspect of the invention is the use of a said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which is not rapidly cleared from the circulation by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof. By using a heterospecific polypeptide construct of the present invention to treat or diagnose an aforementioned disorder, the depletion of said construct is retarded.

[0204] Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time. An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound which remains active in the circulation for extended periods of time. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder requiring a therapeutic or diagnostic compound that is able to circulate in the patients serum for several days, by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof. By using a heterospecific polypeptide construct of the present invention to treat or diagnose an aforementioned disorder, the frequency of treatment is reduced, so resulting in a decreased cost of treatment.

[0205] Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder relating to allergies. An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder relating to allergies. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder relating to allergies, by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, the anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.

[0206] The above aspects and embodiments of the invention also apply when an anti-serum single domain antibody of the aforementioned heterospecific polypeptide constructs corresponds to a sequence represented by SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion.

[0207] The above aspects and embodiments of the invention also apply when a heterospecific polypeptide construct of the invention corresponds to a sequence represented by any of SEQ ID NOS: 5 to 18, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion. Said sequences comprise an anti-TNF-alpha Camelidae VHH.

[0208] The above aspects and embodiments of the invention also apply when an heterospecific polypeptide constructs of the invention corresponds to a sequence represented by any of SEQ ID NOS: 19 to 21 a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion. Said sequences comprise an anti-vWF Camelidae VHH.

[0209] The above aspects and embodiments of the invention also apply when an heterospecific polypeptide constructs of the invention corresponds to a sequence represented by any of SEQ ID NOS: 22 to 24 a homologous sequence thereof, a functional portion thereof. Said sequences comprise an anti-IgE Camelidae VHH.

[0210] The above aspects and embodiments of the invention also apply when an heterospecific polypeptide construct according to the invention corresponds to a sequence represented by any of SEQ ID NOS:25 to 27, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion. Said sequences comprise an anti-Interferon-gamma Camelidae VHH.

[0211] A non-limiting example, in relation to allergies, of a target against which an anti-target single domain antibody may be directed is IgE. During their lifetime, subjects can develop an allergic response to harmless parasites such as Dermatophagoides pteronyssinus, the house dust mite or to substances such as clumps, plastics, metals. This results in an induction of IgE molecules that initiates a cascade of immunological responses. One aspect of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies. In one aspect of the invention, said anti-IgE single domain antibodies prevents the interaction of IgE with their receptor(s) on mast cells and basophils, so blocking initiation of the immunological cascade and a subsequent allergic reaction. In another aspect an anti-serum protein single domain antibody is directed to one of the subject's serum proteins. A heterospecific polypeptide construct as disclosed herein thus reduces or prevents an allergic response due to common or unusual allergens. Furthermore, the construct has a prolonged lifetime in the blood so increasing the therapeutic window. 7

[0212] Tumor necrosis factor alpha (TNF-alpha) is believed to play an important role in various diseases, for example in inflammatory diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. Both TNF-alpha and the receptors (CD120a, CD120b) have been studied in great detail. TNF-alpha in its bioactive form is a trimer and the groove formed by neighboring subunits is important for the cytokine-receptor interaction. Several strategies to antagonize the action of the cytokine have been developed and are currently used to treat various disease states.

[0213] A TNF inhibitor which has sufficient specificity and selectivity to TNF may be an efficient prophylactic or therapeutic pharmaceutical compound for preventing or treating inflammatory diseases. However, it is extremely difficult and a lengthy process to develop a small chemical entitly (NCE) with sufficient potency and selectivity to such target sequence. Antibody-based therapeutics on the other hand have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. In addition, the development time can be reduced considerably when compared to the development of new chemical entities (NCE's). However, conventional antibodies are difficult to elicit against multimeric proteins where the receptor-binding domain of the ligand is embedded in a groove, as is the case with TNF-alpha.

[0214] The heterospecific polypeptide constructs of the present invention, wherein the anti-target single domain antibody is directed against TNF-alpha overcome the problems experienced using peptide therapeutics of the art because of the properties such as stability, size, and reliable expressioin. Furthermore, the inventors have found that, despite presence of a groove in multimeric TNF-alpha, the heterospecific polypeptide constructs are still able to achieve strong binding to TNF-alpha

[0215] Another embodiment of the present invention is a heterospecific polypeptide construct as disclosed herein for use in treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules. An aspect of the invention is the use of said construct for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules. Another aspect of the invention is a method for treating, preventing and/or alleviating the symptoms of a disorder mediated by inflammatory molecules, by administering a heterospecific polypeptide construct as disclosed herein to an individual. According to the present invention, an anti-target single domain antibody of said heterospecific polypeptide is directed against a target involved in a cause or a manifestation of said disorder, or involved in causing symptoms thereof.

[0216] According to one aspect of the invention, a target against which a single domain antibody of a heterospecific polypeptide construct is directed is tumor necrosis factor alpha (TNF-alpha). TNF-alpha is believed to play an important role in various disorders, for example in inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.

[0217] Anti-target single domain antibodies may be directed against whole TNF-alpha or a fragment thereof, or a fragment of a homologous sequence thereof.

[0218] One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibody fused to one or more anti-serum protein single domain antibody, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 5 to 18. The anti-TNF-alpha single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to TNF-alpha.

[0219] One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of inflammatory disorders. TNF-alpha is involved in inflammatory processes, and the blocking of TNF-alpha action can have an anti-inflammatory effect, which is highly desirable in certain disorder states such as, for example, Crohn's disease. Oral delivery of these heterospecific polypeptide construct results in the delivery of such molecules in an active form in the colon at sites that are affected by the disorder. These sites are highly inflamed and contain TNF-alpha producing cells. These heterospecific polypeptide constructs can neutralise the TNF-alpha locally, avoiding distribution throughout the whole body and thus limiting negative side-effects. Genetically modified microorganisms such as Micrococcus lactis are able to secrete antibody fragments. Such modified microorganisms can be used as vehicles for local production and delivery of antibody fragments in the intestine. By using a strain which produces a TNF-alpha-neutralising heterospecific polypeptide construct, inflammatory bowel disorder could be treated.

[0220] Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0221] Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0222] Another aspect of the invention is a method of treating, preventing and/or alleviating disorders relating to inflammatory processes, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0223] Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.

[0224] Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to inflammatory processes.

[0225] It is an aspect of the invention that the anti-TNF-alpha single domain antibodies of the present invention may be derived from VHHs of any class. For example, they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.

[0226] The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 5 to 18, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof. SEQ ID NOS: 5 to 18 comprise anti-TNF alpha Camelidae VHH and anti-mouse serum albumin Camelidae VHH.

[0227] The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-TNF-alpha single domain antibodies fused to one or more anti-serum protein single domain antibodies wherein said anti-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4 (anti-serum protein Camelidae VHHs), a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.

[0228] The inventors have found that a heterospecific polypeptide construct comprising a sequence corresponding to any of SEQ ID NOS: 5 to 18 surprisingly exhibits higher than expected affinity towards its target and prolonged half-life in the circulatory system.

[0229] Platelet-mediated aggregation is the process wherein von Willebrand Factor (vWF)-bound collagen adheres to platelets and/or platelet receptors (examples of both are gpla/lla, gplb, or collagen), ultimately resulting in platelet activation. Platelet activation leads to fibrinogen binding, and finally to platelet aggregation. The ability to disrupt platelet-mediated aggregation has many applications including the treatment of disease as mentioned below. Since the heterospecific polypeptide constructs of the invention effective prevent clotting, and the half-life thereof is controllable, they may be used for surgical procedures, for example, which require an inhibition of platelet-mediated aggregation for a limited time period.

[0230] Monovalent single domain antibodies such as VHHs show surprisingly high platelet aggregation inhibition in experiments to measure platelet aggregation inhibition under high shear: 50% inhibition of platelet aggregation was obtained at a concentration between 4 and 25 nM. In comparison, the Fab fragment derived from a vWF-specific antibody inhibiting the interaction with collagen, 82D6A3, inhibits 50% of platelet aggregation at approximately a twenty-fold higher concentration (Vanhoorelbeke K. et al, Journal of Biological Chemistry, 2003, 278: 37815-37821). These results were unexpected given that the IC50 values for the monovalent VHH's are up to 225 times fold worse in ELISA then the IC50 value of the IgG of 82D6A3.

[0231] This clearly shows that IgG antibodies is not suited to interaction with macromolecules which are starting, or are in the process of aggregating, such as those involved in platelet-mediated aggregation. vWF makes multimers of up to 60 monomers (final multimers of up to 20 million dalton in size). Indeed, it has been shown that not all A3 domains are accessible to 82D6A3 (Dongmei WU, Blood, 2002, 99, 3623 to 3628). Furthermore the large size of conventional antibodies, would restrict tissue penetration, for example, during platelet-mediated aggregation at the site of a damaged vessel wall.

[0232] The structure of single domain antibodies, in particular is unique. For example VHH molecules derived from Camelidae antibodies are among the smallest intact antigen-binding domains known (approximately 15 kDa, or 10 times smaller than a conventional IgG) and hence are well suited towards delivery to dense tissues and for accessing the limited space between macromolecules participating in or starting the process of platelet mediated aggregation.

[0233] To our knowledge, this is the first time that experiments show, that the small size of a VHH is advantageous over a large intact antibody for inhibition of interactions between such large macromolecules.

[0234] Despite the small size of nanobodies, and thus advantages for penetration, it is still surprising that such a small molecule can inhibit interactions between large polymers such as vWF (up to 60 monomers) and collagen and with such a high efficiency. It has been described that only the large multimeric forms of vWF are hemostatically active (Furian, M,. 1996, Ann. Hematol. 72:341-348). Binding of multimeric vWF to collagen occurs with .about.100-fold higher affinity than binding of monomeric vWF fragments.

[0235] The results from the high shear experiments indicate that a lower dose will be needed for administration to patients. Therefore, fewer side effects are expected (such as immunogenicity or bleeding problems).

[0236] It is an aspect of the present invention to provide heterospecific polypeptide constructs which modulate processes which comprise platelet-mediated aggregation such as, for example, vWF-collagen binding, vWF-platelet receptor adhesion, collagen-platelet receptor adhesion, platelet activation, fibrinogen binding and/or platelet aggregation. Said heterospecific polypeptide constructs are derived from single domain antibodies directed towards vWF, vWF A1 or A3 domains, gplb or collagen.

[0237] Anti-target single domain antibodies may be directed against whole vWF, vWF A1 or A3 domains, gplb or collagen or a fragment thereof, or a fragment of a homologous sequence thereof.

[0238] According to one aspect of the invention, a target against which a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is von Willebrand factor (vWF). According to another aspect of the invention, the target is vWF A1 or A3 domains. According to another aspect of the invention, the target is gplb. According to another aspect of the invention, the target is gpla/llA. According to another aspect of the invention, the target is collagen.

[0239] One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-vWF single domain antibodies fused to one or more anti-serum protein VHHs, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 19 to 21. The anti-vWF single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to vWF.

[0240] One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains, gplb or collagen for use in treating, preventing and/or alleviating the symptoms of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof. Said disorders include transient cerebral ischemic attack, unstable angina pectoris, cerebral infarction, myocardial infarction, peripheral arterial occlusive disease, restenosis. Said conditions include those arising from coronary by-pass graft, coronary artery valve replacement and coronary interventions such angioplasty, stenting, or atherectomy.

[0241] One aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0242] Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0243] Another aspect of the invention is a method of treating, preventing and/or alleviating disorders or conditions relating to relating to platelet-mediated aggregation or dysfunction thereof, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies target, wherein the target is any of vWF, vWF A1 or A3 domains or collagen, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0244] Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for use in the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.

[0245] Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains or collagen for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders or conditions relating to platelet-mediated aggregation or dysfunction thereof.

[0246] It is an aspect of the invention that the anti-vWF, anti-vWF A1 or anti-vWF A3 or anti-collagen VHHs of the present invention may be derived from VHHs of any class. For example, they may be derived from the class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.

[0247] The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-vWF single domain antibodies wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 19 to 21, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof. SEQ ID NOS: 19 to 21 comprise anti-vWF VHH and anti-mouse serum albumin VHH.

[0248] The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the target is any of vWF, vWF A1 or A3 domains, gplb or collagen and wherein said anti-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.

[0249] During their lifetime, subjects may develop an allergic response to harmless parasites (e.g. Dermatophagoides pteronyssinus, house dust mite) or substances (clumps, plastics, metals). This results in the induction of IgE molecules that initiate a cascade of immunological responses. One aspect of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies, said heterospecific polypeptide construct preventing the interaction of IgEs with their receptor(s) on mast cells and basophils. As such they prevent the initiation of the immunological cascade, an allergic reaction.

[0250] According to one aspect of the invention, a target against which a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is IgE. Said antibodies may be directed against whole IgE or a fragment thereof, or a fragment of a homologous sequence thereof.

[0251] One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 22 to 24. The anti-IgE single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to IgE.

[0252] Anti-target single domain antibodies may be directed against whole IgE-alpha or a fragment thereof, or a fragment of a homologous sequence thereof.

[0253] One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibody fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of disorders relating to allergies. Said disorders comprise a wide range of IgE-mediated diseases such as hay fever, asthma, atopic dermatitis, allergic skin reactions, allergic eye reactions and food allergies.

[0254] One aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders relating to allergies, wherein said VHH is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0255] Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0256] Another aspect of the invention is a method of treating, preventing and/or alleviating disorders relating to allergies, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0257] Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies.

[0258] Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders relating to allergies.

[0259] It is an aspect of the invention that the anti-IgE single domain antibodies of the present invention may be derived from VHHs of any class. For example, they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. Said VHHs may be derived from Camelidae. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.

[0260] The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies, wherein the heterospecific polypeptides correspond to a sequence represented by any of SEQ ID NOS: 22 to 24 , a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof. SEQ ID NOS: 22 to 24 comprise anti-IgE Camelidae VHH and anti-mouse serum albumin Camelidae VHH.

[0261] The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IgE single domain antibodies fused to one or more anti-serum protein single domain antibodies wherein said ant-serum protein single domain antibodies correspond to any of SEQ ID NOS: 1 to 4 (ant-protein serum Camelidae VHHs), a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.

[0262] A heterospecific polypeptide construct as disclosed herein prevents thus reduces or prevents an allergic response due to common or unusual allergens. Furthermore, the construct has a prolonged lifetime in the blood so increasing the therapeutic window.

[0263] Interferon gamma (IFN-gamma) is believed to play an important role in various disorders, for example in inflammatory disorders such as rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, multiple sclerosis and hyperimmune reactions in the eye. IFN-gamma has also been shown to play a significant role in the pathology of autoimmune diseases. For example, the presence of IFN-gamma has been implicated in rheumatoid arthritis (Brennan et al, Brit. J. Rheum., 31, 293-8 (1992)). Several strategies to antagonize the action of these cytokines have been developed and are currently used to treat various disease states.

[0264] IFN-gamma in its bioactive form is a dimer and the groove formed by the two subunits is important for its biological activity through interaction with the IFN-gamma receptor. An IFN-gamma inhibitor which has sufficient specificity and selectivity to IFN-gamma may be an efficient prophylactic or therapeutic pharmaceutical compound for preventing or treating inflammatory disorders. Diseases associated with IFN-gamma include multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, and psoriatic arthritis (U.S. Pat. No. 6,333,032 Advanced Biotherapy Concepts, Inc.). Other diseases include Crohn's disease and psoriasis (U.S. Pat. No. 6,329,511 Protein Design Labs). Yet other diseases are bowel disease, ulcerative colitis and Crohn's disease (EP0695189 Genentech).

[0265] None of the presently available drugs are completely effective for the treatment of autoimmune disease, and most are limited by severe toxicity. In addition, it is extremely difficult and a lengthy process to develop a new chemical entity (NCE) with sufficient potency and selectivity to such target sequence. Antibody-based therapeutics on the other hand have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. In addition, the development time can be reduced considerably when compared to the development of new chemical entities (NCE's). However, conventional antibodies are difficult to raise against multimeric proteins where the receptor-binding domain of the ligand is embedded in a groove, as is the case with IFN-gamma.

[0266] The heterospecific polypeptide constructs of the present invention, wherein the anti-target single domain antibody is directed against TNF-alpha overcome the problems experienced using peptide therapeutics of the art because of the properties thereof such as stability, size, and reliable expression. Furthermore, the inventors have found that, despite presence of a groove in multimeric IFN-gamma, the heterospecific polypeptide constructs are still able to achieve strong binding to IF NA-gamma.

[0267] According to one aspect of the invention, a target against which one or more anti-target single domain antibodies of a heterospecific polypeptide construct comprising one or more anti-target single domain antibodies fused to one or more anti-serum protein single domain antibodies is directed is interferon-gamma (IFN-gamma). IFN-gamma is secreted by some T cells. In addition to its anti-viral activity, IFN-gamma stimulates natural killer (NK) cells and T helper 1 (Th1 ) cells, and activates macrophages and stimulates the expression of MHC molecules on the surface of cells. Hence, IFN-gamma generally serves to enhance many aspects of immune function, and is a candidate for treatment of disorders where the immune system is over-active e.g. Crohn's disease, autoimmune disorders and organ plant rejection in addition inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.

[0268] One aspect of the present invention relates to a heterospecific polypeptide construct comprising one or more anti-lFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies, the sequences of said heterospecific polypeptide corresponding to any of SEQ ID NOS: 25 to 27. The anti-IFN-gamma single domain antibodies therein are derived from Camelidae heavy chain antibodies (VHHs), which bind to IFN-gamma.

[0269] Anti-target single domain antibodies may be directed against whole IFN-gamma or a fragment thereof, or a fragment of a homologous sequence thereof.

[0270] One embodiment of the present invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in treating, preventing and/or alleviating the symptoms of the disorders wherein the immune system is overactive, as mentioned above. Current therapy consists of intravenous administration of anti-IFN-gamma antibodies. Oral delivery of these heterospecific polypeptide constructs results in the delivery of such molecules in an active form in the colon at sites that are affected by the disorder. These sites are highly inflamed and contain IFN-gamma producing cells. These heterospecific polypeptide constructs can neutralise the IFN-gamma locally, avoiding distribution throughout the whole body and thus limiting negative side-effects. Genetically modified microorganisms such as Micrococcus lactis are able to secrete antibody fragments. Such modified microorganisms can be used as vehicles for local production and delivery of antibody fragments in the intestine. By using a strain which produces a IFN-gamma neutralising heterospecific polypeptide construct, inflammatory bowel disorder could be treated.

[0271] Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the treatment, prevention and/or alleviation of disorders wherein the immune system is overactive, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0272] Another aspect of the invention is the use of a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is over active, wherein said heterospecific polypeptide construct is administered intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0273] Another aspect of the invention is a method of treating, preventing and/or alleviating disorders wherein the immune system is overactive, comprising administering to a subject a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies intravenously, orally, sublingually, topically, nasally, vaginally, rectally or by inhalation.

[0274] Another aspect of the invention is a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies joined to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is overactive.

[0275] Another aspect of the invention is a use of a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein single domain antibodies for use in the preparation of a medicament for the treatment, prevention and/or alleviation of disorders wherein the immune system is over active.

[0276] It is an aspect of the invention that the anti-IFN-gamma single domain antibodies of the present invention may be derived from VHHs of any class. For example, they may be derived from a class of VHHs with high homology to the human VH sequence, or may be derived from any of the other classes of VHHs, including the major class of VHH. These VHHs include the full length Camelidae VHHs, domains and may comprise a human Fc domain if effector functions are needed.

[0277] The above aspect and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IFN-gamma VHHs fused to one or more anti-serum protein single domain antibodies wherein said heterospecific polypeptide corresponds to a sequence represented by any of SEQ ID NOS: 25 to 27, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion. SEQ ID NOS: 25 to 27 comprise anti-IFN-gamma VHH and anti-mouse serum albumin VHH.

[0278] The above aspects and embodiments apply to a heterospecific polypeptide construct comprising one or more anti-IFN-gamma single domain antibodies fused to one or more anti-serum protein VHHs wherein said anti-serum protein VHHs correspond to any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, of a homologous sequence of a functional portion thereof.

[0279] One embodiment of the present invention is a recombinant done comprising nucleic acid encoding a heterospeclfic polypeptide construct according to the invention. In one aspect of the invention, said nucleic acid encodes one or more single domain antibodies each directed to a therapeutic or diagnostic target antigen and one or more single domain antibodies directed to a serum protein, said single domain antibodies linked without intervening linkers, or with one or more peptide linker sequences. According to one aspect of the invention, a linker sequence is any suitable linker sequence known in the art. According to another aspect of the invention, a linker sequence is a naturally occurring sequence. Preferred properties of linkers sequences are that they are not immunogenic or not significantly immunogenic, they can provide sufficient flexibility to the heterospecific polypeptide construct, and are resistant to proteolytic degradation. An example of a linker according to the invention is that disclosed in PCT/EP96/01725 which is derived from the hinge region of VHH.

[0280] According to another aspect of the invention, a done comprises nucleic acid encoding a polypeptide corresponding to a sequence represented by any of SEQ ID NOS: 1 to 4, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion, and nucleic acid encoding one or more anti-target single domain antibodies, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion thereof.

[0281] According to another aspect of the invention, a clone comprises nucleic acid capable of encoding a polypeptide corresponding to a sequence represented by any of SEQ ID NOS:5 to 27, a homologous sequence thereof, a functional portion thereof, or a homologous sequence of a functional portion thereof.

[0282] It is within the scope of the invention that nucleic acid encoding multiple anti-target and/or multiple anti-serum VHHs are present in a clone of the invention.

[0283] By transforming a compatible host with a clone encoding a heterospecific polypeptide construct of the invention, the heterospecific polypeptide construct can be produced in sufficient quantities for use in therapy. Examples of organisms into which said clone may be transformed include, but are not limited to E. coli or Sacchoromyces cerevisiae.

[0284] Another embodiment of the present invention is a method for prolonging the half-life of an anti-target-VHH comprising the step of joining thereto one or more anti-serum albumin single domain antibodies. As already mentioned above, methods for joining are known in the art or may be any future method, for example, they may be fused by chemical coupling, fused at the DNA level etc.

[0285] Treating, preventing and/or alleviating the symptoms of one or more of the disorders mentioned herein generally involves administering to a subject a "therapeutically effective amount" of heterospecific polypeptide construct. By "therapeutically effective amount", "therapeutically effective dose" and "effective amount" means the amount needed to achieve the desired result or results. One of ordinary skill in the art will recognise that the potency and, therefore, an "effective amount" can vary for the various compounds that inhibit a disorder pathway used in the invention. One skilled in the art can readily assess the potency of the compound.

[0286] As used herein, the term "compound" refers to a heterospecific polypeptide construct as disclosed herein, a polypeptide represented by SEQ ID NOS: 5 to 27, a homologous sequence thereof, or a homologue thereof, or a nucleic acid capable of encoding said polypeptide.

[0287] By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0288] The Invention disclosed herein is useful for treating or preventing a condition relating to a disorder as mentioned herein (e.g. allergy and/or inflammation), in a subject and comprising administering a pharmaceutically effective amount of a compound or composition that binds to a component involved in the disorder pathway (e.g. to IgE and/or TNF-alpha in the blood stream), so inhibiting the disorder pathway and the disorder.

[0289] One aspect of the present invention is the use of compounds of the invention for treating or preventing a condition relating to a disorder as mentioned herein (e.g. allergy and/or inflammation), in a subject and comprising administering a pharmaceutically effective amount of a compound in combination with another, such as, for example, aspirin.

[0290] The present invention is not limited to the administration of formulations comprising a single compound of the invention. It is within the scope of the invention to provide combination treatments wherein a formulation is administered to a patient in need thereof that comprises more than one compound of the invention.

[0291] It is well known in the art how to determine the inhibition of a disorder pathway using the standard tests described herein, or using other similar tests. Preferably, the method would result in at least a 10% reduction in an indicator of the disorder, including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, more preferably by 90%. For example, an inhibition of an allergic pathway by inhibition of IgE by a peptide of the invention might result in a 10% reduction in food-specific IgE levels.

[0292] The compound useful in the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient or any animal in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intranasally by inhalation, intravenous, intramuscular, topical or subcutaneous routes.

[0293] The compound of the present invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene for the compound of the present invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells.

[0294] Thus, the present compound may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0295] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0296] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0297] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0298] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0299] For topical administration, the present compound may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0300] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the present compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0301] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0302] Examples of useful dermatological compositons which can be used to deliver the compound to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

[0303] Useful dosages of the compound can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0304] Generally, the concentration of the compound(s) in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

[0305] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the compound varies depending on the target cell, tumor, tissue, graft, or organ.

[0306] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0307] An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill In the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co., Easton, Pa. The dosage can also be adjusted by the individual physician in the event of any complication.

EXAMPLES

Example 1

Immunization of Llamas

[0308] One llama was Immunized with human serum albumin (HSA). The immunization scheme is summarized in Table 1.

Example 2

Repertoire Cloning

[0309] Peripheral blood lymphocytes (PBLs) were isolated by centrifugation on a density gradient (Ficoll-Paque Plus Amersham Biosciences). PBLs were used to exitact total RNA (Chomczynski and Sacchi 1987). cDNA was prepared on 100 .mu.g total RNA with MMLV Reverse Transcriptase (Gibco BRL) using oligo d(T) oligonucleotides. The cDNA was purified with a phenol/chloroform extraction, followed by an ethanol precipitation and subsequently used as template to amplify the VHH repertoire.

[0310] In a first PCR, the repertoire of both conventional (1.6 kb) and heavy-chain (1.3 kb) antibody gene segments were amplified using a leader specific primer (5'-GGCTGAGCTCGGTGGTCCTGGCT-3') (SEQ ID N.sup.o 41) and the oligo d(T) primer (5'-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT-3') (SEQ ID N.sup.o 42). The resulting DNA fragments were separated by agarose gel electrophoresis and the 1.3 kb fragment, encoding heavy-chain antibody segments was purified from the agarose gel. A second PCR was performed using a mixture of FR1 reverse primers and the same oligo d(T) forward primer. The PCR products were digested with Sfil (introduced in the FR1 primer) and BstEll (naturally occurring in FR4). Following gel electrophoresis, the DNA fragment of approximately 400 basepairs were purified from gel and ligated into the corresponding restriction sites of phagemid pAX004 to obtain a library of cloned VHHs after electroporation of Escherichia coli TG1. The size of the library was 1.4.times.10.sup.7 cfu, and all clones contained insert of the correct size.

Example 3

Rescue of the Library, Phage Preparation

[0311] The library was grown at 37.degree. C. in 10 ml 2.times.TY medium containing 2% glucose, and 100 .mu.g/ml ampicillin, until the OD600 nm reached 0.5. M13KO7 phages (10.sup.12) were added and the mixture was incubated at 37.degree. C. for 2.times.30 minutes, first without shaking, then with shaking at 100 rpm. Cells were centrifuged for 10 minutes at 4500 rpm at room temperature. The bacterial pellet was resuspended in 50 ml of 2.times.TY medium containing 100 .mu.g/ml ampicillin and 25 .mu.g/ml kanamycin, and incubated overnight at 37.degree. C. with vigorously shaking at 250 rpm. The overnight cultures were centrifuged for 15 minutes at 10,000 rpm at 4.degree. C. Phages were PEG precipitated (20% poly-ethylene-glycol and 1.5 M NaCl) and centrifuged for 30 minutes at 10,000 rpm. The pellet was resuspended in 20 ml PBS. Phages were again PEG precipitated and centrifuged for 30 minutes at 20,000 rpm and 4.degree. C. The pellet was dissolved in 5 ml PBS-1% casein. Phages were titrated by infection of TG1 cells at OD600 nm=0.5 and plating on LB agar plates containing 100 .mu.g/ml ampicillin and 2% glucose. The number of transformants indicates the number of phages (=pfu). The phages were stored at -80.degree. C. with 15% glycerol.

Example 4

Phage ELISA

[0312] A microtiter plate (Maxisorp) was coated overnight at 4.degree. C. with PBS-1% casein or with 5 .mu.g/ml HSA (human serum albumin). The plate was washed 3 times with PBS-Tween (0.05% Tween20) and blocked for 2 hours at room temperature with 200 .mu.l PBS-1% casein. The plate was washed five times with PBS-Tween. Phages were prepared as described above and applied to the wells in consecutive twofold dilutions. Plates were washed five times with PBS-Tween. Bound phage were detected with a mouse monoclonal antibody anti-M13 conjugated with horse radish peroxidase (HRP) diluted 1/2000 in PBS. The plates were washed five times with PBS-Tween. Staining was performed with ABTS/H.sub.2O.sub.2 and signals were measured after 30 minutes at 405 nm. Results are shown in FIG. 1 and indicate the presence of HSA-specific nanobodies in the library.

Example 5

Selection: First and Second Round of Biopanning

[0313] A well in a microtiterplate was coated with 10 .mu.g/ml mouse serum albumin (MSA), or with PBS containing 1% casein. After overnight incubation at 4.degree. C., the wells were blocked with PBS containing 1 % casein, for 3 hours at room temperature (RT). 200 .mu.l phages was added to the wells. After 2 hours incubation at RT, the wells were washed 10.times. with PBS-Tween and 10.times. with PBS. Bound phages were eluted with 100 .mu.l 0.2 M glycin buffer pH=2.4. Elutions were performed for 20 minutes at room temperature. Eluted phages were allowed to infect exponentially growing E.Coli TG1 cells, and were then plated on LB agar plates containing 100 .mu.g/ml ampicillin and 2% glucose. A second round was performed with the same conditions as described above. Results are summarized in Table 2.

Example 6

Screening of Individual Clones After Biopanning

ELISA: binding to human serum albumin (HSA) and mouse serum albumin (MSA)

[0314] A single colony was used to start an overnight culture in LB containing 2% glucose and 100 .mu.g/ml ampicillin. This overnight culture was diluted 100-fold in TB medium containing 100 .mu.g/ml ampicillin, and incubated at 37.degree. C. until OD600 nm=0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37.degree. C. or overnight at 28.degree. C. Cultures were centrifuged for 20 minutes at 10,000 rpm at 4.degree. C. The pellet was frozen overnight or for 1 hour at -20.degree. C. Next, the pellet was thawed at room temperature for 40 minutes, re-suspended in PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4.degree. C. at 20,000 rpm. The supernatant containing the VHH was used for further analysis.

[0315] A microtiter plate was coated with 5 .mu.g/ml HSA, with 5 .mu./ml mouse serum albumin (MSA) or with PBS-1% casein, overnight at 4.degree. C. Plates were blocked for two hours at room temperature with 300 .mu.l 1% casein in PBS. The plates were washed three times with PBS-Tween. Periplasmic fraction was prepared for 23 individual clones after the first and second round of selection, and allowed to bind to the wells of the microtiterplate. Plates were washed six times with PBS-Tween, after which binding of nanobody was detected by incubation with mouse anti-Histidine monoclonal antibody Serotec MCA 1396 ( 1/1000 dilution) in PBS for 1 hour at RT followed by anti-mouse-alkaline phosphatase conjugate 1/2000 in PBS, also for 1 hour at RT. Staining was performed with the substrate PNPP (p-nitrophenyl-phosphate, 2 mg/ml in 1 M diethanolamine, 1 mM Mg.sub.2SO.sub.4, pH9.8) and the signals were measured after 30 minutes at 405 nm. Results are summarized in Table 3.

Example 7

Hinfl Pattern and Sequencing

[0316] A PCR was performed on positive clones after the second round of panning, with a set of primers binding to a sequence in the vector. The PCR product was digested with the restriction enzyme Hinfl and loaded on a agarose gel. 4 clones were selected with a different Hinfl-pattern for further evaluation. Those clones were sequenced, and results are summarized in Table 4 (SEQ ID NOS: 1, 2 ,3 and 4).

Example 8

Test Cross-reactivity with Albumin of Different Species

[0317] A SDS-PAGE was run for plasma ( 1/10 dilution) from different species (baboon, pig, hamster, human, rat, mouse and rabbit) and blotted on a nitrocellulose membrane. Phages were prepared for clones MSA 21, MSA 24, MSA 210, MSA212 and a control nanobody as described in Example 3. Phages were allowed to bind to the nitrocellulose blotted serum albumins and unbound phages were washed away. Binding was detected with an anti-M13 polyclonal antibody coupled to HRP. DAP was used as a substrate for detection. Results are shown in FIG. 2.

[0318] From these results we can conclude that all 4 binders are cross-reactive between pig, human, mouse (less for MSA212) and hamster serum albumin. MSA 21 is also cross-reactive with rabbit serum albumin. With the irrelevant nanobody no binding was observed (not shown).

[0319] As a control experiment, a SDS-PAGE was run with the different plasma samples diluted 1/100 in PBS. The gel was stained with coomassie. We can conclude from FIG. 3 that albumin levels in all plasma samples are high except for rabbit plasma, with low levels of albumin.

Example 9

Expression and Purification

[0320] Plasmid was prepared for the binders and was transformed into WK6 electrocompetent cells. A single colony was used to start an overnight culture in LB containing 2% glucose and 100 .mu.g/ml ampicillin. This overnight culture was diluted 100-fold in 300 ml TB medium containing 100 .mu.g/ml ampicillin, and incubated at 37.degree. C. until OD600 nm=0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37.degree. C. or overnight at 28.degree. C. Cultures were centrifuged for 20 minutes at 10,000 rpm at 4.degree. C. The pellet was frozen overnight or for 1 hour at -20.degree. C. Next, the pellet was thawed at room temperature for 40 minutes, re-suspended in 20 ml PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4.degree. C. at 20,000 rpm. The supernatant containing the nanobody was loaded on Ni-NTA and purified to homogeneity.

Example 10

ELISA on MSA of the Purified Nanobodies

[0321] A microtiterplate was coated with 5 .mu.g/ml MSA overnight at 4 C. After washing, the plate was blocked for 2 hours at RT with PBS-1% casein. Samples were applied in duplicate starting at a concentration of 2500 nM at 1/3 dilutions and allowed to bind for 2 hours at RT. A polyclonal rabbit anti-nanobody serum was added at 1/1000 (K208) for one hour at RT. Detection was with ant-rabbit alkaline phosphatase conjugate at 1/1000 and staining with PNPP as described in Example 6. Results are shown in FIG. 4.

Example 11

Construction of Bispecific Constructs

[0322] The E. coli production vector pAX11 was constructed to allow the two-step cloning of bivalent or bispecific VHH (FIG. 5).

[0323] The carboxy terminal VHH was cloned first with Pstl and BstEll, while in the second step the other VHH was inserted by Sfil and Notl, which do not cut within the first gene fragment. The procedure avoids the enforcement of new sites by amplification and thus the risk of introducing PCR errors. The middle hinge of llama was used as a linker between the nanobodies. A VHH against human TNF alpha was cloned at the COOH terminal of MSA specific nanobodies. Sequences are summarized in Table 4 (SEQ ID NOS: 5, 6, 7 and 8). Plasmid was prepared and was transformed into WK6 electrocompetent cells. A single colony was used to start an overnight culture in LB containing 2% glucose and 100 .mu.g/ml ampicillin. This overnight culture was diluted 100-fold in 300 .mu.l TB medium containing 100 mg/ml ampicillin, and incubated at 37.degree. C. until OD600 nm=0.5. 1 mM IPTG was added and the culture was incubated for 3 more hours at 37.degree. C.

[0324] Cultures were centrifuged for 20 minutes at 10,000 rpm at 4.degree. C. The pellet was frozen overnight at -20 C. The next morning, the pellet was thawed in the cold room for 40 minutes, re-suspended in 20 ml PBS and shaken on ice for 1 hour. Periplasmic fraction was isolated by centrifugation for 20 minutes at 4.degree. C. at 10,000 rpm. The supernatant was loaded on Ni-NTA and purified to homogeneity. Sequences are shown in Table 4 (SEQ ID NOS: 5, 6, 7 and 8). A extra purification step was needed to remove some degradation product (5%) on gelfiltration.

[0325] Another bispecific VHH against human TNF-alpha (MP7 12 b) is listed in Table 4 (SEQ ID NOS: 15, 16, 17 and 18).

Example 12

Test Bispecific Construct in Sandwich ELISA

[0326] A microtiter plate was coated with 5 .mu.g/ml MSA overnight at 4.degree. C. Plates were blocked for two hours at room temperature with 300 .mu.l 1% casein in PBS. The plates were washed three times with PBS-Tween. Purified protein for the bispecific constructs was allowed to bind to the wells of the microtiterplate at a concentration of 0.4, 0.5, 2.5 and 2.5 .mu.g/ml for MSA21, MSA24, MSA210 and MSA212 respectively. Plates were washed six times with PBS-Tween, Biotinilated TNF was added at a concentration of 10 .mu.g/ml and diluted 3 fold, and allowed to bind for 2 hours at room temperature. Binding was detected by incubation with mouse extravidin alkaline phosphatase conjugate (Sigma) 1/2000 in PBS, for 1 hour at RT. Staining was performed with the substrate PNPP (p-nitrophenyl-phosphate, 2 mg/ml in 1 M diethanolamine, 1 mM Mg.sub.2SO.sub.4, pH9.8) and the signals were measured after 30 minutes at 405 nm. Results are shown in FIG. 6 and indicate that the bispecific construct can bind both antigens simultaneously.

Example 13

Determine Affinity of Albumin Binders In BIACORE

[0327] Affinities for mouse albumin were determined in BIACORE by immobilization of mouse albumin on a CM5 BIAcore chip using EDC-NHS covalent coupling and are summarized in Table 5. The results indicate that the affinity for albumin is retained in the bispecific construct.

Example 14

Optimization of ELISA in Plasma or Blood

[0328] Pharamcokinetic experiments were initiated to compare half life in mice of the TNF-alpha binder TNF3E with MSA21/VHH#3E and MSA24/VHH#3E. Therefore our ELISA had to be optimized to obtain low background values when the samples are in blood or in plasma. A microtiterplate was coated with neutravidin. After overnight incubation at 4 C, the plates were washed and blocked for 2 hours at RT with PBS-1% casein. 1 .mu.g/ml biotinylated TNF-alpha was allowed to bind for 30 minutes at RT and the plate was washed. Samples (monovalent VHH#3E and MSA21/VHH#3E) were applied starting at a concentration of 1 .mu.g/ml, diluted in PBS, 10% plasma or 10% blood and allowed to bind for 2 hours. After washing the plates, a rabbit antiserum was added at a dilution of 1/2000 either recognizing the heavy chain class (K208) or recognizing the conventional class (URL49). After 1 hour incubation, the plates were washed and an anti-rabbit alkaline phosphatase conjugate was added (Sigma) at a dilution of 1/1000. After 1 hour incubation at RT, plates were washed and binding was detected with substrate. Results are shown in FIG. 7. The results clearly show that background values with the rabbit antisera (K208 and URL49) are very low when the samples are diluted in 10% blood or 10% plasma as compared to PBS. The URL49 antiserum only recognizes the MSA21/VHH#3E bispecific nanobody and not monovalent VHH#3E, therefore, this antiserum can be used to test the integrity of our bispecific nanobody upon administration to the mice.

Example 15

Large Scale Expression and Purification of VHH#3E, MSA21/VHH#3E and MSA24/VHH#3E for Pharmacokinetic Studies in Mice

[0329] 3 liter culture was started for monovalent TNF3E and for bispecific MSA21/VHH#3E or MSA24/VHH#3E and purified as described in Example 11. An extra purification step was needed for the removal of endotoxins. Therefore, samples were purified on a Polymyxin column (BIO-RAD). Samples were analyzed for bacterial endotoxin concentration with the LAL-assay (Limulus Amebocyte Lysate, Bio Whittaker). Results are summarized in Table 6.

Example 16

Pharmacokinetics in Mice

[0330] 9 mice (CB57/BI6) for each construct were injected intravenously in the tail with 100 .mu.g nanobody. Blood was retrieved at different time points (3 mice per time point) and serum was prepared. Samples were analyzed by ELISA for the presence of monovalent or bispecific nanobody as described in example 14. K208 was also compared to URL49 for the bispecific constructs to verify the integrity of the molecule. Results are shown in FIGS. 8 to 11.

[0331] We can conclude from the results that the half life of the monovalent nanobody (40-45 minutes) is dramatically improved by making a bispecific nanobody with specificity for albumin MSA21/VHH#3E and MSA24/VHH#3E (half-life 2.5 to 3 days). The bispecific nanobody MSA21/VHH#3E remains intact even after 19 days in the mice as shown in ELISA with URL49 (FIG. 11).

Example 17

Further Extension of Half-life of Nanobodies

[0332] In order to increase the half-life of MSA21/TNF3E and MSA24/TNF3E even further, a trivalent nanobody was prepared by fusing the bivalent MSA21-MSA21 construct to target-specific nanobody TNF3E. The resulting MSA21/MSA21/TNF3E (Table 7, and SEQ ID NO: 9) was tested in vivo according to the method of Example 16.

Example 18

Immunization of Llama002

[0333] 1 llama was immunized with vWF. The immunization scheme is summarized in Table 7.

Example 19

Repertoire Cloning and Phage Preparation

[0334] The library was prepared as described in Example 2. The size of the library was 1.4.times.10.sup.7 cfu, and >90% of the clones contained insert of the correct size. Phages were prepared as described in Example 3.

Example 20

Selection for Binders for vWF Inhibiting the Interaction with Collagen: First and Second Round of Panning

[0335] A well in a microtiterplate was coated with 2 .mu.g/ml vWF or with PBS containing 1% casein. After overnight incubation at 4.degree. C., the wells were blocked with PBS containing 1% casein, for 3 hours at RT. 200 .mu.l phages was added to the wells. After 2 hours incubation at RT, the wells were washed 10.times. with PBS-Tween and 10.times. with PBS. Phages were specifically eluted with 100 .mu.l of 100 .mu.g/ml collagen type III. Elutions were performed for overnight at room temperature. Eluted phages were allowed to infect exponentially growing TG1 cells, and were then plated on LB agar plates containing 100 .mu.g/ml ampicillin and 2% glucose. This experiment was repeated for a second round of panning, under the same conditions as described above. The results from the panning are presented in Tables 8 and 9.

Example 21

Functional Characterization of vWF Binders: Inhibition of Binding of vWF to Collagen by VHH

[0336] A microtiter plate was coated overnight at 4.degree. C. with collagen type III at 25 .mu.g/ml in PBS. The plate was washed five times with PBS-Tween and blocked for 2 hours at room temperature with PBS containing 1% casein. The plate was washed five times with PBS-tween. 100 .mu.l of 2 .mu.g/ml vWF (vWF is pre-incubated at 37.degree. C. for 15 minutes) was mixed with 20 .mu.l periplasmic extract containing a VHH antibody (described in Example 6) and incubated for 90 minutes at room temperature in the wells of the microtiterplate. The plate was washed five times with PBS-tween. An anti-vWF-HRP monoclonal antibody (DAKO) was diluted 3,000-fold in PBS and incubated for 1 hour. The plate was washed five times with PBS-Tween and vWF-binding was detected with ABTS/H.sub.2O.sub.2. Signals were measured after 30 minutes at 405 nm. The results are presented in Table 10, showing that inhibitors are obtained after the first and second round of panning.

Example 22

Expression and Purification of VHH

[0337] Protein was prepared and purified as described in Example 9.

Example 23

ELISA: Binding to vWF

[0338] A microtiter plate was coated with 2 .mu.g/ml vWF, overnight at 4.degree. C. Plates were blocked for two hours at room temperature with 300 .mu.l 1% casein in PBS. The plates were washed three times with PBS-Tween. Dilution series of all purified samples were incubated for 2 hours at RT. Plates were washed six times with PBS-Tween, after which binding of VHH was detected by incubation with mouse anti-myc mAB 1/2000 in PBS for 1 hour at RT followed by anti-mouse-HRP conjugate 1/1000 in PBS, also for 1 hour at RT. Staining was performed with the substrate ABTS/H.sub.2O.sub.2 and the signals were measured after 30 minutes at 405 nm. The binding as a function of concentration of purified VHH is indicated in FIG. 12.

Example 24

Inhibition ELISA with Purified VHH

[0339] Inhibition ELISA was performed as described in Example 20 but with decreasing concentrations of VHH and with human plasma at a dilution of 1/60 instead of with purified vWF. Results are represented in FIG. 13. The concentration of VHH resulting in 50% inhibition (IC50) is given in table 10.

Example 25

Construction and Sequence of Bispecific Constructs

[0340] Bispecific constructs were prepared with the first VHH specific for albumin (MSA21) and the second VHH specific for vWF. Constructs were made as described in Example 11. Sequences are shown in Table 4 (SEQ ID NOS: 19 to 21)

Example 26

Expression and Purification of Bispecific Constructs

[0341] Protein was expressed and purified as described in Example 9. An extra purification step was needed on superdex 75 for removal of some monovalent degradation product (5-10%).

Example 27

Functionality of Both VHHs in the Bispecific Construct

[0342] A microtiterplate was coated with 5 .mu.g/ml mouse serum albumin overnight at 4.degree. C. After washing the plate, wells were blocked for 2 hours with PBS-1% casein. The bispecific proteins were allowed to bind to the wells for 2 hours at RT. After washing, human, dog and pig plasma was added at different dilutions and allowed to bind for 2 hours at RT. Binding of vWF was detected with anti-vWF-HRP from DAKO at 1/3000 dilution. Staining was performed with ABTS/H.sub.2O.sub.2. Results are shown in FIG. 14 and indicate that functionality of both VHHs is retained in the bispecific construct.

Example 28

Inhibition of Binding of vWF to Collagen by the Bispecific Constructs as Compared to the Monovalent VHHs

[0343] Inhibition for binding of vWF to collagen was tested for monovalent as compared to bispecific constructs as described in Example 20. IC50 values are summarized in Table 11. Results indicate that the inhibitory properties of the VHH are retained in the bispecific construct.

Example 29

Construction of a Bispecific Construct Containing a VHH-CDR3 Fragment Fused to an Anti-serum Albumin VHH

[0344] A functional portion, the CDR3 region of MP2F6SR, was amplified by using a sense primer located in the framework 4 region (F6 CRD3 Forward:CTGGCCCCAGAAGTCATACC) (SEQ ID N.sup.o 43) and an anti-sense primer located in the framework 3 region (F6 CDR3 Reverse primer:TGTGCATGTGCAGCAAACC) (SEQ ID N.sup.o 44).

[0345] In order to fuse the CDR-3 fragment with the anti-serum albumin VHH MSA-21, a second round PCR amplification was performed with following primers: TABLE-US-00001 F6 CDR3 Reverse primer Sfi1: (SEQ ID N.degree. 45) GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCATGTGCAGCAAACC F6 CDR3 Forward primer Not1: (SEQ ID N.degree. 46) GTCCTCGCAACTGCGCGGCCGCCTGGCCCCAGAAGTCATACC

[0346] The PCR reactions was performed in 50 ml reaction volume using 50 pmol of each primer. The reaction conditions for the primary PCR were 11 min at 94.degree. C., followed by 30/60/120 sec at 94/55/72.degree. C. for 30 cycles, and 5 min at 72.degree. C. All reaction were performed wit 2.5 mM MgCl2 , 200 mM dNTP and 1.25 U AmpliTaq God DNA Polymerase (Roche Diagnostics, Brussels, Belgium).

[0347] After cleavage of the VHH gene of MSA clones with restriction enzymes Pst1/BstEll the digested products were cloned in pAX11 to obtain clones with a VHH at the C-terminus of the multicloning site. The clones were examined by PCR using vector based primers. From clones yielding a 650 bp product, DNA was prepared and used as acceptor vector to clone the CDR3 of MP2F6SR after cleavage of the PCR product with restriction enzymes Sfi1/Not1 to allow N-terminal expression of CDR3 in fusion with a MSA VHH.

Example 30

Calculation of Homologies Between Anti-target Single Domain Antibodies of the Invention

[0348] The degree of amino acid sequence homology between anti-target single domain antibodies of the invention was calculated using the Bioedit Sequence Alignment Editor. The calculations indicate the proportion of identical residues between all of the sequences as they are aligned by ClustalW. (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research, submitted, June 1994). Table 12 indicates the fraction homology between anti-serum albumin VHHs of the invention. Table 13 indicates the fraction homology between anti-TNF-alpha VHHs of the invention. Table 14 indicates the percentage homology between anti-IFN-gamma VHHs of the invention. Table 15 indicates the fraction homology between anti-vWF VHHs of the invention. TABLE-US-00002 TABLE 1 Immunization scheme according to Example 1 HSA Day of immunization Llama006 0 100 .mu.g 7 100 .mu.g 14 50 .mu.g 21 50 .mu.g 28 50 .mu.g 35 50 .mu.g

[0349] TABLE-US-00003 TABLE 2 results after one and two rounds of panning on mouse serum albumin as described in example 5. First round Second round Pfu mouse serum albumin 2.5 .times. 10.sup.7 2.5 .times. 10.sup.7 Pfu casein 5 .times. 10.sup.3 2.5 .times. 10.sup.3 enrichment 5,000 10,000

[0350] TABLE-US-00004 TABLE 3 Clones were selected after one and two rounds of selection and peripiasmic extracts were prepared. These clones were analyzed in ELISA for binding to human and mouse albumin as described in Example 6. First round Second round ELISA mouse serum albumin 1/16 15/16 ELISA human serum albumin 1/16 15/16 ELISA casein 0/16 0/16

[0351] TABLE-US-00005 TABLE 4 Sequence listing NAME SEQ ID SEQUENCE Anti-mouse serum albumin MSA21 1 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG TQVTVSS MSA24 2 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG TQVTVSS MSA210 3 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSS MSA212 4 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSW VRQAPGKGLEWVSAISADGSDKRYADSVKGRFTISR DNGKKMLTLDMNSLKPEDTAVYYCVIGRGSPASQGT QVTVSS MSAc16 28 AVQLVESGGGLVQAGDSLRLSCVVSGTTFSSAAMGW FRQAPGKEREFVGAIKWSGTSTYYTDSVKGRFTISR DNVKTVYLQMNNLKPEDTGVYTCAADRDRYRDRMGP MTTTDFRFWGQGTQVTVSS MSAcl12 29 QVKLEESGGGLVQTGGSLRLSCAASGRTFSSFAMGW FRQAPGREREFVASIGSSGITTNYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTGLCYCAVNRYGIPYRSG TQYQNWGQGTQVTVSS MSAcl10 30 EVQLEESGGGLVQPGGSLRLSCAASGLTFNDYAMGW YRQAPGKERDMVATISIGGRTYYADSVKGRFTISRD NAKNTVYLQMNSLKPEDTAIYYCVAHRQTVVRGPYL LWGQGTQVTVSS MSAcl14 31 QVQLVESGGKLVQAGGSLRLSCAASGRTFSNYAMGW FRQAPGKEREFVAGSGRSNSYNYYSDSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCAASTNLWPRDRN LYAYWGQGTQVTVSS MSAcl16 32 EVQLVESGGGLVQAGDSLRLSCAASGRSLGIYRMGW FRQVPGKEREFVAAISWSGGTTRYIDSVKGRFTISR DSTKNAVYLQMNSLKPEDTAVYYCAVDSSGRLYWTL STSYDYWGQGTQVTVSS MSAcl19 33 QVQLVEFGGGLVQAGDSLRLSCAASGRSLGIYKMAW FRQVPGKEREFVAAISWSGGTTRYIDSVKGRFTLSR DNTKNMVYLQDMNSLKPDDTAVYYCAVDSSGRLYWT LSTSYDYWGQGTQVTVSS MSAc15 34 EVQLVESGGGLVQAGGSLSLSCAASGRTFSPYTMGW FRQAPGKEREFLAGVTWSGSSTFYGDSVKGRFTASR DSAKNTVTLEMNSLNPEDTAVYYCAAAYGGGLYRDP RSYDYWGRGTQVTVSS MScl11 35 AVQLVESGGGLVQAGGSLRLSCAASGFTLDAWPIAW FRQAPGKEREGVSCIRDGTTYYADSVKGRFTISSDN ANNTVYLQTNSLKPEDTAVYYCAAPSGPATGSSHTF GIYWNLRDDYDNWGQGTQVTVSS MSAcl15 36 EVQLVESGGGLVQAGGSLRLSCAASGFTFDHYTIGW FRQVPGKEREGVSCISSSDGSTYYADSVKGRFTISS DNAKNTVYLQMNTLEPDDTAVYYCAAGGLLLRVEEL QASDYDYWGQGIQVTVSS MSAcl8 37 AVQLVDSGGGLVQPGGSLRLSCTASGFTLDYYAIGW FRQAPGKEREGVACISNSDGSTYYGDSVKGRFTISR DNAKTTVYLQMNSLKPEDTAVYYCATADRHYSASHH PFADFAFNSWGQGTQVTVSS MSAcl7 38 EVQLVESGGGLVQAGGSLRLSCAAYGLTFWRAAMAW FRRAPGKERELVVARNWGDGSTRYADSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCAAVRTYGSATYD IWGQGTQVTVSS MSAcl20 39 EVQLVESGGGLVQDGGSLRLSCIFSGRTFANYAMGW FRQAPGKEREFVAAINRNGGTTNYADALKGRFTISR DNTKNTAFLQMNSLKPDDTAVYYCAAREWPFSTIPS GWRYWGQGTQVTVSS MSAcl4 40 DVQLVESGGGWVQPGGSLRLSCAASGPTASSHAIGW FRQAPGKEREFVVGINRGGVTRDYADSVKGRFAVSR DNVKNTVYLQMNRLKPEDSAIYICAARPEYSFTAMS KGDMDYWGKGTLVTVSS Anti-mouse serum albumin/anti TNF-alpha MSA21/ 5 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW VHH#3E VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS LRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFV ARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMN NLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQV TVSS MSA24/ 6 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW VHH#3E VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS LRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFV ARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMN NLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQV TVSS MSA210/ 7 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VHH#3E VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVA RIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNN LEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVT VSS MSA212/ 8 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSW VHH#3E VRQAPGKGLEWVSAISADGSDKRYADSVKGRFTISR DNGKKMLTLDMNSLKPEDTAVYYCVIGRGSPASQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVA RIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNN LEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVT VSS MSA21/ 9 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW MSA21/ VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR VHH#3E DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS LRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGISS LGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPE DTAVYYCTIGGSLNPGGQGTQVTVSSEPKTPKPQPA AAQVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSG YTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVK GRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDG IPTSRSVESYNYWGQGTQVTVSS MSA210/ 10 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VHH#1 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCATSGFDFSVSWMYWVRQAPGKGLEWVSEINTN GLITKYVDSVKGRFTISRDNAKNTLYLQMDSLIPED TALYYCARSPSGSFRGQGTQVTVSS MSA210/ 11 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VHH#9 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCAASGSIFRVNAMGWYRQVPGNQREFVAIITSG DNLNYADAVKGRFTISTDNVKKTVYLQMNVLKPEDT AVYYCNAILQTSRWSIPSNYWGQGTQVTVSS MSA210/ 12 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VHH#13 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCATSGFTFSDYWMYWVRQAPGKGLEWVSTVNTN GLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSED TAVYYCTKVVPPYSDDSRTNADWGQGTQVTVSS MSA210/ 13 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VHH+1902 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVA RIYWSSGNTYYADSVKGRFAISRDIAKINTVDLTMN NLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQV TVSS MSA210/ 14 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VHH#3 VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQAGGSL RLSCAVSGRTFSAHSVYTMGWFRQAPGKEREFVARI YWSSANTYYADSVKGRFTISRDNAKNTVDLLMNSLK PEDTAVYYCAARDGIPTSRTVGSYNYWGQGTQVTVS S MSA21/ 15 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW VHH#12B VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS LRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNT NGLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSE DTAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS MSA24/ 16 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW VHH#12B VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS LRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNT NGLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSE DTAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS MSA210/ 17 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW VHH#12B VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNTN GLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSED TAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS MSA212/ 18 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSW VHH#12B VRQAPGKGLEWVSAISADGSDKRYADSVKGRFTISR GTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGG SLRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVN TNGLITRYADSVKGRFTISRDNAKYTLYLQMNSLKS EDTAVYYCTKVLPPYSDDSRTNADWGQGTQVTVSS Anti-mouse serum albumin/anti-vWF MSA21/ 19 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW AM-2-75 VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTSRD NAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL RLSCAASGFNFNWYPMSWVRQAPGKGLEWVSTISTY GEPRYADSVKADSPSSETTPTTRCICNEQPETEDTA VYYCARGAGTSSYLPQRGNWDQGTQVTVSS MSA21/ 20 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW AM-4-15- VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTSRD 3 NAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGT QVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQAGGSL RLACAASGSIFSINSMGWYRQAPGKQRELVAHALAD GSASYRDSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNTVPSSVTKGYWGQGTQVTVSS MSA21/ 21 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW 22-4L-16 VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTSRD NAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGT QVTVSSEPKTPKPQPAAAQVQLVESGGGLVQAGGSL RLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWS GGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCVADTGGISWIRTQGYNYWGQGTQVTVSS Anti-mouse serum albumin/anti-IgE MSA21/ 22 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW EV 2H11 VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGS LRLSCAASGVTFSSYAMGWFRQAPGKEREFVASITW TGTGTYYADSVKGRFTISRDHAGTTVYLQMNSLKPE DTAVYYCAVDRRSSTYYLMKGEYDYRGRGTQVTVSS MSA 24/ 23 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW EV 2H11 VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR

DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG TQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGS LRLSCAASGVTFSSYAMGWFRQAPGKEREFVASITW TGTGTYYADSVKGRFTISRDHAGTTVYLQMNSLKPE DTAVYYCAVDRRSSTYYLMKGEYDYRGRGTQVTVSS MSA 210/ 24 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW EV 2H11 VRQAPGKGLEWVSAISSDSGTKYADSVKGRFTISRD NAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGTQ VTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGSLR LSCAASGVTFSSYAMGWFRQAPGKEREFVASITWTG TGTYYADSVKGRFTISRDHAGTTVYLQMNSLKPEDT AVYYCAVDRRSSTYYLMKGEYDYRGRGTQVTVSS Anti-mouse serum albumin/anti-IFN-gamma MSA21/ 25 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTW MP2F6SR VRQAPGKGVEWVSGISSLGDSTLYADSVKGRFTISR DNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQG TQVTVSSEPKTPKPQPAAAQVKLEESGGGLVQAGGS LRLSCAASGRTFNNYNMGWFRQAPGKEREFVAAISW NGGSTYYDDSVKGRFTISRDNANNLVYLQMNSLNFE DTAVYYCACAAANYGIPQYRENRYDFWGQGTQVTVS S MSA24/ 26 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSW MP2F1BR VRQAPGKEPEWVSSISGSGSNTIYADSVKDRFTISR DNAKSTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQG TQVTVSSEPKTPKPQPAAAAVQLVESGGGLVQTGDS LRLSCVASGGTFSRYAMGWFRQAPGKEREFVARIGY SGRSISYATSVEGRFAISRDNAKNTVYLQMNSLKPE DTAVYYCASLVSGTLYQADYWGQGTQVTVSS MSA 210/ 27 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSW MP3HGSRA VRQAPGKGLEWVSAISSDSGTKNYADSVKGRFTISR DNAKKMLFLQMNSLRPEDTAVYYCVIGRGSPSSQGT QVTVSSEPKTPKPQPAAAQVQLQESGGGLVQAGGSL RLSCAASGRTFSIYNMGWFRQAPGKEREFVAGISWN GGSIYYTSSVEGRFTISRDNAENTVYLQMNSLKPED TGVYYCASKGRPYGVPSPRQGDYDYWGQGTQVTVSS

[0352] TABLE-US-00006 TABLE 5 Affinities (koff, kon and KD) for albumin binders as determined by BIACORE as described in Example 13. K.sub.on (10.sup.5 M.sup.-1s.sup.-1) K.sub.off (10.sup.-5s.sup.-1) K.sub.D [nM] MSA21 3.4 420 12 MSA24 6.4 1800 28 MSA212 3.7 9330 250 MSA21/TNF3E 2.3 370 16 MSA24/TNF3E 3.1 630 20 MSA212/TNF3E 0.42 490 120

[0353] TABLE-US-00007 TABLE 6 Results for the LAL-assay for monovalent and bispecific nanobodies after purification on polymyxin as described in Example 15. Monovalent Bispecific Bispecific TNF3E MSA21/TNF3E MSA24/TNF3E Endotoxin units/mg 0.13 Eu/mg 0.75 Eu/mg 2.8 Eu/mg of VHH

[0354] TABLE-US-00008 TABLE 7 Immunization scheme used for llama 002 according to Example 17. Llama002 Day of immunization vWF 0 100 .mu.g 7 100 .mu.g 14 50 .mu.g 21 50 .mu.g 28 50 .mu.g 35 50 .mu.g

[0355] TABLE-US-00009 TABLE 8 Plaque forming units (pfu) after one or two round(s) of panning on vWF as compared to PBS-casein as described in example 19. Pfu vWF (antigen) divided by pfu casein (a specific binding) = enrichment. round Pfu vWF Pfu casein Enrichment First 1 .times. 10.sup.7 2.5 .times. 10.sup.5 40 Second 5 .times. 10.sup.8 2.5 .times. 10.sup.6 200

[0356] TABLE-US-00010 TABLE 9 Number of Inhibitors versus the number of clones tested after the first and the second round of panning as described in Example 20. Number of inhibitors versus round number of clones tested First 4/800 Second 4/96

[0357] TABLE-US-00011 TABLE 10 concentration of VHH (nM) needed to Inhibit binding of vWF to collagen by 50% (IC50) as described in Example 23. Name VHH IC50 (nM) 22-2L-34 10 T76 30 AM-4-15-3 2 22-4L-16 0.5 C37 2 AM-2-75 2

[0358] TABLE-US-00012 TABLE 11 IC50 values for bispecific nanobodies against albumin and against vWF as described in Example 28. IC50 (ng/ml) AM-2-75 100 MSA21/AM-2-75 60 AM-4-15-3 155 MSA21/AM-4-15-3 245 22-4L-16 100 MSA21/22-4L-16 140

[0359] TABLE-US-00013 TABLE 12 Fractional homologies between the amino acid sequences of anti-mouse serum albumin VHHs of the invention. SEQ MSA21 MSA24 MSA210 MSA212 MSA21 1.000 0.834 0.800 0.782 MSA24 -- 1.000 0.782 0.791 MSA210 -- -- 1.000 0.903 MSA212 -- -- -- 1.000

[0360] TABLE-US-00014 TABLE 13 Fractional homologies between anti-TNF-alpha VHHs of the invention. SEQ VHH#1A VHH#7B VHH#2B VHH#3E VHH#3G VHH#10A VHH#2G VHH#1F VHH#1A 1.000 0.601 0.764 0.596 0.622 0.600 0.682 0.629 VHH#7B -- 1.000 0.604 0.635 0.645 0.943 0.653 0.616 VHH#2B -- -- 1.000 0.620 0.645 0.611 0.682 0.661 VHH#3E -- -- -- 1.000 0.875 0.641 0.713 0.689 VHH#3G -- -- -- -- 1.000 0.651 0.779 0.740 VHH#10A -- -- -- -- -- 1.000 0.658 0.614 VHH#2G -- -- -- -- -- -- 1.000 0.741 VHH#1F -- -- -- -- -- -- -- 1.000 VHH#9C -- -- -- -- -- -- -- -- VHH#11E -- -- -- -- -- -- -- -- VHH#10C -- -- -- -- -- -- -- -- VHH#4B -- -- -- -- -- -- -- -- VHH#10D -- -- -- -- -- -- -- -- VHH#12B -- -- -- -- -- -- -- -- VHH#9E -- -- -- -- -- -- -- -- VHH#3F SEQ VHH#9C VHH#11E VHH#10C VHH#4B VHH#10D VHH#12B VHH#9E VHH#3F VHH#1A 0.609 0.601 0.614 0.818 0.642 0.747 0.596 0.604 VHH#7B 0.933 0.933 0.719 0.593 0.614 0.620 0.616 0.324 VHH#2B 0.629 0.620 0.637 0.796 0.634 0.951 0.620 0.645 VHH#3E 0.620 0.643 0.612 0.604 0.648 0.596 0.674 0.682 VHH#3G 0.637 0.637 0.653 0.645 0.689 0.622 0.708 0.716 VHH#10A 0.935 0.934 0.725 0.592 0.612 0.626 0.622 0.637 VHH#2G 0.653 0.669 0.685 0.666 0.746 0.650 0.701 0.717 VHH#1F 0.616 0.616 0.664 0.661 0.714 0.645 0.709 0.717 VHH#9C 1.000 0.941 0.743 0.601 0.622 0.637 0.608 0.624 VHH#11E -- 1.000 0.719 0.601 0.622 0.637 0.608 0.624 VHH#10C -- -- 1.000 0.650 0.606 0.637 0.600 0.632 VHH#4B -- -- -- 1.000 0.611 0.796 0.588 0.629 VHH#10D -- -- -- -- 1.000 0.619 0.674 0.674 VHH#12B -- -- -- -- -- 1.000 0.604 0.637 VHH#9E -- -- -- -- -- -- 1.000 0.654 VHH#3F 1.000

[0361] TABLE-US-00015 TABLE 14 Percentage homologies between anti-IFN-gamma VHHs pf the invention. % Homology MP3D2SRA MP3A3SR MP3C5SR MP3C1SR MP3G8SR MP3D2BR MP3H6SRA MP3B4SRA MP4E4BR MP3D2SRA X 95 66 65 66 82 71 71 71 MP3A3SR -- X 66 66 66 62 72 72 72 MP3C5SR -- -- X 97 98 73 65 65 64 MP3C1SR -- -- -- X 98 72 64 64 64 MP3G8SR -- -- -- -- X 73 65 65 64 MP3D3BR -- -- -- -- -- X 63 63 63 MP3H6SRA -- -- -- -- -- -- X 100 97 MP3B4SRA -- -- -- -- -- -- -- X 97 MP4E4BR -- -- -- -- -- -- -- -- X MP4H8SR -- -- -- -- -- -- -- -- -- MP2F6SR -- -- -- -- -- -- -- -- -- MP3D1BR -- -- -- -- -- -- -- -- -- MP2B5BR -- -- -- -- -- -- -- -- -- MP2C1BR -- -- -- -- -- -- -- -- -- MP4A12SR -- -- -- -- -- -- -- -- -- MP3F4SRA -- -- -- -- -- -- -- -- -- MP3D3BR -- -- -- -- -- -- -- -- -- MP3E5BR -- -- -- -- -- -- -- -- -- MP3C7SRA -- -- -- -- -- -- -- -- -- MP2F1BR -- -- -- -- -- -- -- -- -- MP2C5BR -- -- -- -- -- -- -- -- -- MP2C10BR -- -- -- -- -- -- -- -- -- MP2G5SR -- -- -- -- -- -- -- -- -- MP3B1SRA -- -- -- -- -- -- -- -- -- MP2F10SR -- -- -- -- -- -- -- -- -- MP3A7SRA -- -- -- -- -- -- -- -- -- MP4C10SR -- -- -- -- -- -- -- -- -- MP4D5BR -- -- -- -- -- -- -- -- -- MP3F1SRA -- -- -- -- -- -- -- -- -- MP6D6BR -- -- -- -- -- -- -- -- -- MP6B1BR -- -- -- -- -- -- -- -- -- MP6A8BR -- -- -- -- -- -- -- -- -- MP6B12BR -- -- -- -- -- -- -- -- -- MP6C11BR MP6B10BR % Homology MP4H8SR MP2F6SR MP3D1BR MP2B5BR MP2C1BR MP4A12SR MP3F4SRA MP3D3SR MP3E5BR MP3D2SRA 70 68 69 65 63 64 63 66 67 MP3A3SR 71 70 71 65 63 64 68 66 67 MP3C5SR 63 63 63 60 58 59 64 64 65 MP3C1SR 62 62 62 58 57 58 65 64 64 MP3G8SR 63 63 63 59 58 59 64 64 65 MP3D3BR 62 52 64 59 58 58 62 61 62 MP3H6SRA 97 80 81 67 68 67 75 71 73 MP3B4SRA 97 80 81 67 68 67 75 71 73 MP4E4BR 97 81 82 68 69 68 70 70 71 MP4H8SR X 81 81 66 66 66 72 69 71 MP2F6SR -- X 94 65 68 64 70 67 69 MP3D1BR -- -- X 65 66 65 71 69 71 MP2B5BR -- -- -- X 95 97 63 64 64 MP2C1BR -- -- -- -- X 95 63 64 64 MP4A12SR -- -- -- -- -- X 63 64 64 MP3F4SRA -- -- -- -- -- -- X 64 98 MP3D3BR -- -- -- -- -- -- -- X 98 MP3E5BR -- -- -- -- -- -- -- -- X MP3C7SRA -- -- -- -- -- -- -- -- -- MP2F1BR -- -- -- -- -- -- -- -- -- MP2C5BR -- -- -- -- -- -- -- -- -- MP2C10BR -- -- -- -- -- -- -- -- -- MP2G5SR -- -- -- -- -- -- -- -- -- MP3B1SRA -- -- -- -- -- -- -- -- -- MP2F10SR -- -- -- -- -- -- -- -- -- MP3A7SRA -- -- -- -- -- -- -- -- -- MP4C10SR -- -- -- -- -- -- -- -- -- MP4D5BR -- -- -- -- -- -- -- -- -- MP3F1SRA -- -- -- -- -- -- -- -- -- MP6D6BR -- -- -- -- -- -- -- -- -- MP6B1BR -- -- -- -- -- -- -- -- -- MP6A8BR -- -- -- -- -- -- -- -- -- MP6B12BR -- -- -- -- -- -- -- -- -- MP6C11BR MP6B10BR % Homology MPC7SRA MP2F1BR MP3C5BR MP3C10BR MP2G5SR MP3B1SRA MP2F10SR MP3A7SRA MP4C10SR MP3D2SRA 68 71 70 68 67 63 67 68 60 MP3A3SR 68 72 72 69 67 64 66 67 60 MP3C5SR 66 65 65 65 63 63 64 64 61 MP3C1SR 65 64 63 64 62 63 64 65 60 MP3G8SR 66 65 64 65 63 63 65 65 61 MP3D3BR 63 64 63 63 63 64 63 63 63 MP3H6SRA 75 73 71 73 71 66 75 75 63 MP3B4SRA 75 73 71 73 71 66 75 75 63 MP4E4BR 73 73 71 73 71 66 75 75 63 MP4H8SR 72 71 71 72 71 64 73 73 62 MP2F6SR 71 67 65 73 71 63 71 70 62 MP3D1BR 72 67 65 70 69 63 71 71 62 MP2B5BR 64 65 52 64 52 60 66 63 57 MP2C1BR 64 63 61 66 65 59 66 63 56 MP4A12SR 64 62 60 63 62 59 65 52 56 MP3F4SRA 97 69 67 68 68 62 67 69 60 MP3D3BR 96 70 68 67 67 62 67 67 60 MP3E5BR 98 70 68 68 69 63 68 68 60 MP3C7SRA X 71 69 69 70 63 69 69 61 MP2F1BR -- X 94 66 67 63 68 67 61 MP2C5BR -- -- X 66 67 63 67 65 62 MP2C10BR -- -- -- X 94 62 68 66 59 MP2G5SR -- -- -- -- X 62 67 65 59 MP3B1SRA -- -- -- -- -- X 66 65 91 MP2F10SR -- -- -- -- -- -- X 97 61 MP3A7SRA -- -- -- -- -- -- -- X 61 MP4C10SR -- -- -- -- -- -- -- -- X MP4D5BR -- -- -- -- -- -- -- -- -- MP3F1SRA -- -- -- -- -- -- -- -- -- MP6D6BR -- -- -- -- -- -- -- -- -- MP6B1BR -- -- -- -- -- -- -- -- -- MP6A8BR -- -- -- -- -- -- -- -- -- MP6B12BR -- -- -- -- -- -- -- -- -- MP6C11BR MP6B10BR % Homology MP4D5BR MP3F1SRA MP6D6BR MP6B1BR MP6A8BR MP6B12BR MP8C11BR MP6B10BR MP3D2SRA 72 65 68 67 66 67 76 70 MP3A3SR 73 65 67 67 65 66 77 71 MP3C5SR 67 60 74 63 60 63 70 64 MP3C1SR 67 59 73 63 60 62 70 65 MP3G8SR 66 60 73 63 61 63 71 64 MP3D3BR 65 58 73 64 60 63 68 67 MP3H6SRA 71 69 71 71 68 70 82 70 MP3B4SRA 71 69 71 71 68 70 82 70 MP4E4BR 72 70 71 71 68 70 80 71 MP4H8SR 70 67 69 70 67 70 79 71 MP2F6SR 69 66 67 69 68 67 79 69 MP3D1BR 68 66 67 71 69 69 79 70 MP2B5BR 63 84 65 63 63 62 70 65 MP2C1BR 61 85 65 64 63 62 70 65 MP4A12SR 61 84 64 63 63 62 70 65 MP3F4SRA 72 63 67 68 65 65 76 71 MP3D3BR 70 64 66 66 64 64 75 69 MP3E5BR 72 64 67 68 65 66 77 71 MP3C7SRA 72 64 68 68 66 66 78 71 MP2F1BR 70 64 68 65 64 64 74 67 MP2C5BR 69 63 67 64 62 63 73 67 MP2C10BR 67 66 69 68 64 68 74 73 MP2G5SR 67 65 67 66 64 66 73 73 MP3B1SRA 67 60 67 69 68 69 69 65 MP2F10SR 67 65 71 66 65 67 77 68 MP3A7SRA 68 63 71 65 65 67 77 69 MP4C10SR 64 58 65 64 63 66 66 63 MP4D5BR X 64 69 68 65 67 76 73 MP3F1SRA -- X 65 64 64 63 71 68 MP6D6BR -- -- X 70 65 70 77 73 MP6B1BR -- -- -- X 78 81 76 71 MP6A8BR -- -- -- -- X 75 74 66 MP6B12BR -- -- -- -- -- X 73 68 MP6C11BR X 77 MP6B10BR X

[0362] TABLE-US-00016 TABLE 15 Fractional homologies between anti-vWF VHHs of the Invention. SEQ C37 C37-hum AM-2-75 22-2L-34 22-4L-16 T76 AM-4-15-3 A50 I53 Z29 M53 2A1-4L-79 C37 1.00 0.95 0.99 0.59 0.68 0.63 0.63 0.65 0.59 0.57 0.59 0.57 C37-hum -- 1.00 0.94 0.59 0.68 0.63 0.63 0.65 0.58 0.57 0.60 0.59 AM-2-75 -- -- 1.00 0.60 0.68 0.64 0.64 0.66 0.59 0.57 0.60 0.58 22-2L-34 -- -- -- 1.00 0.77 0.61 0.64 0.71 0.66 0.64 0.64 0.67 22-4L-16 -- -- -- -- 1.00 0.71 0.70 0.80 0.70 0.73 0.69 0.70 T76 -- -- -- -- -- 1.00 0.77 0.68 0.59 0.62 0.61 0.61 AM-4-15-3 -- -- -- -- -- -- 1.00 0.66 0.65 0.61 0.62 0.63 A50 -- -- -- -- -- -- -- 1.00 0.67 0.70 0.66 0.67 I53 -- -- -- -- -- -- -- -- 1.00 0.63 0.69 0.70 Z29 -- -- -- -- -- -- -- -- -- 1.00 0.64 0.64 M53 -- -- -- -- -- -- -- -- -- -- 1.00 0.70 2A1-4L-79 -- -- -- -- -- -- -- -- -- -- -- 1.00 2A1-4L-129 -- -- -- -- -- -- -- -- -- -- -- -- 2A1-4L-34 -- -- -- -- -- -- -- -- -- -- -- -- 2A1-4L-78 -- -- -- -- -- -- -- -- -- -- -- -- 2LA1-15 -- -- -- -- -- -- -- -- -- -- -- -- 3P1-31 -- -- -- -- -- -- -- -- -- -- -- -- 3L-41 -- -- -- -- -- -- -- -- -- -- -- -- 3P2-31 -- -- -- -- -- -- -- -- -- -- -- -- C37-3 -- -- -- -- -- -- -- -- -- -- -- -- C37-4 -- -- -- -- -- -- -- -- -- -- -- -- C37-8 -- -- -- -- -- -- -- -- -- -- -- -- C37-10 -- -- -- -- -- -- -- -- -- -- -- -- SEQ 2A1-4L-129 2A1-4L-34 2A1-4L-78 2LA1-15 3P1-31 3L-41 3P2-31 C37-3 C37-4 C37-8 C37-10 C37 0.61 0.59 0.62 0.61 0.66 0.63 0.60 0.97 0.96 0.93 0.91 C37-hum 0.61 0.60 0.62 0.62 0.66 0.63 0.59 0.97 0.98 0.98 0.96 AM-2-75 0.62 0.60 0.62 0.62 0.67 0.64 0.60 0.96 0.95 0.92 0.92 22-2L-34 0.70 0.70 0.65 0.65 0.66 0.63 0.63 0.59 0.59 0.58 0.58 22-4L-16 0.73 0.72 0.70 0.68 0.73 0.69 0.71 0.67 0.67 0.68 0.68 T76 0.62 0.61 0.65 0.60 0.69 0.65 0.65 0.62 0.62 0.61 0.61 AM-4-15-3 0.65 0.65 0.62 0.67 0.69 0.68 0.62 0.63 0.63 0.62 0.62 A50 0.70 0.67 0.68 0.68 0.69 0.67 0.69 0.64 0.64 0.64 0.64 I53 0.72 0.72 0.64 0.65 0.66 0.65 0.63 0.58 0.58 0.56 0.56 Z29 0.67 0.68 0.71 0.64 0.63 0.61 0.66 0.56 0.56 0.56 0.56 M53 0.70 0.72 0.67 0.60 0.64 0.64 0.69 0.59 0.59 0.58 0.60 2A1-4L-79 0.88 0.85 0.66 0.63 0.64 0.62 0.62 0.57 0.57 0.57 0.57 2A1-4L-129 1.00 0.88 0.70 0.65 0.67 0.64 0.64 0.61 0.61 0.60 0.60 2A1-4L-34 -- 1.00 0.66 0.64 0.65 0.64 0.62 0.58 0.58 0.58 0.58 2A1-4L-78 -- -- 1.00 0.63 0.65 0.62 0.70 0.62 0.62 0.60 0.60 2LA1-15 -- -- -- 1.00 0.65 0.62 0.60 0.60 0.61 0.60 0.60 3P1-31 -- -- -- -- 1.00 0.89 0.67 0.65 0.65 0.64 0.64 3L-41 -- -- -- -- -- 1.00 0.65 0.63 0.63 0.62 0.62 3P2-31 -- -- -- -- -- -- 1.00 0.58 0.58 0.57 0.57 C37-3 -- -- -- -- -- -- -- 1.00 0.99 0.95 0.94 C37-4 -- -- -- -- -- -- -- -- 1.00 0.96 0.95 C37-8 -- -- -- -- -- -- -- -- -- 1.00 0.98 C37-10 -- -- -- -- -- -- -- -- -- -- 1.00

[0363]

Sequence CWU 1

48 1 115 PRT Lama glama 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 2 115 PRT Lama glama 2 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 3 114 PRT Lama glama 3 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser 4 114 PRT Lama glama 4 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asp Gly Ser Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Lys Met Leu Thr 65 70 75 80 Leu Asp Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ala Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser 5 256 PRT Lama glama 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser 145 150 155 160 Gly Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 165 170 175 Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr 180 185 190 Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys 195 200 205 Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val 225 230 235 240 Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 255 6 256 PRT Lama glama 6 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser 145 150 155 160 Gly Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 165 170 175 Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr 180 185 190 Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys 195 200 205 Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val 225 230 235 240 Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 255 7 255 PRT Lama glama 7 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser Gly 145 150 155 160 Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 165 170 175 Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn 195 200 205 Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val Glu 225 230 235 240 Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 255 8 255 PRT Lama glama 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asp Gly Ser Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Lys Met Leu Thr 65 70 75 80 Leu Asp Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ala Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser Gly 145 150 155 160 Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 165 170 175 Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn 195 200 205 Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val Glu 225 230 235 240 Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 255 9 383 PRT Lama glama 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly 145 150 155 160 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val Ser 165 170 175 Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 195 200 205 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr 210 215 220 Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr Val 225 230 235 240 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 245 250 255 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 260 265 270 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser Gly 275 280 285 Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 290 295 300 Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala 305 310 315 320 Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn 325 330 335 Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val Glu 355 360 365 Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 370 375 380 10 241 PRT Lama glama 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Thr Ser Gly Phe Asp Phe Ser Val Ser Trp Met 145 150 155 160 Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Glu 165 170 175 Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Val Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 195 200 205 Met Asp Ser Leu Ile Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg 210 215 220 Ser Pro Ser Gly Ser Phe Arg Gly Gln Gly Thr Gln Val Thr Val Ser 225 230 235 240 Ser 11 247 PRT Lama glama 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Arg Val Asn Ala Met 145 150 155 160 Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu Phe Val Ala Ile 165 170 175 Ile Thr Ser Gly Asp Asn Leu Asn Tyr Ala Asp Ala Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Thr Asp Asn Val Lys Lys Thr Val Tyr Leu Gln Met 195 200 205 Asn Val Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Ile 210 215 220 Leu Gln Thr Ser Arg Trp Ser Ile Pro Ser Asn Tyr Trp Gly Gln Gly 225 230 235 240 Thr Gln Val Thr Val Ser Ser 245 12 249 PRT Lama glama 12 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50

55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr Trp Met 145 150 155 160 Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr 165 170 175 Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu Gln 195 200 205 Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Lys 210 215 220 Val Val Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp Trp Gly 225 230 235 240 Gln Gly Thr Gln Val Thr Val Ser Ser 245 13 255 PRT Lama glama 13 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser Gly 145 150 155 160 Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 165 170 175 Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn 195 200 205 Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val Glu 225 230 235 240 Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 255 14 253 PRT Lama glama 14 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Phe Ser Ala His Ser Val 145 150 155 160 Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 165 170 175 Val Ala Arg Ile Tyr Trp Ser Ser Ala Asn Thr Tyr Tyr Ala Asp Ser 180 185 190 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 195 200 205 Asp Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 210 215 220 Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Thr Val Gly Ser Tyr 225 230 235 240 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 15 250 PRT Lama glama 15 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His Trp 145 150 155 160 Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 165 170 175 Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu 195 200 205 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr 210 215 220 Lys Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp Trp 225 230 235 240 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 16 250 PRT Lama glama 16 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His Trp 145 150 155 160 Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 165 170 175 Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu 195 200 205 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr 210 215 220 Lys Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp Trp 225 230 235 240 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 17 249 PRT Lama glama 17 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His Trp Met 145 150 155 160 Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr 165 170 175 Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu Gln 195 200 205 Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Lys 210 215 220 Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp Trp Gly 225 230 235 240 Gln Gly Thr Gln Val Thr Val Ser Ser 245 18 249 PRT Lama glama 18 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asp Gly Ser Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Lys Met Leu Thr 65 70 75 80 Leu Asp Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ala Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His Trp Met 145 150 155 160 Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr 165 170 175 Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu Gln 195 200 205 Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Lys 210 215 220 Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp Trp Gly 225 230 235 240 Gln Gly Thr Gln Val Thr Val Ser Ser 245 19 246 PRT Lama glama 19 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Trp Tyr Pro Met 145 150 155 160 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr 165 170 175 Ile Ser Thr Tyr Gly Glu Pro Arg Tyr Ala Asp Ser Val Lys Ala Asp 180 185 190 Ser Pro Ser Ser Glu Thr Thr Pro Thr Thr Arg Cys Ile Cys Asn Glu 195 200 205 Gln Pro Glu Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala 210 215 220 Gly Thr Ser Ser Tyr Leu Pro Gln Arg Gly Asn Trp Asp Gln Gly Thr 225 230 235 240 Gln Val Thr Val Ser Ser 245 20 243 PRT Lama glama 20 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ala Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn Ser Met 145 150 155 160 Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala His 165 170 175 Ala Leu Ala Asp Gly Ser Ala Ser Tyr Arg Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met 195 200 205 Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr Val 210 215 220 Pro Ser Ser Val Thr Lys Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr 225 230 235 240 Val Ser Ser 21 250 PRT Lama glama 21 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Ala Met 145 150 155 160 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala 165 170 175 Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 195 200 205 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala 210 215 220 Asp Thr Gly Gly Ile Ser Trp Ile Arg Thr Gln Gly Tyr Asn Tyr Trp 225 230 235 240 Gly Gln Gly Thr Gln Val Thr Val Ser Ser

245 250 22 252 PRT Lama glama 22 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Ser Ser Tyr Ala 145 150 155 160 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 165 170 175 Ser Ile Thr Trp Thr Gly Thr Gly Thr Tyr Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp His Ala Gly Thr Thr Val Tyr Leu 195 200 205 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Val Asp Arg Arg Ser Ser Thr Tyr Tyr Leu Met Lys Gly Glu Tyr Asp 225 230 235 240 Tyr Arg Gly Arg Gly Thr Gln Val Thr Val Ser Ser 245 250 23 252 PRT Lama glama 23 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Ser Ser Tyr Ala 145 150 155 160 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 165 170 175 Ser Ile Thr Trp Thr Gly Thr Gly Thr Tyr Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp His Ala Gly Thr Thr Val Tyr Leu 195 200 205 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Val Asp Arg Arg Ser Ser Thr Tyr Tyr Leu Met Lys Gly Glu Tyr Asp 225 230 235 240 Tyr Arg Gly Arg Gly Thr Gln Val Thr Val Ser Ser 245 250 24 251 PRT Lama glama 24 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Ser Ser Tyr Ala Met 145 150 155 160 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ser 165 170 175 Ile Thr Trp Thr Gly Thr Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp His Ala Gly Thr Thr Val Tyr Leu Gln 195 200 205 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val 210 215 220 Asp Arg Arg Ser Ser Thr Tyr Tyr Leu Met Lys Gly Glu Tyr Asp Tyr 225 230 235 240 Arg Gly Arg Gly Thr Gln Val Thr Val Ser Ser 245 250 25 253 PRT Lama glama 25 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125 Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr Asn 145 150 155 160 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 165 170 175 Ala Ile Ser Trp Asn Gly Gly Ser Thr Tyr Tyr Asp Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Leu Val Tyr Leu 195 200 205 Gln Met Asn Ser Leu Asn Phe Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Cys Ala Ala Asn Pro Tyr Gly Ile Pro Gln Tyr Arg Glu Asn Arg Tyr 225 230 235 240 Asp Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 26 247 PRT Lama glama 26 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Ala 115 120 125 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Asp Ser 130 135 140 Leu Arg Leu Ser Cys Val Ala Ser Gly Gly Thr Phe Ser Arg Tyr Ala 145 150 155 160 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 165 170 175 Arg Ile Gly Tyr Ser Gly Arg Ser Ile Ser Tyr Ala Thr Ser Val Glu 180 185 190 Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 195 200 205 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Ser Leu Val Ser Gly Thr Leu Tyr Gln Ala Asp Tyr Trp Gly Gln Gly 225 230 235 240 Thr Gln Val Thr Val Ser Ser 245 27 252 PRT Lama glama 27 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125 Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ile Tyr Asn Met 145 150 155 160 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Gly 165 170 175 Ile Ser Trp Asn Gly Gly Ser Ile Tyr Tyr Thr Ser Ser Val Glu Gly 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr Leu Gln 195 200 205 Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Ser 210 215 220 Lys Gly Arg Pro Tyr Gly Val Pro Ser Pro Arg Gln Gly Asp Tyr Asp 225 230 235 240 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 28 128 PRT Lama glama 28 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Thr Thr Phe Ser Ser Ala 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Gly Ala Ile Lys Trp Ser Gly Thr Ser Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Val Tyr Thr Cys 85 90 95 Ala Ala Asp Arg Asp Arg Tyr Arg Asp Arg Met Gly Pro Met Thr Thr 100 105 110 Thr Asp Phe Arg Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 29 124 PRT Lama glama 29 Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Phe 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Gly Ser Ser Gly Ile Thr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Leu Cys Tyr Cys 85 90 95 Ala Val Asn Arg Tyr Gly Ile Pro Tyr Arg Ser Gly Thr Gln Tyr Gln 100 105 110 Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 30 120 PRT Lama glama 30 Glu Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Asn Asp Tyr 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Asp Met Val 35 40 45 Ala Thr Ile Ser Ile Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Ala His Arg Gln Thr Val Val Arg Gly Pro Tyr Leu Leu Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 31 123 PRT Lama glama 31 Gln Val Gln Leu Val Glu Ser Gly Gly Lys Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ser Gly Arg Ser Asn Ser Tyr Asn Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Thr Asn Leu Trp Pro Arg Asp Arg Asn Leu Tyr Ala Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 32 125 PRT Lama glama 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Leu Gly Ile Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Gly Thr Thr Arg Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Thr Lys Asn Ala Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Asp Ser Ser Gly Arg Leu Tyr Trp Thr Leu Ser Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 33 125 PRT Lama glama 33 Gln Val Gln Leu Val Glu Phe Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Leu Gly Ile Tyr 20 25 30 Lys Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Gly Thr Thr Arg Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Thr Lys Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Asp Ser Ser Gly Arg Leu Tyr Trp Thr Leu Ser Thr Ser Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 34 124 PRT Lama glama 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Pro Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Gly Val Thr Trp Ser Gly Ser Ser Thr Phe Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ala Ser Arg Asp Ser Ala Lys Asn Thr Val Thr 65 70 75 80 Leu Glu Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Tyr Gly Gly Gly Leu Tyr Arg Asp Pro Arg Ser Tyr Asp 100 105 110 Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 35 131 PRT Lama glama 35 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Ala Trp 20 25

30 Pro Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Arg Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Ser Asp Asn Ala Asn Asn Thr Val Tyr Leu Gln 65 70 75 80 Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 85 90 95 Pro Ser Gly Pro Ala Thr Gly Ser Ser His Thr Phe Gly Ile Tyr Trp 100 105 110 Asn Leu Arg Asp Asp Tyr Asp Asn Trp Gly Gln Gly Thr Gln Val Thr 115 120 125 Val Ser Ser 130 36 126 PRT Lama glama 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp His Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Glu Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Leu Leu Leu Arg Val Glu Glu Leu Gln Ala Ser Asp 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr Val Ser Ser 115 120 125 37 128 PRT Lama glama 37 Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Cys Ile Ser Asn Ser Asp Gly Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ala Asp Arg His Tyr Ser Ala Ser His His Pro Phe Ala Asp 100 105 110 Phe Ala Phe Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 38 120 PRT Lama glama 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Tyr Gly Leu Thr Phe Trp Arg Ala 20 25 30 Ala Met Ala Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Val Ala Arg Asn Trp Gly Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Val Arg Thr Tyr Gly Ser Ala Thr Tyr Asp Ile Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 39 123 PRT Lama glama 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Asp Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Phe Ser Gly Arg Thr Phe Ala Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Arg Asn Gly Gly Thr Thr Asn Tyr Ala Asp Ala Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Ala Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Glu Trp Pro Phe Ser Thr Ile Pro Ser Gly Trp Arg Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 40 125 PRT Lama glama 40 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Pro Thr Ala Ser Ser His 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Val Gly Ile Asn Arg Gly Gly Val Thr Arg Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala Val Ser Arg Asp Asn Val Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Ser Ala Ile Tyr Ile Cys 85 90 95 Ala Ala Arg Pro Glu Tyr Ser Phe Thr Ala Met Ser Lys Gly Asp Met 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 41 23 DNA Lama glama 41 ggctgagctc ggtggtcctg gct 23 42 45 DNA Lama glama 42 aactggaaga attcgcggcc gcaggaattt tttttttttt ttttt 45 43 20 DNA Lama glama 43 ctggccccag aagtcatacc 20 44 19 DNA Lama glama 44 tgtgcatgtg cagcaaacc 19 45 46 DNA Lama glama 45 gtcctcgcaa ctgcggccca gccggcctgt gcatgtgcag caaacc 46 46 42 DNA Lama glama 46 gtcctcgcaa ctgcgcggcc gcctggcccc agaagtcata cc 42 47 253 DNA Lama glama 47 aagcttgcat gcaaattcta tttcaaggag acagtcataa tgaaatacct attgcctacg 60 gcagccgctg gattgttatt actcgcggcc cagccggcca tggggcctaa taggcggccg 120 cacaggtgca gctgcaggag tcataatgag ggacccaggt caccgtctcc tcagaacaaa 180 aactcatctc agaagaggat ctgaatgggg ccgcacatca tcatcatcat cattaatgag 240 aattcactgg ccg 253 48 61 PRT Lama glama 48 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Gly Pro Ala Ala Ala Gln Val Gln Leu Gln Glu 20 25 30 Ser Gly Thr Gln Val Thr Val Ser Ser Glu Gln Lys Leu Ile Ser Glu 35 40 45 Glu Asp Leu Asn Gly Ala Ala His His His His His His 50 55 60

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