Easy To Use Patents Search & Patent Lawyer Directory

At Patents you can conduct a Patent Search, File a Patent Application, find a Patent Attorney, or search available technology through our Patent Exchange. Patents are available using simple keyword or date criteria. If you are looking to hire a patent attorney, you've come to the right place. Protect your idea and hire a patent lawyer.


Search All Patents:



  This Patent May Be For Sale or Lease. Contact Us

  Is This Your Patent? Claim This Patent Now.



Register or Login To Download This Patent As A PDF




United States Patent Application 20170369573
Kind Code A1
GAUTHIER; LAURENT December 28, 2017

MONOMERIC FC DOMAINS

Abstract

Monomeric Fc-domains are provided, as well as multimeric and single chain proteins comprising the monomeric Fc-domains. The proteins have utility in the treatment of disease. Included, inter glia, are multi specific polypeptides.


Inventors: GAUTHIER; LAURENT; (MARSEILLE, FR)
Applicant:
Name City State Country Type

INNATE PHARMA

MARSEILLE

FR
Family ID: 1000002870192
Appl. No.: 15/541025
Filed: January 4, 2016
PCT Filed: January 4, 2016
PCT NO: PCT/EP2016/050032
371 Date: June 30, 2017


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
62099634Jan 5, 2015

Current U.S. Class: 1/1
Current CPC Class: C07K 16/2803 20130101; C07K 16/468 20130101; G06K 9/6267 20130101; G06K 9/66 20130101; G06T 11/003 20130101; G06T 7/0012 20130101; G06T 2207/30096 20130101; C07K 2317/41 20130101; C07K 2317/526 20130101; C07K 2317/64 20130101; C07K 2317/71 20130101; C07K 2317/92 20130101; G06T 2207/10081 20130101; G06T 7/11 20170101
International Class: C07K 16/28 20060101 C07K016/28; G06K 9/62 20060101 G06K009/62; G06T 7/11 20060101 G06T007/11; G06T 7/00 20060101 G06T007/00; G06T 11/00 20060101 G06T011/00; C07K 16/46 20060101 C07K016/46; G06K 9/66 20060101 G06K009/66

Claims



1-36. (canceled)

37. A polypeptide chain capable of binding to human neonatal Fc receptor (FeRn), comprising a first antigen binding domain (ABD) and, optionally, a second ABD (ABD.sub.2) and a first and a second CH3 domain separated by a flexible linker, wherein the polypeptide chain has a domain arrangement selected from: (ABD)-linker-CH2-CH3-linker-CH3; (ABD.sub.1)-linker-CH2-CH3-linker-CH3-linker-(ABD.sub.2); and (ABD.sub.1)-linker-CH1-CH2-CH3-linker-CH3-linker-(ABD.sub.2).

38. The polypeptide chain of claim 37, wherein the first and second CH3 domain are separated by a linker of sufficient length to permit the first and second CH3 domain to associate with one another via non-covalent interactions.

39. The polypeptide chain of claim 37, wherein the amino acid sequence separating the first and second CH3 domains is a peptide linker having between 10 and 30 residues.

40. The polypeptide chain of claim 37, wherein the amino acid sequence separating the first and second CH3 domains is a peptide linker having the formula (G.sub.4S).sub.x wherein x is 2, 3, 4, 5 or 6.

41. The polypeptide chain of claim 37, wherein the polypeptide substantially lacks binding via its Fc domain to one or more human Fey receptors selected from the group consisting of: CD16, CD32A, CD32B and CD64.

42. The polypeptide chain of claim 37, wherein the CH2 domain comprises an amino acid substitution to decrease binding to a human Fey receptor.

43. The polypeptide chain of claim 37, wherein each ABD comprises the hypervariable regions, optionally the heavy and light chain CDRs, of an antibody.

44. The polypeptide chain of claim 37, wherein each ABD comprises an immunoglobulin heavy chain variable domain and a light chain variable domain.

45. The polypeptide chain of claim 37, wherein one of the antigen binding domains binds to a cancer antigen.

46. The polypeptide chain of claim 37, wherein one of the antigen binding domains binds to a cell surface receptor on an immune effector cell.

47. The polypeptide chain of claim 37, wherein one of the antigen binding domains binds to a cell surface receptor on an immune effector cell and the second antigen binding domain binds to a cancer, viral or bacterial antigen.

48. The polypeptide chain of claim 47, wherein the cell surface receptor on an immune effector cell is a member of the immunoglobulin superfamily.

49. The polypeptide chain of claim 47, wherein the cell surface receptor on an immune effector cell is a member of the natural cytotoxicity receptor family or the NK cell lectin-like receptor family.

50. The polypeptide chain of claim 47, wherein the cell surface receptor on an immune effector cell is selected from the group consisting of: activating KIR, NKG2D, NKp30, NKp44, NKp46, CD8 and CD3.

51. A protein comprising a polypeptide chain of claim 37, and one or more further polypeptide chains that associate therewith.

52. A pharmaceutical composition comprising a polypeptide chain of claim 37, and a pharmaceutically acceptable carrier.

53. A method of treating a cancer, infectious disease or an inflammatory or autoimmune disease in a subject comprising administering to the subject a composition of claim 37.

54. A method of treating a cancer, infectious disease or an inflammatory or autoimmune disease in a subject comprising administering to the subject a composition of claim 51.
Description



CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/099,634, filed Jan. 31, 2015; which is incorporated herein by reference in its entirety; including any drawings.

REFERENCE TO THE SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled "NTECH2 PCT_ST25 txt", created Dec. 31, 2015, which is 100 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] Monomeric Fc-domains are provided, as well as multimeric and single chain proteins comprising the monomeric Fc-domains. The proteins have utility in the treatment of disease. Included, inter alia, are multispecific polypeptides.

BACKGROUND

[0004] Antibody fragments such as Fab, Fv, scFv, VH and VHH, and more new antibody fragment formats are under development that have decreased size and may provide improved tissue penetration, for example for treating solid tumors. New formats have also been developed for bispecific antibodies binding two different epitopes offering opportunities for increasing specificity, broadening potency, and utilizing novel mechanisms of action that cannot be achieved with a traditional monoclonal antibody. However, many of these formats have very short half-lives compared to full-size IgG.

[0005] The Fc domain increases the half-life of an IgG through its unique pH-dependent association with the neonatal Fc receptor (FcRn). After internalization, the Fc domain of IgG can bind to FcRn in the acidic environment of the endosome, so that the IgG is then cycled onto the cell surface and re-released into circulation. This biological system protects IgG from degradation and results in a long serum half-life. Fusions of an Fc domain and a therapeutic molecule have an extended half-life. In addition, since the Fc fragment of IgG consists of a tightly packed homodimer, two therapeutic proteins are present in each molecule. One approach to produce smaller monomeric Fc fusion involved introducing mutations into the CH3 domain to prevent CH3-CH3 homodimerization (see, e.g., WO2013/138643 and WO2011/063348). However, introduction of mutations involves risk of immunogenicity when administered to humans.

[0006] There remains therefore a need to produce new protein formats that permit the use of monomeric Fc domains.

SUMMARY OF THE INVENTION

[0007] The present invention arises from the discovery of a Fc-polypeptide construct with two immunoglobulin CH3 domains arranged in tandem that yields functional proteins in which the Fc domain remains monomeric and interacts with the neonatal Fc receptor (FcRn). The constructs permit a range of single chain and multi-chain (multimeric) proteins to be constructed, including but not limit to single chain and multimeric bispecific binding proteins, e.g., antibodies.

[0008] The two CH3 domains within a single polypeptide chain (referred to as "tandem CH3 domains") can be incorporated into any Fc-domain containing molecule, e.g. a molecule that comprises other amino acid sequences or protein domains on the same polypeptide chain and/or on additional polypeptide chains that multimerize with the tandem CH3 domain-containing polypeptide chain. While the Fc domain remains monomeric (e.g., not an Fc dimer formed from two Fc domains that undergo CH3-CH3 dimerization), the protein that contains the tandem CH3 domain may be monomeric or multimeric (containing more than one polypeptide chains, e.g., a dimer, trimer, tetramer, pentamer).

[0009] A tandem CH3 domain-containing polypeptide chain may typically comprise a CH2 domain linked to one of the two CH3 domains, and optionally further a protein domain of interest, e.g., an immunoglobulin variable region such as a heavy chain variable region (VH) and/or light chain variable region (VL), and/or generally any antigen binding domain. The domain can for example be fused directly to the CH2 or CH3 domain or via intervening sequence of amino acid residues such as a peptide linker, a hinge, a CH1 domain or a CK domain, and the like. Providing a suitable linker (e.g. any amino acid sequence or other flexible molecule) within the tandem CH3 domain between the two CH3 domains permits the CH3 domains to associate with one another by non-covalent interactions. The two CH3 domains are therefore not available for CH3-CH3 dimerization with other polypeptide chains, thereby avoiding homo-dimerization among CH3 domain-containing molecules. In one example, tandem CH3 domains are incorporated into a polypeptide that further comprises a CH2 domain, resulting in an Fc domain that remains monomeric. The single polypeptide chain having a monomeric Fc domain can be used as such or can be combined with further polypeptide chain to give rise to a multimeric polypeptide having a monomeric Fc domain.

[0010] These Fc-domain containing molecules disclosed herein have the advantage of accommodating native CH3 domains. The molecules can also be easily produced. They can furthermore harbor more than one functional protein domain, e.g. an antigen binding domain, permitting a wide range of Fc-domain containing molecules to be produced. In particular, bispecific antibodies can be produced that have a monomeric Fc domain that binds the neonatal Fc (FcRn) receptor.

[0011] In one aspect provided is a tandem CH3 domain. In one aspect provided is an Fc domain comprising a CH2 domain and the tandem CH3 domain. In one aspect provided is a polypeptide chain comprising the tandem CH3 domain or the Fc domain. In one aspect provided is a multimeric polypeptide comprising the polypeptide chain comprising such tandem CH3 domain. In one embodiment, the two CH3 domains of the tandem CH3 domain are separated by a flexible peptide linker. In one embodiment, the two CH3 domains of the tandem CH3 domain associate with one another via non-covalent interactions. In one embodiment, the two CH3 domains associate with one another via the CH3 dimerization interface of each CH3 domain. In one embodiment, the tandem CH3 domain is incapable of further CH3-CH3 dimerization (e.g., with a CH3 domain of a further polypeptide chain).

[0012] In one embodiment, provided is a polypeptide chain comprising a first and a second CH3 domain, wherein the two CH3 domains associate with one another via non-covalent interactions. In one embodiment, the two CH3 domains associate with one another via the CH3 dimerization interface of each CH3 domain. In one embodiment, the polypeptide chain does not dimerize with another polypeptide chain comprising an Fc domain.

[0013] In one embodiment, provided is a multimeric (e.g. a dimeric, trimeric, tetrameric, pentameric) protein having a monomeric Fc domain, the protein comprising a polypeptide chain comprising an Fc domain comprising a CH2 domain and a tandem CH3 domain. In one embodiment, the two CH3 domains of the Fc domain associate with one another via non-covalent interactions.

[0014] In one embodiment of any aspect herein, a first CH3 domain is connected to a second CH3 domain by a linker. The tandem CH3 domains can thus be placed on the same polypeptide chain so as to have the domain arrangement as follows:

[0015] --CH3-linker-CH3-

[0016] The linker will be a flexible linker (e.g. peptide linker). In one embodiment the linker permits the CH3 domains to associate with one another by non-covalent interactions. In one embodiment, the linker is a peptide linker having 10-50 amino acid residues. In one embodiment, the linker has the formula (G.sub.4S).sub.x. Optionally, x is 2, 3, 4, 5 or 6.

[0017] In any of the embodiments, each CH3 domain is independently a human full-length and/or native CH3 domain, or a fragment or modified CH3 domain which retains a functional CH3 dimerization interface (e.g. thereof that retains the ability to undergo CH3-CH3 dimerization with another CH3 domain).

[0018] In any of the embodiments herein, an exemplary CH3 domain for use in a tandem CH3 domain or Fc domain comprises an amino acid sequence of SEQ ID NO: 1, or a sequence at least 70%, 80%, 90%, 95% or 98% identical thereto.

[0019] In any of the embodiments herein, an exemplary tandem CH3 domain comprises an amino acid sequence of SEQ ID NO: 2, or a sequence at least 70%, 80%, 90%, 95% or 98% identical thereto.

[0020] In one aspect provided is a polypeptide comprising a monomeric Fc domain comprising a native CH3 domain (i.e., which retains a functional CH3 dimerization interface). In one aspect the polypeptide is a monomeric polypeptide. In one aspect the polypeptide is a multimeric polypeptide comprising a monomeric Fc domain. In one embodiment, the tandem CH3 domain is incapable of further CH3-CH3 dimerization (i.e. with a further CH3 domain of a polypeptide).

[0021] In one aspect of any embodiment, provided is an isolated single chain polypeptide comprising a tandem CH3 domain. Also provided is a single chain Fc domain molecule comprising a tandem CH3 domain, such as a fusion protein including the monomeric Fc domain molecule or tandem CH3 domain. In one embodiment, the Fc domain molecule comprises a soluble polypeptide fused to an Fc domain. In one embodiment, the Fc domain molecule comprises an extracellular domain (ECD) of a cell surface receptor fused to an Fc domain, optionally via intervening sequences of amino acid residues (e.g. a linker, a protein domain, etc.). One example of Fc fusion polypeptide is a polypeptide with the domain arrangement, from N-terminus (left) to C-terminus (right):

[0022] (ECD)-CH2-CH3-linker-CH3.

[0023] In one embodiment the fusion protein is a monomeric or multimeric antibody-derived polypeptide. These molecules include a further polypeptide fused at the N- or C-terminus of the Fc domain or CH3 domain. In some non-limiting examples, the heterologous protein is a protein of interest (shown as (P) in the domain arrangement below), an antigen binding domain (ABD), a immunoglobulin variable domain, a cytokine, or a receptor polypeptide. A protein (P) can be any desired sequence of amino acid residues, e.g. 1-20, 20-50, 10-100, 1-200, 1-500, or 20-500 amino acid residues. Tandem CH3 domains can be comprised in polypeptide chains having one the following domain arrangements, which polypeptide chains can remain monomeric or can associate with further polypeptide chains to form multimeric polypeptides with monomeric Fc domains:

[0024] (P)-CH2-CH3-linker-CH3,

[0025] (ABD)-CH2-CH3-linker-CH3,

or

[0026] (scFv)-CH2-CH3-linker-CH3.

[0027] If desired, a heterologous polypeptide can be fused to both the N- and C-terminus of the Fc domain or CH3 domain, e.g. a tandem CH3 domains can be comprised in polypeptide chain together with a first and second protein of interest (shown as (P.sub.1 and P.sub.2) in the domain arrangement below), according to an exemplary domain arrangement:

[0028] (P.sub.1)-CH2-CH3-linker-CH3-(P.sub.2).

[0029] Examples of multimeric antibody-derived polypeptides with monomeric Fc domains include polypeptides with domain arrangements below, where the polypeptide chains can associate with one another by CH1-CK heterodimerization (linkage shown between CH1 and CK), forming interchain non-covalent bonds, and optionally further disulfide bonds) between respective complementary CH1 and CK domains.

[0030] One example is shown as follows:

##STR00001##

wherein the variable region in one chain is a VH and the variable region in the other chain is a VL, such that the VH and VL form an antigen binding domain that binds an antigen of interest, and wherein the CH1 or CK constant region of one chain is a CH1 and the CH1 or CK constant region in the other chain is a CK, such that the CH1 and CK in the respective chains associate by non-covalent bonds (and optionally further disulfide bonds). Each polypeptide chain may optionally comprise one or more domains at its N- and/or C-terminus. In one embodiment, a hinge domain is placed between the CH1 or CK domain and the CH2 domain.

[0031] Examples of protein domain arrangements include:

##STR00002##

[0032] Each polypeptide chain may optionally comprise one or more domains at its N- and/or C-terminus. In one embodiment, a hinge domain is placed between the CH1 or CK domain and the CH2 domain. In one embodiment of any polypeptide herein, a VH or VK domain, or a CH1 or CK (or CA) domain, will be fused to an Fc domain (e.g. or a CH2 domain thereof) via a hinge region.

[0033] The antibodies are also particularly well adapted for use as a multispecific (e.g. bispecific) binding protein. For example, the bispecific protein can bind a first antigen on a target cell to be eliminated and a second antigen on an immune effector cell (e.g. an NK cell and/or a T cell), where the effector cells are directed to the target cell, e.g. a cancer cell. The multispecific antibody retains FcRn binding yet lacks Fc.gamma.R binding such that the multispecific antibody has attractive in vivo pharmacodynamics yet is selective for the particular effector cells of interest (i.e. the cells expressing the second antigen), thereby reducing toxicity mediated by immune cells other than those expressing the particular effector cell surface antigen to which the antibody binds. The multispecific polypeptide is capable, for example, of directing antigen-of-interest-expressing effector cells to lyse a target cell expressing a target antigen, e.g. cancer antigen, viral antigen, etc. The multispecific antibody is particularly effective when binding both effector cell surface protein and a second antigen (an antigen expressed by a target cell) in monovalent fashion.

[0034] In one embodiment, provided is a multispecific (e.g. bispecific) polypeptide that binds a first and a second antigen each in monovalent fashion, comprising: a first antigen binding domain (ABD.sub.1) that specifically binds to a first antigen of interest, a second antigen binding domain (ABD.sub.2) that specifically binds a second antigen of interest, and a first and a second human CH3 domain separated by a flexible linker. In one embodiment, the first and second human CH3 domain separated by a flexible linker is interposed between the first and second antigen binding domains. In one embodiment, a human CH2 domain is placed between an ABD and a CH3 domain. In one embodiment, the first and second antigen binding domains each comprise an immunoglobulin variable heavy domain and a variable light domain. In one embodiment, the first and second antigen binding domains are each a scFv.

[0035] Examples include polypeptides with domain arrangements below, where the polypeptide chains can associate with one another by CH1-CK heterodimerization (linkage shown between CH1 and CK), forming interchain non-covalent bonds, and optionally further disulfide bonds) between respective complementary CH1 and CK domains.

[0036] One example is shown as follows:

##STR00003##

wherein each of the two scFv forms an antigen binding domain that binds an antigen of interest (e.g. two different antigens), and wherein the CH1 or CK constant region of one chain is a CH1 and the CH1 or CK constant region in the other chain is a CK, such that the CH1 and CK in the respective chains associate by non-covalent bonds (and optionally further disulfide bonds). Each polypeptide chain may optionally comprise one or more domains at its N- and/or C-terminus. In one embodiment, a hinge domain is placed between the CH1 or CK domain and the CH2 domain.

[0037] In one embodiment of any multispecific polypeptide, each CH3 domain (or fragment thereof) has the amino acid of a native human CH3 domain (or fragment thereof). Optionally, the multispecific polypeptide (and/or its Fc domain) has decreased binding to a human Fc.gamma. receptor, e.g., compared to a full length wild type human IgG1 antibody. In one embodiment, the first and second antigen binding domains are separated by an Fc domain that comprises, from N- to C-terminal, a human CH2 domain, a first human CH3 domain, a flexible amino acid linker, and a second human CH3 domain.

[0038] In one embodiment of any multispecific polypeptide, the two CH3 domains associate with one another via non-covalent interactions. In one embodiment, the two CH3 domains associate with one another via the CH3 dimerization interface of each CH3 domain. In one embodiment, the tandem CH3 domain is incapable of further CH3-CH3 dimerization (i.e. with a further CH3 domain or polypeptide comprising such).

[0039] In one embodiment, the multispecific polypeptide (and/or its Fc domain) is capable of binding to human neonatal Fc receptor (FcRn).

[0040] In one embodiment, the multispecific polypeptide does not form a dimer with another Fc-derived polypeptide (e.g. does not form a homodimer with another identical polypeptide).

[0041] In one examples of bispecific polypeptides, tandem CH3 domains can be comprised in a polypeptide chain having the following domain arrangement, which polypeptide chain can remain monomeric or can associate with further polypeptide chains to form multimeric polypeptides with monomeric Fc domains:

[0042] (ABD.sub.1)-CH2-CH3-linker-CH3-(ABD.sub.2)

[0043] Optionally, the polypeptide further comprises linking amino acids between the aforementioned domains. In one embodiment, a CH1 or CK domain or fragment thereof, a hinge region or fragment thereof, and/or a linker peptide can be placed between ABD.sub.1 and CH2. In one embodiment, a CH1 or CK domain or fragment thereof, a hinge region or fragment thereof, and/or a linker peptide can be placed between CH3 and ABD.sub.2.

[0044] Examples of multimeric antibody-derived bispecific polypeptides with monomeric Fc domains include polypeptides with domain arrangements below, where the polypeptide chains can associate with one another by CH1-CK heterodimerization (linkage shown between CH1 and CK), forming interchain disulfide bonds between respective hinge domains and/or between complementary CH1 and CK domains:

##STR00004##

[0045] In one aspect of any embodiment herein, ABD.sub.1 and/or ABD.sub.2 each independently comprise a scFv, an affibody, a V.sub.H domain, a V.sub.L domain, a single domain antibody (nanobody) such as a V-NAR domain or a V.sub.HH domain. Optionally, ABD.sub.1 and ABD.sub.2 each comprise a scFv, an affibody, a V.sub.H domain, a V.sub.L domain, or a single domain antibody (nanobody) such as a V-NAR domain or a V.sub.HH domain. Optionally, each ABD.sub.1 and ABD.sub.2 are each a human or humanized scFv, an affibody, a V.sub.H domain, a V.sub.L domain, a single domain antibody (nanobody) such as a V-NAR domain or a V.sub.HH domain.

[0046] In one aspect of any embodiment herein, the polypeptide may (or may not) further comprise additional protein domains at the C- and/or N-terminus, e.g. a further ABD, variable domain, scFv, etc.

[0047] In one embodiment, the polypeptide (and/or its Fc domain) has decreased binding (i.e. via its Fc domain) to a human Fc.gamma. receptor (e.g. CD16, CD32A, CD32B and/or CD64), e.g., compared to a full length wild type human IgG1 antibody. In one embodiment, the polypeptide has decreased (e.g. partial or complete loss of) antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP), FcR-mediated cellular activation (e.g. cytokine release through FcR cross-linking), and/or FcR-mediated platelet activation/depletion, as mediated by immune effector cells that do not express an antigen of interest bound by the antigen binding domains of the polypeptide (i.e. in the absence of cells that express an antigen of interest bound by the antigen binding domains), compared, e.g., to the same polypeptide having a wild-type Fc domain of human IgG1 isotype.

[0048] An antigen binding domain, when present, may comprise an antibody heavy chain variable domain and a light chain variable domain, optionally the first antigen binding domain comprises a scFv.

[0049] In one embodiment, a first or second antigen binding domain, when present, each independently comprises an antibody heavy chain variable domain and a light chain variable domain, optionally the first antigen binding domain comprises a scFv.

[0050] In one embodiment, the tandem CH3 domain is substantially incapable of forming a CH3-CH3 dimer with another Fc-derived polypeptide in solution. For example, the tandem CH3 domain does not form a dimer with another identical polypeptide, or does not form a dimer with a wild-type human IgG heavy chain.

[0051] In one embodiment, a CH2 domain comprises an amino acid modification that decreases binding to a human Fc.gamma. receptor, compared to a wild-type CH2 domain. In one embodiment, the Fc polypeptide comprises N-linked glycosylation at residue N297. In one embodiment, the Fc polypeptide lacks N-linked glycosylation or has modified N-linked glycosylation at residue N297. In one embodiment, the Fc polypeptide comprises an N297X mutation (EU numbering), wherein X is any amino acid other than asparagine.

[0052] In one aspect of any of the embodiments herein, a polypeptide is a bispecific polypeptide that comprises a first antigen binding domain that bind a cancer antigen and a second antigen binding domain that binds an antigen expressed by an immune effector cell, wherein the protein has a higher binding affinity (monovalent) for a cancer antigen (or a viral or bacterial antigen) than for an antigen expressed by an immune effector cell. Such polypeptide will provide for advantageous pharmacological properties. The polypeptide will more closely mimic (albeit with better specificity) a natural antibody that has high avidity for target antigen and lower affinity for Fc receptors. In one aspect of any of the embodiments of the invention, the polypeptide has a Kd for binding (monovalent) to an antigen expressed by immune effector cell of less than 10.sup.-6 M, optionally less than 10.sup.-7 M, optionally less than 10.sup.-9 M, or optionally less than 10.sup.-9M. In one aspect of any of the embodiments of the invention, the polypeptide has a Kd for binding (monovalent) to an antigen expressed by immune effector cell of less than 10.sup.-7 M, optionally less than 10.sup.-9 M, optionally less than 10.sup.-9 M, or optionally less than 10.sup.-10 Min one embodiment, the polypeptide has a Kd for binding (monovalent) to a cancer, viral or bacterial antigen that is less than (i.e. has better binding affinity than) the Kd for binding (monovalent) to the antigen expressed by immune effector cell. For example, the polypeptide has a Kd for binding (monovalent) to a cancer, viral or bacterial antigen that is at least 5-fold less, optionally at least 10-fold less, than the Kd for binding (monovalent) to the antigen expressed by immune effector cell

[0053] In one embodiment of any of the polypeptides herein, the multispecific polypeptide is capable of directing effector cells (e.g. a T cell, an NK cell) expressing one of first or second antigen of interest to lyse a target cell expressing the other of said first of second antigen of interest (e.g. a cancer cell).

[0054] In one aspect of any of the embodiments herein, provided is a recombinant nucleic acid encoding a polypeptide chain of any of the proteins of the disclosure. In one aspect of any of the embodiments herein, provided is a recombinant host cell comprising a nucleic acid encoding a polypeptide chain of any of the proteins of the disclosure, optionally wherein the host cell produces a protein of the disclosure with a yield (final productivity after purification) of at least 1, 2, 3 or 4 mg/L. Also provided is a kit or set of nucleic acids comprising a recombinant nucleic acid encoding a first polypeptide chain of the disclosure, a recombinant nucleic acid encoding a second polypeptide chain of the disclosure, and, optionally, a recombinant nucleic acid encoding a third, fourth and/or fifth polypeptide chain of the disclosure. Also provided are methods of making monomeric or multimeric proteins of the disclosure.

[0055] In one embodiment, the invention provides methods of making a protein (e.g. any heterodimeric, trimeric, tetrameric or pentameric protein), comprising:

[0056] a) providing a first nucleic acid encoding a polypeptide chain comprising a tandem CH3 domain as described herein;

[0057] b) optionally providing a further nucleic acid encoding a further polypeptide chain described herein (e.g. a chain that associates with the chain of (a) via non-covalent and/or covalent interactions); and

[0058] c) expressing said first (and optionally further) nucleic acids in a host cell to produce a protein comprising said first (and optionally the further) polypeptide chain, respectively; and recovering a monomeric (or optionally heterodimeric, heterotrimeric, heterotetrameric or pentameric) protein. Optionally step (c) comprises loading the protein produced onto an affinity purification support, optionally an affinity exchange column, optionally a Protein-A support or column, and collecting the heterodimeric protein; and/or loading the protein produced (e.g., the protein collected following loading onto an affinity exchange or Protein A column) onto an ion exchange column; and collecting the monomeric (or optionally heterodimeric heterotrimeric, heterotetrameric or pentameric) fraction.

[0059] In one embodiment of any of the proteins herein, provided is a composition comprising a plurality of tandem-CH3 domain containing proteins described herein, wherein at least 70%, 80%, 90%, 95% or 98% of the tandem-CH3 domain containing proteins have a monomeric Fc domain (do not have dimeric Fc domains upon dimerization with a second polypeptide). In one embodiment, at least 70%, 80%, 90%, 95% or 98% of the tandem-CH3 domain containing proteins are monomers (i.e. single chain polypeptides). In one embodiment, at least 70%, 80%, 90%, 95% or 98% of the tandem-CH3 domain containing proteins are dimers. In one embodiment, at least 70%, 80%, 90%, 95% or 98% of the tandem-CH3 domain containing proteins are trimers. In one embodiment, at least 70%, 80%, 90%, 95% or 98% of the tandem-CH3 domain containing proteins are tetramers.

[0060] Any of the methods can further be characterized as comprising any step described in the application, including notably in the "Detailed Description of the Invention"). The invention further relates to an antibody obtainable by any of present methods. The disclosure further relates to pharmaceutical or diagnostic formulations of the antibodies of the present invention. The disclosure further relates to methods of using antibodies in methods of treatment or diagnosis.

[0061] These and additional advantageous aspects and features of the invention may be further described elsewhere herein.

BRIEF DESCRIPTION OF THE FIGURES

[0062] FIGS. 1A, 1B and 1C show examples of monomeric multispecific polypeptides comprising a monomeric Fc domain with tandem CH3 domains.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0063] As used in the specification, "a" or "an" may mean one or more. As used in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0064] Where "comprising" is used, this can optionally be replaced by "consisting essentially of", or optionally by "consisting of".

[0065] As used herein, the term "antigen binding domain" refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one embodiment, said domain can comprise a hypervariable region, optionally a VH and/or VL domain of an antibody chain, optionally at least a VH domain. In another embodiment, the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain. In another embodiment, the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.

[0066] The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). An "antibody fragment" comprises a portion of a full-length antibody, e.g. antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab).sub.2, F(ab').sub.2, F(ab).sub.3, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201.

[0067] The term "antibody derivative", as used herein, comprises a full-length antibody or a fragment of an antibody, e.g. comprising at least antigen-binding or variable regions thereof, wherein one or more of the amino acids are chemically modified, e.g., by alkylation, PEGylation, acylation, ester formation or amide formation or the like. This includes, but is not limited to, PEGylated antibodies, cysteine-PEGylated antibodies, and variants thereof.

[0068] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[0069] By "framework" or "FR" residues as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).

[0070] By "constant region" as defined herein is meant an antibody-derived constant region that is encoded by one of the light or heavy chain immunoglobulin constant region genes. By "constant light chain" or "light chain constant region" as used herein is meant the region of an antibody encoded by the kappa (Ckappa) or lambda (Clambda) light chains. The constant light chain typically comprises a single domain, and as defined herein refers to positions 108-214 of Ckappa, or Clambda, wherein numbering is according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). By "constant heavy chain" or "heavy chain constant region" as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full length IgG antibodies, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.

[0071] By "Fab" or "Fab region" as used herein is meant the polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a polypeptide, multispecific polypeptide or ABD, or any other embodiments as outlined herein.

[0072] By "single-chain Fv" or "scFv" as used herein are meant antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0073] By "Fv" or "Fv fragment" or "Fv region" as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.

[0074] By "Fc" or "Fc region", as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains C.gamma.2 (CH2) and C.gamma.3 (CH3) and the hinge between C.gamma.1 and C.gamma.2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226, P230 or A231 to its carboxyl-terminus, wherein the numbering is according to the EU index. Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below. By "Fc polypeptide" or "Fc-derived polypeptide" as used herein is meant a polypeptide that comprises all or part of an Fc region. Fc polypeptides include but is not limited to antibodies, Fc fusions and Fc fragments.

[0075] The "hinge", "hinge region" or "hinge domain" of an IgG is generally defined as including Glu216 and terminating at Pro230 of human IgG1 according to the Kabat system but functionally, the flexible portion of the chain may be considered to include additional residues termed the upper and lower hinge regions, such as from Glu216 to Gly237 (Roux et al., 1998 J Immunol 161:4083) and the lower hinge has been referred to as residues 233 to 239 of the Fc region. As used herein, the "hinge" encompasses the upper and lower hinge regions. Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S--S binds. Although boundaries may vary slightly, as numbered according to the Kabat system, the CH1 domain is adjacent to the VH domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule and includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain, e.g., from about EU positions 118-215. The Fc domain extends from amino acid 231 to amino acid 447; the CH2 domain is from about Ala231 to Lys340 or Gly341 and the CH3 from about Gly341 or Gln342 to Lys447. The residues of the IgG heavy chain constant region of the CH1 region terminate at Lys. The EU numbering system is conventionally used in the art (see generally, Kabat et al, Sequences of Protein of Immunological Interest, NIH Publication No. 91-3242, US Department of Health and Human Services (1991)).

[0076] By "variable region" as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including Vkappa and Vlambda) and/or VH genes that make up the light chain (including kappa and lambda) and heavy chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (VL and VH) consists of a "framework" or "FR" region interrupted by three hypervariable regions referred to as "complementarity determining regions" or "CDRs". The extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1983)), and as in Chothia. The framework regions of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen.

[0077] The term "specifically binds to" means that an antibody or polypeptide can bind preferably in a competitive binding assay to the binding partner, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0078] The term "affinity", as used herein, means the strength of the binding of an antibody or polypeptide to an epitope. The affinity of an antibody is given by the dissociation constant K.sub.D, defined as [Ab].times.[Ag]/[Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant K.sub.A is defined by 1/K.sub.D. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore.TM. SPR analytical device).

[0079] By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. An example of amino acid modification herein is a substitution. By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W refers to a variant of a parent polypeptide, in which the tyrosine at position 50 is replaced with tryptophan. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or "parent" polypeptide. The polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.

[0080] "Conservative" amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0081] The term "identity" or "identical", when used in a relationship between the sequences of two or more polypeptides, refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0082] Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.

[0083] An "isolated" molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition). Commonly, a composition of a polypeptide will exhibit 98%, 98%, or 99% homogeneity for polypeptides in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.

[0084] In the context herein, "treatment" or "treating" refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context. For example, "treatment" of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas "treatment" of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.

[0085] As used herein, "NK cells" refers to a sub-population of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and/or NKp46 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self" MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells. Within the context herein "active" NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells. For instance, an "active" NK cell can be able to kill cells that express a ligand for an activating NK receptor and/or fail to express MHC/HLA antigens recognized by a KIR on the NK cell. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell K S and Colonna M). Human Press. pp. 219-238 (2000).

[0086] As used herein, "T cells" refers to a sub-population of lymphocytes that mature in the thymus, and which display, among other molecules T cell receptors on their surface. T cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including the TCR, CD4 or CD8, the ability of certain

[0087] T cells to kill tumor or infected cells, the ability of certain T cells to activate other cells of the immune system, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify T cells, using methods well known in the art. Within the context herein, "active" or "activated" T cells designate biologically active T cells, more particularly T cells having the capacity of cytolysis or of stimulating an immune response by, e.g., secreting cytokines. Active cells can be detected in any of a number of well-known methods, including functional assays and expression-based assays such as the expression of cytokines such as TNF-alpha.

Producing Polypeptides

[0088] The invention herein relates to a tandem CH3 domain and to polypeptides that comprise a tandem CH3 domain. The tandem CH3 domain can be configured within a single polypeptide chain so that the two CH3 domains interact by forming CH3-CH3 dimers. Such CH3 domains will then not be available for further dimerization with other CH3-domain containing polypeptides, notably Fc-domain-containing polypeptides. Any amino acid sequence, polypeptide domain or polypeptide of interest, e.g. a "parent", "starting" or "nonvariant" polypeptide, can be used as starting point or as a source of amino acid sequence to be incorporated into a tandem CH3 domain-containing polypeptide using techniques available in the art for generating polypeptides. The tandem CH3 domain-containing polypeptide can for example comprise a human Fc region comprising a CH2 domain linked to a CH3 domain, wherein the CH3 domains is one of the tandem CH3 domains.

[0089] The parent polypeptide may be any polypeptide comprising an Fc region, for example an Fc fusion protein comprising an Fc domain (e.g. a dimeric Fc domain). Examples herein comprise an Fc domain and an antigen binding domain from an antibody (e.g. a variable domain).

[0090] The Fc domains can be useful for extending the half-life of any protein of interest, or otherwise to provide a desired functionality conferred by the Fc domain, e.g. recognition by a secondary reagent, binding to a solid support for purification, etc. Thus, the Fc domain molecules can include any heterologous polypeptide. The fused partner can be any other proteinaceous molecule of interest, such as an enzyme, a detectable molecule, a ligand that activates upon interaction with a cell-surface receptor, a peptidic antigen (Ag) against a challenging pathogen or a `bait` protein to identify binding partners assembled in a protein microarray.

[0091] Examples of proteins include labels, receptors, growth factors, ligands, enzymes, erythropoietin, chemokines, antigen binding domain from a non-immunoglobulin scaffold, therapeutic proteins and hormones. In some non-limiting examples, the heterologous protein is a human interferon, erythropoietin (EPO), soluble tumor necrosis factor receptor, CTLA-4, soluble interleukin (IL)-4 receptor, or Factor IX. Examples of Fc-domain containing molecules that can be used as monomeric or multimeric proteins with monomeric Fc domain and modified by incorporating a tandem CH3 domain include: CTLA-4 fused to the Fc of human IgG1 (Nulojix (belatacept), for the treatment of organ rejection); VEGFR1/VEGFR2 fused to the Fc of human IgG1 (Eylea (aflibercept), for the treatment of age related macular degeneration); IL-1 R fused to the Fc of human IgG1 (Arcalyst (rilonacept), for the treatment of cryopyrin-associated periodic syndromes); Thrombopoietin-binding peptide fused to the Fc of human IgG1 (NPlate (romiplostim), for the treatment of thrombocytopenia in chronic immune thrombocytopenic purpura patients); Mutated CTLA-4 fused to the Fc of human IgG1 (Orencia (abatacept), for the treatment of rheumatoid arthritis); LFA-3 fused to the Fc of human IgG1 (Amevive (alefacept), for the treatment of psoriasis and transplant rejection); and TNFR fused to the Fc of human IgG1 (Enbrel (etanercept), for the treatment of rheumatoid arthritis).

[0092] Exemplary methods for generating Fc-polypeptides that are antibodies are described in more detail in the following sections. The antigen binding domains used in the polypeptides described herein can be readily derived a variety of immunoglobulin or non-immunoglobulin scaffolds, for example affibodies based on the Z-domain of staphylococcal protein A, engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalins, DARPins (desiged ankyrin repeat domains, multimerized LDLR-A module, avimers or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference.

[0093] Antigen binding domains are commonly derived from antibodies (immunoglobulin chains), for example in the form of associated VL and VH domains found on two polypeptide chains, or single chain antigen binding domains such as scFv, a V.sub.H domain, a V.sub.L domain, a dAb, a V-NAR domain or a V.sub.HH domain. An antigen binding domain (e.g., ABD.sub.1 and ABD.sub.2) can also be readily derived from antibodies as a Fab.

[0094] Typically, antibodies are initially obtained by immunization of a non-human animal, e.g., a mouse, with an immunogen comprising a polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, for which it is desired to obtain antibodies (e.g. a human polypeptide). The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988), the entire disclosure of which is herein incorporated by reference).

[0095] Human antibodies may also be produced by using, for immunization, transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods. For example, a XenoMouse (Abgenix, Fremont, Calif.) can be used for immunization. A XenoMouse is a murine host that has had its immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse, are already humanized. The XenoMouse is described in U.S. Pat. No. 6,162,963, which is herein incorporated in its entirety by reference.

[0096] Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in (Ward et al. Nature, 341 (1989) p. 544, the entire disclosure of which is herein incorporated by reference). Phage display technology (McCafferty et al (1990) Nature 348:552-553) can be used to produce antibodies from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. See, e.g., Griffith et al (1993) EMBO J. 12:725-734; U.S. Pat. No. 5,565,332; U.S. Pat. No. 5,573,905; U.S. Pat. No. 5,567,610; U.S. Pat. No. 5,229,275). When combinatorial libraries comprise variable (V) domain gene repertoires of human origin, selection from combinatorial libraries will yield human antibodies.

[0097] Additionally, a wide range of antibodies are available in the scientific and patent literature, including DNA and/or amino acid sequences, or from commercial suppliers. Antibodies will typically be directed to a pre-determined antigen. Examples of antibodies include antibodies that recognize an antigen expressed by a target cell that is to be eliminated, for example a proliferating cell or a cell contributing to pathology. Examples include antibodies that recognize tumor antigens, microbial (e.g. bacterial) antigens or viral antigens.

[0098] Antigen binding domains (ABDs) can be selected based on the desired cellular target, and may include for example cancer antigens, bacterial or viral antigens, etc. As used herein, the term "bacterial antigen" includes, but is not limited to, intact, attenuated or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Examples include gram-positive bacterial antigens and gram-negative bacterial antigens. In some embodiments the bacterial antigen is derived from a bacterium selected from the group consisting of Helicobacter species, in particular Helicobacter pyloris; Borelia species, in particular Borelia burgdorferi; Legionella species, in particular Legionella pneumophilia; Mycobacteria s species, in particular M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae; Staphylococcus species, in particular Staphylococcus aureus; Neisseria species, in particular N. gonorrhoeae, N. meningitidis; Listeria species, in particular Listeria monocytogenes; Streptococcus species, in particular S. pyogenes, S. agalactiae; S. faecalis; S. bovis, S. pneumonias; anaerobic Streptococcus species; pathogenic Campylobacter species; Enterococcus species; Haemophilus species, in particular Haemophilus influenzue; Bacillus species, in particular Bacillus anthracis; Corynebacterium species, in particular Corynebacterium diphtheriae; Erysipelothrix species, in particular Erysipelothrix rhusiopathiae; Clostridium species, in particular C. perfringens, C. tetani; Enterobacter species, in particular Enterobacter aerogenes, Klebsiella species, in particular Klebsiella 1S pneumoniae, Pasturella species, in particular Pasturella multocida, Bacteroides species; Fusobacterium species, in particular Fusobacterium nucleatum; Streptobacillus species, in particular Streptobacillus moniliformis; Treponema species, in particular Treponema pertenue; Leptospira; pathogenic Escherichia species; and Actinomyces species, in particular Actinomyces israelli.

[0099] As used herein, the term "viral antigen" includes, but is not limited to, intact, attenuated or killed whole virus, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Sources of a viral antigen include, but are not limited to viruses from the families: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses); Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), Hepatitis C; Norwalk and related viruses, and astroviruses). Alternatively, a viral antigen may be produced recombinantly.

[0100] As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably and refer to antigens (other than NKp46 or CD16) that are differentially expressed by cancer cells or are expressed by non-tumoral cells (e.g. immune cells) having a pro-tumoral effect (e.g. an immunosuppressive effect), and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, or expressed at lower levels or less frequently, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Still other cancer antigens can be expressed on immune cells capable of mediating a pro-tumoral effect, e.g. a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell.

[0101] The cancer antigens are usually normal cell surface antigens which are either over-expressed or expressed at abnormal times, or are expressed by a targeted population of cells. Ideally the target antigen is expressed only on proliferative cells (e.g., tumor cells) or pro-tumoral cells (e.g. immune cells having an immunosuppressive effect), however this is rarely observed in practice. As a result, target antigens are in many cases selected on the basis of differential expression between proliferative/disease tissue and healthy tissue. Example of cancer antigens include: Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Cripto, CD4, CD20, CD30, CD19, CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), a Siglec family member, for example CD22 (Siglec2) or CD33 (Siglec3), CD79, CD138, CD171, PSCA, L1-CAM, PSMA (prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, Melanotransferin, Mud 6 and TMEFF2. Examples of cancer antigens also include Immunoglobulin superfamily (IgSF) such as cytokine receptors, Killer-Ig Like Receptor, CD28 family proteins, for example, Killer-Ig Like Receptor 3DL2 (KIR3DL2), B7-H3, B7-H4, B7-H6, PD-L1, IL-6 receptor. Examples also include MAGE, MART-1/Melan-A, gp100, major histocompatibility complex class I-related chain A and B polypeptides (MICA and MICB), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-0017-1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), nectins (e.g. nectin-4), major histocompatibility complex class I-related chain A and B polypeptides (MICA and MICB), proteins of the UL16-binding protein (ULBP) family, proteins of the retinoic acid early transcript-1 (RAET1) family, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens, GAGE-family of tumor antigens, anti-Mullerian hormone Type II receptor, delta-like ligand 4 (DLL4), DR5, ROR1 (also known as Receptor Tyrosine Kinase-Like Orphan Receptor 1 or NTRKR1 (EC 2.7.10.1), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptors, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor, members of the human EGF-like receptor family, e.g., HER-2/neu, HER-3, HER-4 or a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, Muc-1, CA125, integrin receptors, .alpha.v.beta.3 integrins, .alpha.5.beta.1 integrins, .alpha.llb.beta.3-integrins, PDGF beta receptor, SVE-cadherin, IL-8 receptor, hCG, IL-6 receptor, CSF1R (tumor-associated monocytes and macrophages), .alpha.-fetoprotein, E-cadherin, .alpha.-catenin, .beta.-catenin and .gamma.-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is not intended to be exhaustive. In one aspect, the antigen of interest is an antigen (e.g. any one of the antigens listed above) capable of undergoing intracellular internalization, for example when bound by an conventional human IgG1 antibody, either in the presence of absence of Fc.gamma. receptor cells.

[0102] In one embodiment, the antigen target is a polypeptide present on an immune cell capable of mediating a pro-tumoral effect, e.g. a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell.

[0103] In exemplary embodiments of a multispecific molecule comprising an ABD.sub.1 and ABD.sub.2, one of such ABD may be bind an antigen expressed by a target cell that is to be eliminated (e.g., a tumor antigen, microbial (e.g. bacterial) antigen, viral antigen, or antigen expressed on an immune cell that is contributing to inflammatory or autoimmune disease, and the other of ABD.sub.1 or ABD.sub.2 will bind to an antigen expressed on an immune cell, for example an immune effector cell, e.g. a cell surface receptor of an effector cells such as a T or NK cell. Examples of antigens expressed on immune cells, optionally immune effector cells, include antigens expressed on a member of the human lymphoid cell lineage, e.g. a human T cell, a human B cell or a human natural killer (NK) cell, a human monocyte, a human neutrophilic granulocyte or a human dendritic cell. Advantageously, such cells will have either a cytotoxic or an apoptotic effect on a target cell that is to be eliminated (e.g., that expresses a tumor antigen, microbial antigen, viral antigen, or antigen expressed on an immune cell that is contributing to inflammatory or autoimmune disease). Especially advantageously, the human lymphoid cell is a cytotoxic T cell or NK cell which, when activated, exerts a cytotoxic effect on the target cell. According to this embodiment, then, the cytotoxic activity of the human effector cells are recruited. According to another embodiment, the human effector cell is a member of the human myeloid lineage.

[0104] An ABD of a multispecific polypeptide may bind to an antigen expressed on an immune cell such as an NK and/or T cell receptor, e.g. any molecule on the surface of NK cells or T cells, respectively, that can serve to direct the NK or T cell to the intended target cell to be eliminated. Examples include, e.g., members of the immunoglobulin superfamily, members of the killer-cell immunoglobulin-like receptor (KIR) family, the leukocyte immunoglobulin-like receptors (LILR) family, or the lectin family or the NK cell lectin-like receptor family. Activity can be measured for example by bringing target cells and effector cells into contact in presence of the multispecific polypeptide.

[0105] Optionally the immune cell receptor is an activating receptor, e.g. an activating NK cell or T cell receptor. As used herein, the terms "activating NK cell receptor" and "activating T cell receptor" refers to any molecule on the surface of NK cells or T cells, respectively, that, when stimulated, causes a measurable increase in any property or activity known in the art as associated with NK cell or T cell activity, respectively, such as cytokine (for example IFN-.gamma. or TNF-.alpha.) production, increases in intracellular free calcium levels, the ability to lyse target cells in a redirected killing assay as described, e.g. elsewhere in the present specification, or the ability to stimulate NK cell or T cell proliferation, respectively. The term "activating NK receptor" includes activating or co-activating receptors and includes but is not limited to such as forms of KIR proteins (for example KIR2DS receptors, KIR2DS2, KIR2DS4), NKG2 receptors, NKp30, NKp46, IL-2R, IL-12R, IL-15R, IL-18R and IL-21R. One important family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46.

[0106] Activation of cytotoxic T cells may for example occur via binding of the CD3 antigen as effector antigen on the surface of the cytotoxic T cell by a multispecific (e.g. bispecific) polypeptide of this embodiment. The human CD3 antigen is present on both helper T cells and cytotoxic T cells. Human CD3 denotes an antigen which is expressed on T cells as part of the multimolecular T cell complex and which comprises three different chains: CD3-epsilon, CD3-delta and CD3-gamma.

[0107] Exemplary lymphoid cell-associated effector antigens bound by a multispecific polypeptide are the human CD8 antigen, the human CD16 antigen, the human NKG2D antigen, the human NKp46 antigen, the human NKp30 antigen, the human CD2 antigen, the human CD28 antigen or the human CD25 antigen. The human CD8 antigen, expressed on effector T cells, can be used to redirect such T cells toward a target cell of interest, without for example affecting CD4 T cells (that could be associated with unwanted cytokine production and associated toxicity) or TReg cells. The human NKp30 and particularly the NKp46 antigen are expressed primarily on NK cells and thereby permit the selective recruitment of NK cell effector activity. In one embodiment, the antigen expressed on an immune or immune effector cell is selected from a Fc.gamma.RI (CD64), Fc.gamma.RIIA (CD32), and an Fc.gamma.RIII (CD16) polypeptide.

[0108] The ABD(s) which are incorporated into the Fc polypeptides can be tested for any desired activity prior to inclusion in a polypeptide. Once appropriate antigen binding domains having desired specificity and/or activity are identified, DNA encoding each of the or ABD can be placed, in suitable arrangements, in an appropriate expression vector(s), together with DNA encoding any elements such as CH2 and CH3 domains, linkers and any other optional elements (e.g. DNA encoding a linker or hinge region) for transfection into an appropriate host. The host is then used for the recombinant production of the Fc polypeptide.

[0109] An ABD derived from an antibody will generally comprise at minimum a hypervariable region sufficient to confer binding activity. It will be appreciated that an ABD may comprise or be fused to other amino acids or functional domains as may be desired, including but not limited to linker elements (e.g. linker peptides, CH1, CK or CA domains, hinges, or fragments thereof, each of which can be placed between an ABD and a CH2 or CH3 domain, or between other domains as needed). In one example an ABD comprises an scFv, a V.sub.H domain and a V.sub.L domain, or a single domain antibody (nanobody or dAb) such as a V-NAR domain or a V.sub.HH domain. Exemplary antibody formats are further described herein and an ABD can be selected based on the desired format.

[0110] In any embodiment, an antigen binding domain can be obtained from a humanized antibody in which residues from a complementary-determining region (CDR) of a human antibody are replaced by residues from a CDR of the original antibody (the parent or donor antibody, e.g. a murine or rat antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. The CDRs of the parent antibody, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted in whole or in part into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536. An antigen binding domain can thus have non-human hypervariable regions or CDRs and human frameworks region sequences (optionally with backmutations).

[0111] Protein domains, e.g. ABDs or ECDs, will be arranged in an expression vector so as to produce the Fc-polypeptides having the desired domains operably linked to one another. The Fc-polypeptide can then be produced in an appropriate host cell or by any suitable synthetic process. The host cell may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of generating mammalian glycosylation on antibodies, e.g. a prokaryotic cell or organism, such as natural or engineered E. coli spp., Klebsiella spp., or Pseudomonas spp.

[0112] Fc-derived polypeptide chains can be constructed that comprise: (a) a protein domain of interest (e.g., an ECD of a cell surface receptor, a VH and/or VL, an antigen binding domain (ABD.sub.1) that binds to a first antigen of interest), (b) an Fc domain comprising a CH2 domain (or fragment thereof) and a tandem CH3 domain comprising a first CH3 domain and a second CH3 domain separated from the first CH3 domain by an intervening sequences of amino acid residues (e.g. a flexible peptide linker). Optionally the CH2 domain and the domain of interest are separated by a linker, a CH1 or CK domain, and/or a hinge region. Optionally the Fc domain (i) does not undergo CH3-CH3 dimerization with another Fc-derived polypeptide, (ii) is capable of binding to human FcRn and/or (iii) has decreased binding to a human Fc.gamma. receptor compared to a wild type human IgG1 Fc domain. Examples include polypeptides having a domain arrangement of one of the following:

[0113] (ECD)-CH2-CH3-linker-CH3,

[0114] (ECD)-CH1-CH2-CH3-linker-CH3,

[0115] (VH or VL)-CH2-CH3-linker-CH3,

[0116] (VH or VL)-CH1-CH2-CH3-linker-CH3,

[0117] (ABD.sub.1)-CH1-CH2-CH3-linker-CH3,

or

[0118] (ABD.sub.1)-CH2-CH3-linker-CH3.

[0119] Multispecific, e.g. bispecific, Fc-derived polypeptide chains can be constructed that comprise: (a) a first protein domain of interest (e.g., an ECD of a cell surface receptor, a VH and/or a VL, an antigen binding domain (ABD.sub.1) that binds to a first antigen of interest); (b) an Fc domain comprising a CH2 domain (of fragment thereof) and a tandem CH3 domain comprising a first CH3 domain and a second CH3 domain separated from the first CH3 domain by an intervening amino acid sequence (e.g. a flexible peptide linker), and (c) a second protein domain of interest (e.g., an ECD of a cell surface receptor, a VH and/or a VL, an antigen binding domain (ABD.sub.2) that binds to a second antigen of interest). Each of the first and second protein domain of interest can independently be any desired amino acid sequence, e.g. an antigen binding domain, a functional peptide such as a detectable tag, an enzymatic recognition tag or a purification tag, a receptor polypeptide, an enzyme, a cytokine, etc. Examples of a polypeptide having a domain arrangement are as follows:

[0120] (domain of interest.sub.1)-CH2-CH3-linker-CH3-(domain of interest.sub.2),

[0121] (domain of interest.sub.1)-CH1-CH2-CH3-linker-CH3-(domain of interest.sub.2),

[0122] (ABD.sub.1)-CH2-CH3-linker-CH3-(ABD.sub.2),

[0123] (ABD.sub.1)-CH1-CH2-CH3-linker-CH3-(ABD.sub.2),

[0124] (ECD)-CH2-CH3-linker-CH3-(ABD),

[0125] (ECD)-CH1-CH2-CH3-linker-CH3-(ABD),

[0126] (ABD)-CH2-CH3-linker-CH3-(ECD),

or

[0127] (ABD)-CH1-CH2-CH3-linker-CH3-(ECD).

[0128] Optionally a protein domain of interest is fused to the adjacent CH2 domain via a linker peptide, a CH1 or CK domain, and/or a hinge region. Optionally the CH3 domain is fused to the adjacent CH3 domain via a linker peptide. Optionally the Fc domain (i) is not capable of CH3-CH3 dimerization with another Fc-derived polypeptide, (ii) is capable of binding to human FcRn and/or (iii) has decreased binding to a human Fc.gamma. receptor compared to a wild type human IgG1 Fc domain.

[0129] The tandem CH3 domain-containing portions of the above examples can comprise for example a domain arrangement as follows (from N-terminus to C-terminus): CH2-CH3-peptide linker-CH3-.

[0130] The Fc domain or the Fc polypeptides can thus optionally be described as being or comprising a native human Fc domain comprising a CH2 domain and a CH3 domain, where the CH3 domain is fused at its free terminus to a peptide linker, which is in turn fused at its free terminus to a second CH3 domain.

[0131] Various domain arrangements can be envisaged. For example a tandem CH3 domain-containing Fc portion can be fused to the C-terminus of a tandem ABD or scFv. Non-limiting examples include polypeptide having any of the following domain arrangements (from N-terminus to C-terminus):

[0132] (ABD.sub.1)-(ABD.sub.2)-CH2-CH3-linker-CH3,

[0133] (ABD.sub.1)-(ABD.sub.2)-CH1-CH2-CH3-linker-CH3,

[0134] or

[0135] (scFv.sub.1)-(scFv.sub.2)-CH2-CH3-linker-CH3.

[0136] Other examples using scFvs include proteins having the following domain arrangement (from N-terminus to C-terminus):

[0137] (scFv.sub.1)-CH2-CH3-linker-CH3-(scFv.sub.2)

[0138] or

[0139] (scFv.sub.1)-CH1-CH2-CH3-linker-CH3-(scFv.sub.2).

[0140] Further examples of multispecific proteins include multimeric proteins, including proteins and domain arrangements having monomeric Fc domain shown in PCT application no. WO2015/197593, the disclosure of which is incorporated by reference. For example, a heterodimer can for example have the configuration as follows (see also Examples of such proteins shown as formats 11 and 12 shown in FIG. 1C):

##STR00005##

wherein one of V.sub.a-1 of the first polypeptide chain and V.sub.b-1 of the second polypeptide chain is a light chain variable domain and the other is a heavy chain variable domain, and wherein one of V.sub.a-2 and V.sub.b-2 is a light chain variable domain and the other is a heavy chain variable domain.

[0141] The resulting heterodimer can in another example have the configuration as follows (see also Examples of such proteins shown as format 10 shown in FIG. 1B):

##STR00006##

wherein one of V.sub.a-1 of the first polypeptide chain and V.sub.b-1 of the second polypeptide chain is a light chain variable domain and the other is a heavy chain variable domain, and wherein one of V.sub.a-2 and V.sub.b-2 is a light chain variable domain and the other is a heavy chain variable domain.

[0142] Further examples include trimers. An exemplary trimer can have the configuration as follows (also shown as format 17 in FIG. 1C):

##STR00007##

[0143] In any Fc polypeptide, a CH1 or CK (or CA) domain may be present between a protein or domain of interest, e.g., ABD, ECD, and a CH2 domain, or the CH1 or CK (or CA) domain may be absent between the domain of interest and a CH2 domain. In one embodiment, a peptide linker is present between a protein or domain of interest and a CH2 domain. In one embodiment, a hinge region or fragment thereof is present between a CH1 and a CH2 domain, for example a modified hinge which lacks one or both of the cysteines involved in interchain bridges in native antibody heavy chains. The first and second ABDs can be linked together by a linker of sufficient length to enable the ABDs to fold in such a way as to permit binding to the respective antigen for which the ABD is intended to bind. Suitable peptide linkers for use in linking ABD.sub.1 to ABD.sub.2, or for use in linking an ABD to a CH2 or CH3 are known in the art, see, e.g. WO2007/073499, the disclosure of which is incorporated herein by reference. Examples of linker sequences include (G.sub.4S).sub.x wherein x is an integer (e.g. 1, 2, 3, 4, or more). The Fc polypeptide can thus for example have the structure (ABD.sub.1-peptide linker-ABD.sub.2-peptide linker-CH2-CH3-peptide linker-CH3). For example, the polypeptide may comprise, as a fusion product, the structure (scFv.sub.1-peptide linker-scFv.sub.2-peptide linker-CH2-CH3), wherein each element is fused to the following element. Another example of an Fc polypeptide has the structure: (ABD.sub.1-peptide linker-CH2-CH3-peptide linker-CH3-peptide linker-ABD.sub.2).

[0144] The arrangements for purposes of illustration are shown from N-terminus (on the left) to C-terminus. Domains can be referred to as fused to one another (e.g. a domain can be said to be fused to the C-terminus of the domain on its left, and/or a domain can be said to be fused to the N-terminus of the domain on its right). Domains can be fused to one another directly or via intervening amino acid sequences.

[0145] As shown in certain examples, a multispecific antibody can be prepared in which the two antigen binding domains are positioned on opposite termini of an Fc domain. This protein can provide for conformations that can provide better binding to target antigens on different cells. This configuration also permits a wider range of antibody variable regions to be used; some antibody binding domains that do not remain functional in tandem scFv format will remain functional in single scFv form. Thus, in one aspect the polypeptide can comprise: (a) a first antigen binding domain (ABD.sub.1) that binds to a first antigen of interest, (b) a tandem CH3 domain-containing portion comprising at least a portion of a human Fc domain comprising a first CH3 domain, and a second CH3 domain separated from the first CH3 domain by an intervening amino acid sequence, wherein the tandem CH3 domain-containing portion is fused (optionally via intervening sequences of amino acid residues) to the C-terminus of the first antigen binding domain, and (c) a second antigen binding domain (ABD.sub.2) that binds a second antigen of interest is fused (optionally via intervening amino acid sequences) to the C-terminus of the tandem CH3 domain-containing portion.

[0146] In one aspect of any embodiment, the first antigen binding domain and/or the second antigen binding domain comprise a scFv, optionally where the scFv comprises human framework amino acid sequences.

[0147] An exemplary CH3 domain for use within a tandem CH3 domain can comprise an amino acid sequence of SEQ ID NO: 1, or a sequence at least 70%, 80%, 90%, 95% or 98% identical thereto:

TABLE-US-00001 (SEQ ID NO: 1) G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G

[0148] An exemplary tandem CH3 with a flexible peptide linker (underlined) is shown below. An exemplary tandem CH3 domain can thus comprise an amino acid sequence of SEQ ID NO: 2, or a sequence at least 70%, 80%, 90%, 95% or 98% identical thereto:

TABLE-US-00002 (SEQ ID NO: 2) G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G G G G G S G G G G S G G G G S G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G

[0149] In one embodiment, a peptide linker used to link an ABD (e.g. an scFv, a VH or VL domain) to a CH2 or CH3 comprises a CH1 domain or a fragment of a CH1 domain. For example, a CH1 domain, or an N-terminal amino acid sequence of a CH1 domain can be fused to an ABD (e.g. an scFv, a VH or VL domain, etc.) in order to mimic as closely as possible the natural structure of an antibody. In one embodiment, the linker comprises a N-terminal CH1 amino acid sequence of between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, between 2-14 residues, between 2-16 residues, between 2-18 residues, between 2-20 residues, between 2-22 residues, between 2-24 residues, between 2-26 residues, between 2-28 residues, between 2-30 residues, between 10-20 residues, between 10-22 residues, between 10-24 residues, between 2-26 residues, between 10-28 residues, between 12-20 residues, between 15-30 residues, between 15-20 residues or between 10-30 residues. In one embodiment linker comprises or consists of the amino acid sequence RTVA.

[0150] When an ABD is an scFv, the VH domain and VL domains (VL or VH domains or fragments thereof that retain binding specificity) that form a scFv are linked together by a linker of sufficient length to enable the ABD to fold in such a way as to permit binding to the antigen for which the ABD is intended to bind. Examples of linkers include, for example, linkers comprising glycine and serine residues, e.g., the amino acid sequence GEGTSTGS(G.sub.2S).sub.2GGAD. In one specific embodiment, the VH domain and VL domains of an scFv are linked together by the amino acid sequence (G.sub.45).sub.3.

[0151] Peptide linkers used to link the two CH3 domains to one another can be any amino acid sequence of sufficient length to enable the two CH3 domains to fold in such a way as to permit them to associate by non-covalent interactions, notably to undergo CH3-CH3 dimerization. Examples of linkers include, for example, linkers comprising glycine and serine residues, e.g., the amino acid sequence (G.sub.4S).sub.x, wherein x is between 1 and 10, optionally between 2 and 6, e.g. (G.sub.4S).sub.3.

[0152] Any of the peptide linkers used to link the two CH3 domains to one another may comprise a length of at least 5 residues, optionally at least 10 residues, at least 15 residues, at least 20 residues, at least 25 residues, at least 30 residues or more. In other embodiments, the linkers comprises a length of between 5-10 residues, between 4-12 residues, between 5-14 residues, between 5-16 residues, between 5-18 residues, between 5-20 residues, between 5-22 residues, between 5-24 residues, between 5-26 residues, between 5-28 residues, between 5-30 residues, between 10-20 residues, between 10-22 residues, between 10-24 residues, between 2-26 residues, between 10-28 residues, between 12-20 residues, between 15-30 residues, between 15-20 residues or between 10-30 residues.

[0153] In one embodiment, the hinge region will be a fragment of a hinge region (e.g. a truncated hinge region without cysteine residues) or may comprise one or amino acid modifications to remove (e.g. substitute by another amino acid, or delete) a cysteine residue, optionally both cysteine residues in a hinge region. Removing cysteines can be useful to prevent formation of disulfide bridges in a monomeric polypeptide.

[0154] Constant region domains can be derived from any suitable antibody. Of particular interest are the heavy chain domains, including, the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as in Kabat. "CH2" refers to positions 237-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat. By "hinge" or "hinge region" or "antibody hinge region" is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. References to amino acid residue within constant region domains found within the polypeptides shall be, unless otherwise indicated or as otherwise dictated by context, with reference to Kabat, in the context of an IgG antibody.

[0155] CH3 domains that can serve in the present antibodies can be derived from any suitable antibody. Such CH3 domains can serve as the basis for a modified CH3 domain. Optionally the CH3 domain is of human origin. In certain embodiments herein, the CH3 domains can be native human CH3 domains, while in other embodiments the CH domains can comprise one or more amino acid modifications (e.g. amino acid substitutions). The modifications will generally avoid disrupting the CH3 dimerization interface, such that the tandem CH3 domains will retain the ability to associate with one another. Optionally, the CH3 domain modifications will additionally not interfere with the ability of the Fc-derived polypeptide to bind to neonatal Fc receptor (FcRn), e.g. human FcRn.

[0156] CH2 domains can be readily obtained from any suitable antibody. Optionally the CH2 domain is of human origin. A CH2 may or may not be linked (e.g. at its N-terminus) to a hinge of linker amino acid sequence. In one embodiment, a CH2 domain is a naturally occurring human CH2 domain of IgG1, 2, 4 or 4 subtype. In one embodiment, a CH2 domain is a fragment of a CH2 domain (e.g. at least 10, 20, 30, 40 or 50 amino acids).

[0157] In one embodiment, a CH2 domain, when present in a polypeptide described herein, will retain binding to a neonatal Fc receptor (FcRn), particularly human FcRn.

[0158] In one embodiment, a CH2 domain, when present in a polypeptide described herein, and the polypeptides described herein, will confer decreased or lack of binding to a Fc.gamma. receptor, notably Fc.gamma.RIIIA (CD16). Polypeptides that comprise a CH2 domain that do not bind CD16 will not be capable of activating or mediating ADCC by cells (e.g. NK cells, T cells) that do not express the effector cell antigen of interest (e.g. NKp46, CD3, etc.).

[0159] In one embodiment, the polypeptides described herein and their Fc domain(s) and/or a CH2 domain thereof, will have decreased or will substantially lack antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP), FcR-mediated cellular activation (e.g. cytokine release through FcR cross-linking), and/or FcR-mediated platelet activation/depletion.

[0160] In one embodiment, a CH2 domain in a polypeptide will have substantial loss of binding to activating Fc.gamma. receptors, e.g., Fc.gamma.RIIIA (CD16), Fc.gamma.RIIA (CD32A) or CD64, or to an inhibitory Fc receptor, e.g., Fc.gamma.RIIB (CD32B). In one embodiment, a CH2 domain in a polypeptide will furthermore have substantial loss of binding to the first component of complement (Clq).

[0161] Optionally the CH2 domain further comprises substitutions into human IgG1 of IgG2 residues to further reduce binding to Fc.gamma. receptors. It has been shown that substitutions at positions 233-236 (EU numbering) and IgG4 residues at positions 327, 330 and 331 (EU numbering) were shown to greatly reduce binding to Fc.gamma. receptors and thus ADCC and CDC. Furthermore, Idusogie et al. (2000) J Immunol. 164(8):4178-84 demonstrated that alanine substitution at different positions, including K322, significantly reduced complement activation. In one embodiment, a CH2 domain that retains binding to human FcRn but has reduction of binding to Fc.gamma. receptors will lack or have modified N-linked glycosylation, e.g. at residue N297 (Kabat EU). In one embodiment, a CH2 domain that loses binding to a Fc.gamma. receptor will comprise at least one amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region.

[0162] In one embodiment the monomeric polypeptide will bind to FcRn, with a 1:1 ratio (1 FcRn for each monomeric polypeptide).

[0163] The monomeric Fc domain-containing polypeptides can retain partial FcRn binding (compared, e.g., to a wild type full length human IgG1 antibody). Optionally the monomeric polypeptide is capable of binding to human FcRn with intermediate affinity, e.g. retains binding to FcRn but has decreased binding to a human FcRn receptor compared to a full-length wild type human IgG1 antibody. The Fc moiety may further comprise one or more amino acid modifications, e.g. in the CH2 domain, that modulates binding to one or more Fc.gamma. receptors.

[0164] In one embodiment of any of the polypeptides herein, a tandem-CH3 domain containing polypeptide is present substantially in monomeric form (i.e. as a single polypeptide chain). This may be the case where the tandem-CH3 domain containing polypeptide a final product, or when the tandem-CH3 domain containing polypeptide is a production intermediate in the case where the polypeptide is a multimeric (e.g. dimeric) polypeptide.

Uses of Compounds

[0165] In one aspect, provided are the use of any of the compounds defined herein for the manufacture of a pharmaceutical preparation for the treatment or diagnosis of a mammal being in need thereof. Provided also are the use any of the compounds defined above as a medicament or an active component or active substance in a medicament. In a further aspect provided is a method for preparing a pharmaceutical composition containing a compound as defined above, to provide a solid or a liquid formulation for administration orally, topically, or by injection. Such a method or process at least comprises the step of mixing the compound with a pharmaceutically acceptable carrier.

[0166] In one aspect, provided is a method to treat, prevent or more generally affect a predefined condition by exerting a certain effect, or detect a certain condition using a compound herein, or a (pharmaceutical) composition comprising a compound disclosed herein.

[0167] The polypeptides described herein can be used to prevent or treat disorders that can be treated with antibodies, such as cancers, solid and non-solid tumors, hematological malignancies, infections such as viral infections, and inflammatory or autoimmune disorders.

[0168] In one embodiment, the antigen of interest is expressed on the surface of a malignant cell of a type of cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors.

[0169] In one embodiment, polypeptides described herein can be used to prevent or treat a cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma. Other exemplary disorders that can be treated according to the invention include hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors.

[0170] In one aspect, the methods of treatment comprise administering to an individual a multispecific polypeptide in a therapeutically effective amount. A therapeutically effective amount may be any amount that has a therapeutic effect in a patient having a disease or disorder (or promotes, enhances, and/or induces such an effect in at least a substantial proportion of patients with the disease or disorder and substantially similar characteristics as the patient).

[0171] The multispecific polypeptides can be included in kits. The kits may optionally further contain any number of polypeptides and/or other compounds, e.g., 1, 2, 3, 4, or any other number of multispecific polypeptide and/or other compounds. It will be appreciated that this description of the contents of the kits is not limiting in any way. For example, the kit may contain other types of therapeutic compounds. Optionally, the kits also include instructions for using the polypeptides, e.g., detailing the herein-described methods.

[0172] Also provided are pharmaceutical compositions comprising the compounds as defined above. A compound may be administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition. The form depends on the intended mode of administration and therapeutic or diagnostic application. The pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the compounds to the patient. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as (sterile) water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, alcohol, fats, waxes, and inert solids A pharmaceutically acceptable carrier may further contain physiologically acceptable compounds that act for example to stabilize or to increase the absorption of the compounds Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions.

[0173] The compounds can be administered parenterally. Preparations of the compounds for parenteral administration must be sterile. Sterilization is readily accomplished by filtration through sterile filtration membranes, optionally prior to or following lyophilization and reconstitution. The parenteral route for administration of compounds is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, or intralesional routes. The compounds may be administered continuously by infusion or by bolus injection. A typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile 0.9% NaCl or 5% glucose optionally supplemented with a 20% albumin solution and 1 mg to 10 g of the compound, depending on the particular type of compound and its required dosing regimen. Methods for preparing parenterally administrable compositions are well known in the art.

EXAMPLES

Example 1

Construction of Bispecific Monomeric Tandem CH3 Fc Polypeptides

Materials and Methods

[0174] Different constructs were made for use in the preparation of a bispecific Fc-polypeptide based on a scFv specific for human tumor antigen CD19 (anti-CD19 scFv) and a scFV specific for human activating receptor NKp46 on NK cells (anti-NKp46 scFv).

[0175] Shown below in Table 1 are the light chain and heavy chain variable regions amino acid sequences for the anti-CD19 component of the proteins.

TABLE-US-00003 TABLE 1 Anti-CD19 Sequence Anti-CD19-VK DNA SEQ ID NO: 3 Anti-CD19-VK amino acid SEQ ID NO: 4 Anti-CD19-VH DNA SEQ ID NO: 5 Anti-CD19-VH amino acid SEQ ID NO: 6

[0176] Shown below in Table 2 are the light chain and heavy chain variable regions and scFv amino acid sequences for the anti-NKp46 component of the proteins.

TABLE-US-00004 TABLE 2 Anti-NKp46 scFV sequence (VHVK)/- stop NKp46-3 VH amino acid SEQ ID NO: 7 NKp46-3 VL amino acid SEQ ID NO: 8 NKp46-3 scFv amino acid SEQ ID NO: 9

Cloning and Production of the Recombinant Proteins

[0177] Coding sequences were generated by direct synthesis and/or by PCR. PCR were performed using the PrimeSTAR MAX DNA polymerase (Takara, #R045A) and PCR products were purified from 1% agarose gel using the NucleoSpin gel and PCR clean-up kit (Macherey-Nagel, #740609.250). Once purified the PCR product were quantified prior to the In-Fusion ligation reaction performed as described in the manufacturer's protocol (ClonTech, #ST0345). The plasmids were obtained after a miniprep preparation run on an EVO200 (Tecan) using the Nucleospin 96 plasmid kit (Macherey-Nagel, #740625.4). Plasmids were then sequenced for sequences confirmation before to transfecting the CHO cell line.

[0178] CHO cells were grown in the CD-CHO medium (Invitrogen) complemented with phenol red and 6 mM GlutaMax. The day before the transfection, cells are counted and seeded at 175.000 cells/ml. For the transfection, cells (200.000 cells/transfection) are prepared as described in the AMAXA SF cell line kit (AMAXA, #V4XC-2032) and nucleofected using the DS137 protocol with the Nucleofector 4D device. All the tranfections were performed using 300 ng of verified plasmids. After transfection, cells are seeded into 24 well plates in pre-warmed culture medium. After 24H, hygromycine B was added in the culture medium (200 .mu.g/ml). Protein expression is monitored after one week in culture. Cells expressing the proteins are then sub-cloned to obtain the best producers. Sub-cloning was performed using 96 flat-bottom well plates in which the cells are seeded at one cell per well into 200 .mu.l of culture medium complemented with 200 .mu.g/ml of hygromycine B. Cells were left for three weeks before to test the clone's productivity.

[0179] Recombinant proteins which contain a IgG1-Fc fragment are purified using Protein-A beads (-rProteinA Sepharose fast flow, GE Healthcare, ref.: 17-1279-03). Briefly, cell culture supernatants were concentrated, clarified by centrifugation and injected onto Protein-A columns to capture the recombinant Fc containing proteins. Proteins were eluted at acidic pH (citric acid 0.1M pH3), immediately neutralized using TRIS-HCL pH8.5 and dialyzed against 1.times. PBS. Recombinant scFv which contain a "six his" tag were purified by affinity chromatography using Cobalt resin. Other recombinant scFv were purified by size exclusion chromatography (SEC).

Format 3 (F3): CD19-F3-NKp46-3

[0180] The domain structure of F3 polypeptides is shown in FIG. 1A. The DNA and amino acid sequences for the Fc portion comprised a tandem CH3 domain in which the two CH3 domains on the same polypeptide chain associated with one another, thereby preventing dimerization between different bispecific proteins.

[0181] The single chain polypeptide has domains arranged (N- to C-terminal) as follows: (VK-VH).sup.anti-CD19-CH2-CH3-CH3-(VH-VK).sup.anti-NKp46

[0182] The (VK-VH) units were made up of a VH domain, a linker and a VK unit (scFv).

Proteins were cloned, produced and purified as above. Bispecific protein was purified from cell culture supernatant by affinity chromatography using prot-A beads and analysed and purified by SEC. The protein showed a high production yield of 3.4 mg/L and with a simple SEC profile. The amino acid sequence for the F3 protein is shown in SEQ ID NO: 10.

Format 4 (F4): CD19-F4-NKp46-3

[0183] The protein having domain arrangement scFvCD19-CH2 (N297S)-CH3-CH3-scFvNkp46-3 shown in FIG. 1A as "Format 4" was constructed. The DNA and amino acid sequences for the Fc portion comprised a tandem CH3 domain as in Format F3, however additionally comprising a N297S mutation to prevent N-linked glycosylation and abolish Fc.gamma.R binding. Proteins were cloned, produced and purified as above. Bispecific proteins was purified from cell culture supernatant by affinity chromatography using prot-A beads and analysed and purified by SEC. The protein showed a good production yield of 1 mg/L and with a simple SEC profile. The amino acid sequence for the F4 protein with NKp46-3 variable domains is shown in SEQ ID NO: 11.

Format 9 (F9): CD19-F9-NKp46-3

[0184] The F9 polypeptide is a trimeric polypeptide having a central polypeptide chain and two polypeptide chains each of which associate with the central chain via CH1-CK dimerization. The domain structure of the trimeric F9 protein is shown in FIG. 1B, wherein the bonds between the CH1 and CK domains are interchain disulfide bonds. The two antigen binding domains have a F(ab) structure permitting the use of antibodies irrespective of whether they remain functional in scFv format. The DNA and amino acid sequences for the Fc portion comprised a tandem CH3 domain as in Format F4 and a CH2 domain comprising a N297S substitution. Three variants of F9 proteins were produced: (a) cysteine residues in the hinge region left intact (wild-type, referred to as F9A), (b) cysteine residues in the hinge region replaced by serine residues (F9B), and (c) a linker sequence GGGSS replacing residues DKTHTCPPCP in the hinge (F9C). Variants F9B and F9C provided advantages in production by avoiding formation of homodimers of the central chain. The heterotrimer is made up of:

[0185] (1) a first (central) polypeptide chain having domains arranged as follows (N- to C-terminal):

[0186] VH.sup.anti-CD19-CH1-CH2-CH3-CH3-VH.sup.anti-NKp46-CK

and

[0187] (2) a second polypeptide chain having domains arranged as follows (N- to C-terminal): VK.sup.anti-NKp46-CH1

and

[0188] (3) a third polypeptide chain having domains arranged as follows (N- to C-terminal):

[0189] VK.sup.anti-CD19-CK

[0190] Proteins were cloned, produced and purified as above. Bispecific proteins was purified from cell culture supernatant by affinity chromatography using prot-A beads and analysed and purified by SEC. The protein showed a high production yield of 8.7 mg/L (F9A) and 3.0 mg/L (F9B), and with a simple SEC profile.

[0191] The amino acid sequences of the three chains of the F9 protein variant F9A are shown in the SEQ ID NOS: 12, 13 and 14. The amino acid sequences of the three chains of the F9 protein variant F9B are shown in the SEQ ID NOS: 12, 15 and 14. The amino acid sequences of the three chains of the F9 protein variant F9C are shown in the SEQ ID NOS: 12, 16 and 14.

Format 10 (F10): CD19-F10-NKp46-3

[0192] The F10 polypeptide is a dimeric protein having a central polypeptide chain and a second polypeptide chain which associates with the central chain via CH1-CK dimerization. The domain structure of the dimeric F10 proteins is shown in FIG. 1B wherein the bonds between the CH1 and CK domains are interchain disulfide bonds. One of the two antigen binding domains has a Fab structure, and the other is a scFv. The DNA and amino acid sequences for the Fc portion comprised a tandem CH3 domain as in Format F4 and a CH2 domain with a N297S substitution. Additionally, three variants of F10 proteins were produced: (a) cysteine residues in the hinge region left intact (wild-type, referred to as F10A), (b) cysteine residues in the hinge region replaced by serine residues (F10B, and (c) a linker sequence GGGSS (SEQ ID NO: 28) replacing residues DKTHTCPPCP (SEQ ID NO: 29) in the hinge (F10C). Variants F10B an F10C provided advantages in production by avoiding formation of homodimers of the central chain. The (VK-VH) unit was made up of a VH domain, a linker and a VK unit (scFv). The heterodimer is made up of:

[0193] (1) a first (central) polypeptide chain having domains arranged as follows (N- to C-terminal):

[0194] VH.sup.anti-CD19-CH1-CH2-CH3-CH3-(VH-VK).sup.anti-NKp46

and

[0195] (2) a second polypeptide chain having domains arranged as follows (N- to C-terminal): VK.sup.anti-CD19-CK.

[0196] Proteins were cloned, produced and purified as above. Bispecific proteins was purified from cell culture supernatant by affinity chromatography using prot-A beads and analysed and purified by SEC. The protein showed a good production yield of 2 mg/L (F10A) and with a simple SEC profile. The amino acid sequences of the two chains of the F10A protein variant are shown in the SEQ ID NO: 17 (second chain) and SEQ ID NO: 18 (first chain). The amino acid sequences of the two chains of the F10B protein variant are shown in the SEQ ID NO: 17 (second chain) and SEQ ID NO: 19 (first chain). The amino acid sequences of the two chains of the F10C protein variant are shown in the SEQ ID NO: 17 (second chain) and SEQ ID NO: 20 (first chain).

Format 11 (F11): CD19-F11-NKp46-3

[0197] The domain structure of F11 polypeptides is shown in FIG. 1C. The heterodimeric protein is similar to F10 but the structures of the antigen binding domains are reversed. One of the two antigen binding domains has a Fab-like structure, and the other is a scFv. The heterodimer is made up of

[0198] (1) a first (central) polypeptide chain having domains arranged as follows (N- to C-terminal):

[0199] (VK-VH).sup.anti-CD19-CH2-CH3-CH3-VH.sup.anti-NKp46-CK

and

[0200] (2) a second polypeptide chain having domains arranged as follows (N- to C-terminal): VK.sup.anti-NKp46CH1.

[0201] Proteins were cloned, produced and purified as above. Bispecific proteins was purified from cell culture supernatant by affinity chromatography using prot-A beads and analysed and purified by SEC. The protein showed a good production yield of 2 mg/L and with a simple SEC profile. The amino acid sequences of the two chains of the F11 protein are shown in SEQ ID NO: 21 (chain 1) and SEQ ID NO: 22 (chain 2).

Format 12 (F12): CD19-F12-NKp46-3

[0202] The domain structure of the dimeric F12 polypeptides is shown in FIG. 1C, wherein the bonds between the CH1 and CK domains are disulfide bonds. The heterodimeric protein is similar to F11 but the CH1 and CK domains within the F(ab) structure are inversed. The heterodimer is made up of:

[0203] (1) a first (central) polypeptide chain having domains arranged as follows (N- to C-terminal):

[0204] (VK-VH).sup.anti-CD19-CH2-CH3-CH3-VH.sup.anti-NKp46-CH1

and

[0205] (2) a second polypeptide chain having domains arranged as follows (N- to C-terminal): VK.sup.anti-NKp46-CK.

[0206] Proteins were cloned, produced and purified as above. Bispecific proteins was purified from cell culture supernatant by affinity chromatography using prot-A beads and analysed and purified by SEC. The protein showed a good production yield of 2.8 mg/L and with a simple SEC profile. The DNA and amino acid sequences for the F12 protein are shown below. The amino acid sequences of the two chains of the F12 protein are shown in SEQ ID NO: 23 (chain 1) and SEQ ID NO: 24 (chain 2).

Format 17 (F17): CD19-F17-NKp46-3

[0207] The domain structure of the trimeric F17 polypeptides is shown in FIG. 1C, wherein the bonds between the CH1 and CK domains are disulfide bonds. The heterodimeric protein is similar to F9 but the VH and VK domains, and the CH1 and CK, domains within the C-terminal F(ab) structure are each respectively inversed with their partner. The heterotrimer is made up of:

[0208] (1) a first (central) polypeptide chain having domains arranged as follows (N- to C-terminal):

[0209] VH.sup.anti-CD19-CH1-CH2-CH3-CH3-VK.sup.anti-NKp46-CH1

and

[0210] (2) a second polypeptide chain having domains arranged as follows (N- to C-terminal): VH.sup.anti-NKp46-CK

and

[0211] (3) a third polypeptide chain having domains arranged as follows (N- to C-terminal): VK.sup.anti-CD19-CK

[0212] Additionally, three variants of F17 proteins were produced: (a) cysteine residues in the hinge region left intact (wild-type, referred to as F17A), (b) cysteine residues in the hinge region replaced by serine residues (F10B, and (c) a linker sequence GGGSS (SEQ ID NO: 28) replacing residues DKTHTCPPCP (SEQ ID NO: 29) in the hinge (F17C). Proteins were cloned, produced and purified as above. The amino acid sequences of the three chains of the F17B protein are shown in SEQ ID NOS: 25, 26 and 27.

Example 2

NKp46 Binding Affinity by Bispecific Proteins by Surface Plasmon Resonance (SPR)

Biacore T100 General Procedure and Reagents

[0213] SPR measurements were performed on a Biacore T100 apparatus (Biacore GE Healthcare) at 25.degree. C. In all Biacore experiments HBS-EP+ (Biacore GE Healthcare) and NaOH 10 mM served as running buffer and regeneration buffer respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Protein-A was purchase from (GE Healthcare). Human NKp46 recombinant proteins were cloned, produced and purified at Innate Pharma.

Immobilization of Protein-A

[0214] Protein-A proteins were immobilized covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5. The chip surface was activated with EDC/NHS (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Protein-A was diluted to 10 .mu.g/ml in coupling buffer (10 mM acetate, pH 5.6) and injected until the appropriate immobilization level was reached (i.e. 2000 RU). Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

Binding Study

[0215] The bispecific proteins were tested as different formats F3, F4, F9, F10, F11 having the anti-NKp46 variable regions from the NKp46-3 antibody, and compared to the NKp46-3 antibody as a full-length human IgG1.

[0216] Bispecific proteins at 1 .mu.g/mL were captured onto Protein-A chip and recombinant human NKp46 proteins were injected at 5 .mu.g/mL over captured bispecific antibodies. For blank subtraction, cycles were performed again replacing NKp46 proteins with running buffer.

Affinity Study

[0217] Monovalent affinity study was done following a regular Capture-Kinetic protocol recommended by the manufacturer (Biacore GE Healthcare kinetic wizard). Seven serial dilutions of human NKp46 recombinant proteins, ranging from 6.25 to 400 nM were sequentially injected over the captured Bi-Specific antibodies and allowed to dissociate for 10 min before regeneration. The entire sensorgram sets were fitted using the 1:1 kinetic binding model.

Results

[0218] SPR showed that all of the bispecific polypeptides of formats F3, F4, F9, F10 and F11 retained binding to NKp46. Monovalent affinities and kinetic association and dissociation rate constants are shown below in the table 3 below.

TABLE-US-00005 TABLE 3 Bispecific mAb ka (1/Ms) kd (1/s) KD (M) CD19-F3-NKp46-3 3.905E+5 0.01117 28E-09 CD19-F4-NKp46-3 3.678E+5 0.01100 30E-09 CD19-F9A-NKp46-3 2.070E+5 0.00669 32E-09 CD19-F10A-NKp46-3 2.607E+5 0.00754 29E-09 CD19-F11A-NKp46-3 3.388E+5 0.01044 30E-09 NKp46-3 IgG1 2.224E+5 0.00433 20E-09

Example 3

NKp46 Mechanism of Action

[0219] NKp46.times.CD19 bispecific proteins having an arrangement according to the F3 format described in Example 1 were compared to rituximab (anti-CD20 ADCC inducing antibody), and a human IgG1 isotype control antibody for functional ability to direct CD16-/NKp46+ NK cell lines to lyse CD19-positive tumor target cells.

[0220] Briefly, the cytolytic activity of the CD16-/NKp46+ human NK cell line KHYG-1 was assessed in a classical 4-h .sup.51Cr-release assay in U-bottom 96 well plates. Daudi or B221 cells were labelled with .sup.51Cr (50 .mu.Ci (1.85 MBq)/1.times.10.sup.6 cells), then mixed with KHYG-1 at an effector/target ratio equal to 50:1, in the presence of test antibodies at dilution range starting from 10.sup.-7 mol/L with 1/5 dilution (n=8 concentrations)

[0221] After brief centrifugation and 4 hours of incubation at 37.degree. C., 50 .mu.L of supernatant were removed and transferred into a LumaPlate (Perkin Elmer Life Sciences, Boston, Mass.), and .sup.51Cr release was measured with a TopCount NXT beta detector (Perkin Elmer Life Sciences, Boston, Mass.). All experimental conditions were analyzed in triplicate, and the percentage of specific lysis was determined as follows: 100.times.(mean cpm experimental release-mean cpm spontaneous release)/(mean cpm total release-mean cpm spontaneous release). Percentage of total release is obtained by lysis of target cells with 2% Triton X100 (Sigma) and spontaneous release corresponds to target cells in medium (without effectors or Abs).

[0222] Results

[0223] In the KHYG-1 hNKp46 NK experimental model, each NKp46.times.CD19 bispecific protein induced specific lysis of Daudi or B221 cells by human KHYG-1 hNKp46 NK cell line, while rituximab and human IgG1 isotype control (IC) antibodies did not.

Example 4

Binding of Different Bispecific Formats to FcRn

[0224] Affinity of different antibody formats for human FcRn was studied by Surface Plasmon Resonance (SPR) by immobilizing recombinant FcRn proteins covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5. A chimeric full length anti-CD19 antibody having human IgG1 constant regions and NKp46.times.CD19 bispecific proteins having an arrangement according to the F3, F4, F9, F10, or F11 formats described in Example 1 were tested; for each analyte, the entire sensorgram was fitted using the steady state or 1:1 SCK binding model.

Biacore T100 General Procedure and Reagents

[0225] SPR measurements were performed on a Biacore T100 apparatus (Biacore GE

[0226] Healthcare) at 25.degree. C. In all Biacore experiments Acetate Buffer (50 mM Acetate pH5.6, 150 mM NaCl, 0.1% surfactant p20) and HBS-EP+ (Biacore GE Healthcare) served as running buffer and regeneration buffer respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Recombinant mouse FcRn was purchase from R&D Systems.

Immobilization of FcRn

[0227] Recombinant FcRn proteins were immobilized covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5. The chip surface was activated with EDC/NHS (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). FcRn proteins were diluted to 10 .mu.g/ml in coupling buffer (10 mM acetate, pH 5.6) and injected until the appropriate immobilization level was reached (i.e. 2500 RU). Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

Affinity Study

[0228] Monovalent affinity study was done following the Single Cycle Kinetic (SCK) protocol. Five serial dilutions of soluble analytes (antibodies and bi-specific molecules) ranging from 41.5 to 660 nM were injected over the FcRn (without regeneration) and allowed to dissociate for 10 min before regeneration. For each analyte, the entire sensorgram was fitted using the 1:1 SCK binding model.

Results

[0229] Results are shown in Table 4 below. The bispecific proteins with monomeric Fc domains (F3, F4, F9, F10, F11) displayed binding to FcRn. Human IgG1/K Anti-CD19 antibody having dimeric Fc domains displayed a KD (nM) of 7.8.

TABLE-US-00006 TABLE 4 Antibody/Bispecific SPR method KD nM CD19-F3- NKp46-3 Steady State 474.4 CD19-F4- NKp46-3 Steady State 711.7 CD19-F9A- NKp46-3 Steady State 858.5 CD19-F10A- NKp46-3 Steady State 432.8 CD19-F11- NKp46-3 Steady State 595.5

[0230] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e. g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

[0231] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0232] The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

[0233] The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of`," "consists essentially of" or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0234] This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

[0235] All publications and patent applications cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0236] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Sequence CWU 1

1

291106PRThomo sapiens 1Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100 105 2227PRThomo sapiens 2Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly 225 3333DNAmus musculus 3gacattcagc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60atctcctgca aggccagcca aagtgttgat tatgatggtg atagttattt gaactggtac 120caacagatac caggacagcc acccaaactc ctcatctatg atgcatccaa tctagtatct 180gggattccac ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggaga aggtggatgc tgcaacctat cactgtcagc aaagtactga ggacccttgg 300acgttcggtg gaggcaccaa gctggaaatc aaa 3334111PRTmus musculus 4Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 5372DNAmus musculus 5caggttcagc tgcagcagtc tggggctgag ctggtgcggc ctgggtcctc agtgaagatt 60tcctgcaaag catctggcta cgcattcagt agctactgga tgaactgggt gaagcagagg 120cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga tactaactac 180aacggaaagt tcaagggcaa ggccacactg actgcagacg aatcctccag cacagcctac 240atgcagctca gcagcctggc ctctgaggac tctgcggtct atttctgtgc aagacgagaa 300acgaccactg tcgggcgtta ttactatgct atggactact ggggtcaagg aaccacagtc 360accgtctcct ca 3726124PRTmus musculus 6Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 7116PRTmus musculus 7Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 8107PRTmus musculus 8Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 9245PRTmus musculus 9Ser Thr Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val 1 5 10 15 Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr 20 25 30 Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser 35 40 45 Leu Glu Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr 50 55 60 Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser 65 70 75 80 Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala 85 90 95 Val Tyr Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Val Met Thr Gln 130 135 140 Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser 145 150 155 160 Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln 165 170 175 Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser 180 185 190 Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp 195 200 205 Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr 210 215 220 Tyr Cys Gln Asn Gly His Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr 225 230 235 240 Lys Leu Glu Leu Lys 245 10837PRTArtificialChimeric mus musculus homo sapiens 10Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val 130 135 140 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160 Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175 Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln 195 200 205 Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 210 215 220 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Ser Ser Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 465 470 475 480 Ser Gly Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 485 490 495 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 500 505 510 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 515 520 525 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 530 535 540 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 545 550 555 560 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 565 570 575 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 580 585 590 Ser Thr Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val 595 600 605 Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr 610 615 620 Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser 625 630 635 640 Leu Glu Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr 645 650 655 Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser 660 665 670 Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala 675 680 685 Val Tyr Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln 690 695 700 Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser 705 710 715 720 Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Val Met Thr Gln 725 730 735 Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser 740 745 750 Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln 755 760 765 Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser 770 775 780 Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp 785 790 795 800 Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr 805 810 815 Tyr Cys Gln Asn Gly His Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr 820 825 830 Lys Leu Glu Leu Lys 835 11837PRTartificialChimeric mus musculus homo sapiens 11Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val 130 135 140 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160 Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175 Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln 195 200 205 Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 210 215 220 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Ser Ser Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265

270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 465 470 475 480 Ser Gly Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 485 490 495 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 500 505 510 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 515 520 525 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 530 535 540 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 545 550 555 560 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 565 570 575 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 580 585 590 Ser Thr Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val 595 600 605 Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr 610 615 620 Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser 625 630 635 640 Leu Glu Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr 645 650 655 Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser 660 665 670 Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala 675 680 685 Val Tyr Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln 690 695 700 Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser 705 710 715 720 Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Val Met Thr Gln 725 730 735 Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser 740 745 750 Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln 755 760 765 Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser 770 775 780 Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp 785 790 795 800 Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr 805 810 815 Tyr Cys Gln Asn Gly His Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr 820 825 830 Lys Leu Glu Leu Lys 835 12218PRTartificialChimeric mus musculus homo sapiens 12Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 13801PRTArtificialChimeric mus musculus homo sapiens 13Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 465 470 475 480 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 485 490 495 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 500 505 510 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 515 520 525 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 530 535 540 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 545 550 555 560 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Thr 565 570 575 Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 580 585 590 Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr 595 600 605 Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 610 615 620 Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln 625 630 635 640 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 645 650 655 Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 660 665 670 Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 675 680 685 Thr Leu Thr Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile 690 695 700 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 705 710 715 720 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 725 730 735 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 740 745 750 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 755 760 765 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 770 775 780 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 785 790 795 800 Cys 14214PRTartificialchimeric mus musculus homo sapiens 14Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ala Ser Thr Lys Gly 100 105 110 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 115 120 125 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 130 135 140 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 145 150 155 160 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 165 170 175 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 180 185 190 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 195 200 205 Ser Cys Asp Lys Thr His 210 15801PRTartificialChimeric mus musculus homo sapiens 15Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 465 470

475 480 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 485 490 495 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 500 505 510 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 515 520 525 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 530 535 540 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 545 550 555 560 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Thr 565 570 575 Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 580 585 590 Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr 595 600 605 Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 610 615 620 Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln 625 630 635 640 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 645 650 655 Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 660 665 670 Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 675 680 685 Thr Leu Thr Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile 690 695 700 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 705 710 715 720 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 725 730 735 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 740 745 750 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 755 760 765 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 770 775 780 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 785 790 795 800 Cys 16796PRTartificialChimeric mus musculus homo sapiens 16Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Gly Gly Gly Ser Ser Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 465 470 475 480 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 485 490 495 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 500 505 510 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 515 520 525 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 530 535 540 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 545 550 555 560 Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Thr Gly Ser Glu Val Gln 565 570 575 Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys 580 585 590 Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Met His 595 600 605 Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly Ile 610 615 620 Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys 625 630 635 640 Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu 645 650 655 Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg 660 665 670 Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 675 680 685 Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 690 695 700 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 705 710 715 720 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 725 730 735 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 740 745 750 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 755 760 765 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 770 775 780 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 785 790 795 17218PRTartificialchimeric mus musculus homo sapiens 17Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 18819PRTartificialChimeric mus musculus homo sapiens 18Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 465 470 475 480 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 485 490 495 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 500 505 510 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 515 520 525 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 530 535 540 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 545 550 555 560 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Thr 565 570 575 Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 580 585 590 Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr 595 600 605 Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 610 615 620 Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln 625 630 635 640 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 645 650 655 Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 660 665 670 Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 675 680 685 Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly 690 695 700 Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Val Met Thr Gln Ser Pro 705 710 715 720 Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg 725 730 735 Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser 740 745 750 His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser 755 760 765 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr 770 775 780 Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys 785 790 795 800 Gln Asn Gly His Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 805 810 815 Glu Leu Lys 19819PRTartificialChimeric mus musculus homo sapiens 19Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 465 470 475 480 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 485 490 495 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 500 505 510 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 515 520 525 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 530 535 540 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 545 550 555 560 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Thr 565 570 575 Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 580 585 590 Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr 595 600 605 Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 610 615 620 Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln 625 630 635 640 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 645 650 655 Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 660 665 670 Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 675 680 685 Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly 690 695 700 Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Val Met Thr Gln Ser Pro 705 710 715 720 Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg 725 730 735 Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser 740 745 750 His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser 755 760 765 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr 770 775 780 Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys 785 790 795 800 Gln Asn Gly His Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 805 810 815 Glu Leu Lys 20815PRTartificialChimeric mus musculus homo sapiens 20Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Gly Gly Gly Ser Ser Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 465 470 475 480 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 485 490 495 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 500 505 510 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 515 520 525 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 530 535 540 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 545 550 555 560 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Thr Gly Ser Glu Val 565 570 575 Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val 580 585 590 Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Met 595 600 605 His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly 610 615 620 Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly 625 630 635 640 Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu 645 650 655 Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 660 665 670 Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 675 680 685 Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 690 695 700 Gly Gly Val Asp Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser 705 710 715 720 Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 725 730 735 Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 740 745 750 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 755 760 765 Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn 770 775 780 Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His 785 790 795 800 Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 805 810 815 21819PRTartificialChimeric mus musculus homo sapiens 21Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val 130 135 140 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160 Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175 Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln 195 200 205 Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 210 215 220 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Ser Ser Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 465 470 475 480 Ser Gly Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 485 490 495 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 500 505 510 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 515 520 525 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 530 535 540 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 545 550 555 560 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 565 570 575 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 580 585 590 Ser Thr Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val 595 600 605 Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr 610 615 620 Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser 625 630 635 640 Leu Glu Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr 645 650 655 Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser 660 665 670 Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala 675 680 685 Val Tyr Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln 690 695 700 Gly Thr Thr Leu Thr Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val 705 710 715 720 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 725 730 735 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 740 745 750 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 755 760 765 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 770

775 780 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 785 790 795 800 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 805 810 815 Gly Glu Cys 22214PRTartificialChimeric mus musculus homo sapiens 22Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ala Ser Thr Lys Gly 100 105 110 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 115 120 125 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 130 135 140 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 145 150 155 160 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 165 170 175 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 180 185 190 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys 195 200 205 Ser Cys Asp Lys Thr His 210 23819PRTartificialChimeric mus musculus homo sapiens 23Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 115 120 125 Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val 130 135 140 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160 Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln 165 170 175 Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180 185 190 Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln 195 200 205 Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 210 215 220 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Ser Ser Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Ser Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 465 470 475 480 Ser Gly Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 485 490 495 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 500 505 510 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 515 520 525 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 530 535 540 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 545 550 555 560 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 565 570 575 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 580 585 590 Ser Thr Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val 595 600 605 Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr 610 615 620 Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser 625 630 635 640 Leu Glu Trp Ile Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr 645 650 655 Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser 660 665 670 Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala 675 680 685 Val Tyr Tyr Cys Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln 690 695 700 Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 705 710 715 720 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 725 730 735 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 740 745 750 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 755 760 765 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 770 775 780 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 785 790 795 800 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 805 810 815 Lys Thr His 24214PRTartificialChimeric mus musculus homo sapiens 24Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 25218PRTartificialChimeric mus musculus homo sapiens 25Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 26792PRTartificialChimeric mus musculus homo sapiens 26Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ser 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 465 470 475 480 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 485 490 495 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 500 505 510 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 515 520 525 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 530 535 540 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 545 550 555 560 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Thr 565 570 575 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr 580 585 590 Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser 595 600 605 Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu 610 615 620 Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe 625 630 635 640 Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val 645 650 655 Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe 660 665 670 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ala Ser Thr 675 680 685 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 690 695 700 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 705 710 715 720 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 725 730 735 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 740 745 750 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 755 760 765 Asn Val Asn His

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 770 775 780 Pro Lys Ser Cys Asp Lys Thr His 785 790 27223PRTartificialChimeric mus musculus homo sapiens 27Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 115 120 125 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 130 135 140 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 145 150 155 160 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 165 170 175 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 180 185 190 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 195 200 205 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 285PRTartificiallinker 28Gly Gly Gly Ser Ser 1 5 2910PRThuman 29Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10

* * * * *

File A Patent Application

  • Protect your idea -- Don't let someone else file first. Learn more.

  • 3 Easy Steps -- Complete Form, application Review, and File. See our process.

  • Attorney Review -- Have your application reviewed by a Patent Attorney. See what's included.