Modified human igm constant regions for modulation of complement-dependent cytolysis effector function

Abstract

To provide IgM heavy chain constant regions that can reduce or eliminate complement fixation and resultant complement dependent cytotoxicity (CDC activity).SOLUTION: The present invention provides a modified human IgM constant region comprising one or more amino acid substitutions relative to a wild-type human IgM constant region, wherein at least one amino acid substitution is at a position in the Cμ3 domain ranging from T302 to K322 in an amino acid sequence consisting of a specific sequence, and wherein a modified IgM antibody comprising the modified IgM constant region and a heavy chain variable region specific for a target antigen exhibits reduced complement-dependent cytotoxicity (CDC) of cells expressing the target antigen relative to a corresponding wild-type human IgM antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 483,087, filed April 7, 2017, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Complement-dependent cytotoxicity is an effector function that antibodies use to engage components of the innate immune system to destroy invading microorganisms. Therapeutic antibodies can engage the complement system to attack cells targeted by the antibody, e.g., tumor cells. The complement cascade begins with the binding of the first component, C1q, to ​​the Fc region of IgG or the Fc Mu region of IgM. Hexamerization of antibodies results in the formation of a hexameric C1q complex that further triggers downstream complement components to form the membrane attack complex (MAC), which causes cell death by perforation of the cell membrane and the resulting osmotic shock. Antibodies of the IgM isotype are multimeric (pentamers and hexamers) and are particularly well suited for C1q binding and multimerization. Indeed, IgM antibodies can fix complement 30-100 times better than the corresponding IgG antibodies.

[0003] For applications where IgM antibodies are used to multimerize cell surface receptors to affect downstream signaling, however, it is not always desirable to retain this strong complement fixing ability. For example, IgM antibodies may be used to activate T cells by engaging members of the TNF receptor superfamily, such as CD40, OX40, or GITR. In either case, downstream signaling driven by multimerization can be used to trigger strong agonistic activity and proliferation of T cells. However, if the complement fixing activity of IgM antibodies remains intact, it can potentially oppose the agonistic activity of IgM antibodies on these targets. Thus, there is a need in the art to identify mutations in the IgM heavy chain constant region that can reduce or eliminate complement fixation and the resulting complement dependent cytotoxicity (CDC activity).

[0004] Mutations that increase or decrease CDC activity in mouse IgM have been identified. See, for example, Arya, S., et al., J. Immunol. 152:1206-1212 (1994), Wright, J. F., et al., J. Biol. Chem. 263:11221-11226 (1988), and Wright, J. F., et al., J. Biol. Chem. 265:10506-10513 (1989). There remains a need in the art to identify and characterize modified human IgM antibodies with altered CDC activity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Arya, S., et al., J. Immunol. 152:1206-1212 (1994) [Non-Patent Document 2] Wright, J, F., et al., J. Biol. Chem. 263:11221-11226 (1988) [Non-Patent Document 3] Wright, JF, et al., J. Biol. Chem. 265:10506-10513 (1989) Summary of the Invention

[0006] overview The present disclosure provides a modified human IgM constant region comprising one or more amino acid substitutions relative to a wild-type human IgM constant region, wherein at least one amino acid substitution is present at a position in the Cμ3 domain spanning from T302 of SEQ ID NO:1 to K322 of SEQ ID NO:1, and a modified IgM antibody comprising the modified IgM constant region and a heavy chain variable region specific for a target antigen exhibits reduced complement dependent cytotoxicity (CDC) of cells expressing the target antigen relative to a corresponding wild-type human IgM antibody.

[0007] In certain aspects, the modified human IgM constant region comprises at least one amino acid substitution present at amino acid T302, C303, T304, V305, T306, H307, T308, D309, L310, P311, S312, P313, L314, K315, Q316, T317, I318, S319, R320, P321, and / or K322 of SEQ ID NO: 1. For example, the modified human IgM constant region can comprise at least one amino acid substitution present at position L310 of SEQ ID NO: 1, position P311 of SEQ ID NO: 1, position P313 of SEQ ID NO: 1, position K315 of SEQ ID NO: 1, or any combination thereof.

[0008] In certain embodiments, at least one amino acid substitution can be at position L310 of SEQ ID NO:1, e.g., L310 of SEQ ID NO:1 can be replaced by alanine (L310A), serine (L310S), aspartic acid (L310D), or glycine (L310G). In certain embodiments, L310 of SEQ ID NO:1 can be replaced by alanine (L310A), e.g., the modified IgM constant region can comprise SEQ ID NO:15. In certain embodiments, L310 of SEQ ID NO:1 can be replaced by aspartic acid (L310D), e.g., the modified IgM constant region can comprise SEQ ID NO:23.

[0009] In certain embodiments, at least one amino acid substitution can be at position P311 of SEQ ID NO:1, e.g., P311 of SEQ ID NO:1 can be replaced with alanine (P311A), serine (P311S) or glycine (P311G). In certain embodiments, P311 of SEQ ID NO:1 can be replaced with alanine (P311A), e.g., the modified IgM constant region can comprise SEQ ID NO:2.

[0010] In certain embodiments, at least one amino acid substitution can be at position P313 of SEQ ID NO:1, e.g., P313 of SEQ ID NO:1 can be replaced with alanine (P313A), serine (P313S), or glycine (P313G). In certain embodiments, P313 of SEQ ID NO:1 can be replaced with serine (P313S), e.g., the modified IgM constant region can comprise SEQ ID NO:3.

[0011] In certain embodiments, at least one amino acid substitution can be at position K315 of SEQ ID NO:1, e.g., K315 of SEQ ID NO:1 can be replaced by alanine (K315A), serine (K315S), aspartic acid (K315D), glutamine (K315Q), or glycine (P313G). In certain embodiments, K315 of SEQ ID NO:1 can be replaced by alanine (K315A), e.g., the modified IgM constant region can comprise SEQ ID NO:16. In certain embodiments, K315 of SEQ ID NO:1 can be replaced by aspartic acid (K315D), e.g., the modified IgM constant region can comprise SEQ ID NO:24. In certain embodiments, K315 of SEQ ID NO:1 can be replaced with glutamine (K315Q), for example, the modified IgM constant region can comprise SEQ ID NO:25.

[0012] In certain embodiments, a modified human IgM constant region as provided herein can include two or more amino acid substitutions, for example, a modified human IgM constant region as provided herein can include amino acid substitutions at two or more of positions L310, P311, P313, or K315 of SEQ ID NO:1.

[0013] In certain aspects, a modified human IgM constant region as provided herein can comprise amino acid substitutions at positions P311 and P313 of SEQ ID NO:1. For example, P311 of SEQ ID NO:1 can be substituted with alanine (P311A), serine (P311S), or glycine (P311G), and P313 of SEQ ID NO:1 can be substituted with alanine (P313A), serine (P313S), or glycine (P313G). In certain aspects, P311 of SEQ ID NO:1 can be substituted with alanine (P311A) and P313 of SEQ ID NO:1 can be substituted with serine (P313S), for example, a modified IgM constant region can comprise SEQ ID NO:4.

[0014] In certain aspects, a modified human IgM constant region as provided herein can comprise amino acid substitutions at positions L310 and K315 of SEQ ID NO: 1. For example, L310 can be substituted with alanine (L310A) or serine (L310S) and K315 can be substituted with alanine (K315A) or serine (K315S), e.g., the modified IgM constant region can comprise SEQ ID NO: 17 or SEQ ID NO: 18.

[0015] In certain aspects, a modified human IgM constant region as provided herein can comprise amino acid substitutions at positions L310 and P311 of SEQ ID NO: 1. For example, L310 can be replaced by alanine (L310A) and P311 can be replaced by alanine (P311A), e.g., the modified IgM constant region can comprise SEQ ID NO: 19.

[0016] In certain aspects, a modified human IgM constant region as provided herein can comprise amino acid substitutions at positions L310 and P313 of SEQ ID NO: 1. For example, L310 can be substituted with alanine (L310A) and P313 can be substituted with serine (P313S), e.g., the modified IgM constant region can comprise SEQ ID NO:20.

[0017] In certain aspects, a modified human IgM constant region as provided herein can comprise amino acid substitutions at positions P311 and K315 of SEQ ID NO: 1. For example, P311 can be replaced by alanine (P311A) and K315 can be replaced by alanine (K315A), e.g., the modified IgM constant region can comprise SEQ ID NO:21.

[0018] In certain aspects, a modified human IgM constant region as provided herein can comprise amino acid substitutions at positions P313 and K315 of SEQ ID NO: 1. For example, P313 can be substituted with serine (P313S) and K315 can be substituted with alanine (K315A), e.g., the modified IgM constant region can comprise SEQ ID NO:22.

[0019] In certain embodiments, the maximum CDC activity achieved in a dose-response assay by a target-specific IgM antibody comprising a modified human IgM constant region as provided herein is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a corresponding wild-type IgM antibody that is identical except for the modified human IgM constant region.

[0020] In certain embodiments, the 50% CDC activity (EC 50) is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to a corresponding wild-type IgM antibody that is identical except for the modified human IgM constant region.

[0021] The present disclosure further provides modified human IgM antibodies, the modified human IgM antibodies comprising a modified human IgM constant region as provided herein and further comprising a heavy chain variable region (VH) positioned amino terminal to the modified human IgM constant region, the modified human IgM antibodies specifically bind to a target antigen and exhibit reduced complement dependent cytotoxicity (CDC) of cells expressing the target antigen compared to a corresponding wild-type human IgM antibody. In certain aspects, the modified human IgM antibodies can be pentameric or hexameric antibodies each comprising five or six bivalent IgM binding units, each binding unit comprising two IgM heavy chains each comprising a VH positioned amino terminal to the modified human IgM constant region and two immunoglobulin light chains each comprising a light chain variable domain (VL) positioned amino terminal to a human immunoglobulin light chain constant region. In certain embodiments, a modified human IgM antibody as provided herein can be a pentamer further comprising a J chain or a functional fragment or a functional variant thereof. In certain embodiments, the J chain can comprise amino acids 23-158 of SEQ ID NO:7 or a functional fragment or a functional variant thereof. In certain embodiments, the J chain or a fragment or variant thereof can be a modified J chain further comprising a heterologous polypeptide, wherein the heterologous polypeptide is fused directly or indirectly to the J chain or a fragment or variant thereof. In certain embodiments, the heterologous polypeptide can be fused to the J chain or a fragment thereof via a peptide linker, for example, the peptide linker comprises at least 5 amino acids but not more than 25 amino acids. In certain embodiments, the peptide linker is The heterologous polypeptide may comprise, consist essentially of, or consist of TIFF2024177479000001.tif4128. In certain embodiments, the heterologous polypeptide may be fused to the N-terminus of the J chain or a fragment or variant thereof, to the C-terminus of the J chain or a fragment or variant thereof, or to both the N-terminus and C-terminus of the J chain or a fragment or variant thereof. In certain embodiments, the heterologous polypeptide may include a binding domain, e.g., an antibody or antigen-binding fragment thereof, e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment, or any combination thereof. In certain embodiments, the modified J chain may include an scFv fragment. In certain embodiments, the heterologous polypeptide may specifically bind to CD3ε. In certain embodiments, the modified J chain can include the amino acid sequence SEQ ID NO:9 (V15J) or SEQ ID NO:11 (J15V). In certain embodiments, the modified J chain can further include a signal peptide, for example, the modified J chain can include the amino acid sequence SEQ ID NO:8 (V15J) or SEQ ID NO:10 (J15V). In certain embodiments, the modified human IgM antibodies as provided herein can direct T cell-mediated killing of cells expressing a target antigen with a level of activity equivalent to that of a corresponding IgM antibody that is identical to the modified IgM antibody except for the modified IgM constant region. In certain embodiments, the cell expressing the target antigen is a eukaryotic cell.

[0022] The present disclosure further provides a polynucleotide comprising a nucleic acid sequence encoding a modified human IgM constant region as provided herein or a nucleic acid sequence encoding a heavy chain polypeptide subunit of a modified human IgM antibody as provided herein. Also provided is a composition comprising the provided polynucleotide. In certain embodiments, the composition further comprises a nucleic acid sequence encoding a light chain polypeptide subunit. In certain embodiments, the light chain polypeptide subunit comprises a human antibody light chain constant region or a fragment thereof fused to the C-terminus of the VL. In certain embodiments, the nucleic acid sequence encoding the heavy chain polypeptide subunit and the nucleic acid sequence encoding the light chain polypeptide subunit can be on separate vectors. In certain embodiments, the nucleic acid sequence encoding the heavy chain polypeptide subunit and the nucleic acid sequence encoding the light chain polypeptide subunit can be on a single vector. In certain embodiments, the composition further comprises a nucleic acid sequence encoding a J chain or a functional fragment or a functional variant thereof. In certain embodiments, the J chain or a fragment or variant thereof is a modified J chain further comprising a heterologous polypeptide, the heterologous polypeptide being fused directly or indirectly to the J chain or a fragment thereof. In certain embodiments, the nucleic acid sequences encoding the heavy chain polypeptide subunits, the nucleic acid sequences encoding the light chain polypeptide subunits, and the nucleic acid sequences encoding the J chain can be present on a single vector. In certain embodiments, the nucleic acid sequences encoding the heavy chain polypeptide subunits, the nucleic acid sequences encoding the light chain polypeptide subunits, and the nucleic acid sequences encoding the J chain can each be present on separate vectors. Any such vector or vectors are further provided by the present disclosure. The present disclosure further provides a host cell comprising the provided polynucleotides, compositions, vector, or vectors. In certain embodiments, the host cell is capable of expressing a modified human IgM constant region as provided herein, or a modified human IgM antibody as provided herein, or any functional fragment thereof.The present disclosure further provides a method of making a modified human IgM constant region as provided herein, or a modified human IgM antibody as provided herein, the method comprising culturing a provided host cell and recovering the constant region or antibody. [The present invention 1001] A modified human IgM constant region comprising one or more amino acid substitutions compared to a wild-type human IgM constant region, A modified human IgM constant region, wherein at least one amino acid substitution is present at a position in the Cμ3 domain spanning from T302 of SEQ ID NO:1 to K322 of SEQ ID NO:1, and a modified IgM antibody comprising the modified IgM constant region and a heavy chain variable region specific for a target antigen exhibits reduced complement dependent cytotoxicity (CDC) of cells expressing the target antigen compared to a corresponding wild-type human IgM antibody. [The present invention 1002] The modified human IgM constant region of the present invention 1001, wherein the at least one amino acid substitution is present at amino acids T302, C303, T304, V305, T306, H307, T308, D309, L310, P311, S312, P313, L314, K315, Q316, T317, I318, S319, R320, P321, and / or K322 of SEQ ID NO:1. [The present invention 1003] The modified human IgM constant region of the present invention 1002, wherein the at least one amino acid substitution is present at position L310 of SEQ ID NO:1, position P311 of SEQ ID NO:1, position P313 of SEQ ID NO:1, position K315 of SEQ ID NO:1, or any combination thereof. [The present invention 1004] The modified human IgM constant region of the present invention 1003, wherein said amino acid substitution is at position L310 of SEQ ID NO:1. [The present invention 1005] The modified human IgM constant region of the present invention 1004, wherein L310 of SEQ ID NO:1 is replaced with alanine (L310A), serine (L310S), aspartic acid (L310D), or glycine (L310G). [The present invention 1006] The modified human IgM constant region of the present invention 1005, wherein L310 of SEQ ID NO:1 is replaced with alanine (L310A). [The present invention 1007] The modified human IgM constant region of the present invention 1006, comprising SEQ ID NO:15. [The present invention 1008] The modified human IgM constant region of the present invention 1005, wherein L310 of SEQ ID NO:1 is replaced with aspartic acid (L310D). [The present invention 1009] The modified human IgM constant region of the present invention 1008, comprising SEQ ID NO:23. [The present invention 1010] The modified human IgM constant region of the present invention 1003, wherein said amino acid substitution is at position P311 of SEQ ID NO:1. [The present invention 1011] 10. The modified human IgM constant region of the present invention 1010, wherein P311 of SEQ ID NO:1 is replaced with alanine (P311A), serine (P311S), or glycine (P311G). [The present invention 1012] 1011. A modified human IgM constant region of the present invention, wherein P311 of SEQ ID NO:1 is replaced with alanine (P311A). [The present invention 1013] The modified human IgM constant region of the present invention 1012, comprising SEQ ID NO:2. [The present invention 1014] The modified human IgM constant region of the present invention 1003, wherein said amino acid substitution is at position P313 of SEQ ID NO:1. [The present invention 1015] The modified human IgM constant region of the present invention 1014, wherein P313 of SEQ ID NO:1 is replaced with alanine (P313A), serine (P313S), or glycine (P313G). [The present invention 1016] The modified human IgM constant region of the present invention 1015, wherein P313 of SEQ ID NO:1 is replaced with serine (P313S). [The present invention 1017] The modified human IgM constant region of the present invention 1016, comprising SEQ ID NO:3. [The present invention 1018] The modified human IgM constant region of the present invention 1003, wherein said amino acid substitution is at position K315 of SEQ ID NO:1. [The present invention 1019] The modified human IgM constant region of the present invention 1018, wherein K315 of SEQ ID NO:1 is replaced with alanine (K315A), serine (K315S), aspartic acid (K315D), glutamine (K315Q), or glycine (P313G). [The present invention 1020] The modified human IgM constant region of the present invention 1019, wherein K315 of SEQ ID NO:1 is replaced with alanine (K315A). [The present invention 1021] The modified human IgM constant region of the present invention 1020, comprising SEQ ID NO:16. [The present invention 1022] The modified human IgM constant region of the present invention 1019, wherein K315 of SEQ ID NO:1 is replaced with aspartic acid (K315D). [The present invention 1023] The modified human IgM constant region of the present invention 1022, comprising SEQ ID NO:24. [The present invention 1024] The modified human IgM constant region of the present invention 1019, wherein K315 of SEQ ID NO:1 is replaced with glutamine (K315Q). [The present invention 1025] The modified human IgM constant region of the present invention 1024, comprising SEQ ID NO:25. [The present invention 1026] The modified human IgM constant region of the present invention 1003, comprising amino acid substitutions at positions P311 and P313 of SEQ ID NO:1. [The present invention 1027] The modified human IgM constant region of the present invention 1026, wherein P311 of SEQ ID NO:1 is replaced with alanine (P311A), serine (P311S), or glycine (P311G), and P313 of SEQ ID NO:1 is replaced with alanine (P313A), serine (P313S), or glycine (P313G). [The present invention 1028] 1027. A modified human IgM constant region of the present invention, wherein P311 of SEQ ID NO:1 is replaced with alanine (P311A) and P313 of SEQ ID NO:1 is replaced with serine (P313S). [The present invention 1029] The modified human IgM constant region of the present invention 1028, comprising SEQ ID NO:4. [The present invention 1030] The modified human IgM constant region of the present invention 1003, comprising amino acid substitutions at positions L310 and K315 of SEQ ID NO:1, wherein L310 is replaced with alanine (L310A) or serine (L310S) and K315 is replaced with alanine (K315A) or serine (K315S). [The present invention 1031] The modified human IgM constant region of the present invention 1030, comprising SEQ ID NO:17 or SEQ ID NO:18. [The present invention 1032] 1003. A modified human IgM constant region of the present invention, comprising amino acid substitutions at positions L310 and P311 of SEQ ID NO:1, wherein L310 is replaced with alanine (L310A) and P311 is replaced with alanine (P311A). [The present invention 1033] The modified human IgM constant region of the present invention 1032, comprising SEQ ID NO:19. [The present invention 1034] The modified human IgM constant region of the present invention 1003, comprising amino acid substitutions at positions L310 and P313 of SEQ ID NO:1, wherein L310 is replaced with alanine (L310A) and P313 is replaced with serine (P313S). [The present invention 1035] The modified human IgM constant region of the present invention 1034, comprising SEQ ID NO:20. [The present invention 1036] 1003. A modified human IgM constant region of the present invention, comprising amino acid substitutions at positions P311 and K315 of SEQ ID NO:1, wherein P311 is replaced with alanine (P311A) and K315 is replaced with alanine (K315A). [The present invention 1037] The modified human IgM constant region of the present invention 1036, comprising SEQ ID NO:21. [The present invention 1038] The modified human IgM constant region of the present invention 1003, comprising amino acid substitutions at positions P313 and K315 of SEQ ID NO:1, wherein P313 is replaced with serine (P313S) and K315 is replaced with alanine (K315A). [The present invention 1039] The modified human IgM constant region of the present invention 1038, comprising SEQ ID NO:22. [The present invention 1040] The modified human IgM constant region of any of claims 1001 to 1039, wherein the maximum CDC activity achieved in a dose-response assay by a target-specific IgM antibody comprising the modified human IgM constant region is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a corresponding wild-type IgM antibody that is identical except for the modified human IgM constant region. [The present invention 1041] The modified human IgM constant region of any of the present inventions 1001 to 1039, wherein the antibody concentration of a target-specific IgM antibody comprising the modified human IgM constant region that results in 50% CDC activity (EC50) is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, and at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold, compared to a corresponding wild-type IgM antibody that is identical except for the modified human IgM constant region. [The present invention 1042] A modified human IgM antibody comprising a modified human IgM constant region of any one of claims 1001 to 1041 and a heavy chain variable region (VH) located amino-terminal to the modified human IgM constant region, which specifically binds to a target antigen and exhibits reduced complement-dependent cytotoxicity (CDC) of cells expressing the target antigen compared to a corresponding wild-type human IgM antibody. [The present invention 1043] A modified human IgM antibody of the invention 1042, which is a pentameric or hexameric antibody comprising five or six bivalent IgM binding units, respectively, each binding unit comprising two IgM heavy chains, each comprising a VH located amino terminal to said modified human IgM constant region, and two immunoglobulin light chains, each comprising a light chain variable domain (VL) located amino terminal to a human immunoglobulin light chain constant region. [The present invention 1044] A modified human IgM antibody of the invention 1043, which is a pentamer and further comprises a J chain or a functional fragment or a functional variant thereof. [The present invention 1045] 1044. The modified human IgM antibody of the invention, wherein the J chain or a fragment thereof comprises amino acids 23 to 158 of SEQ ID NO:7 or a functional fragment or a functional variant thereof. [The present invention 1046] The modified human IgM antibody of the present invention 1044 or 1045, wherein the J chain or a fragment or variant thereof is a modified J chain further comprising a heterologous polypeptide, the heterologous polypeptide being fused directly or indirectly to the J chain or a fragment or variant thereof. [The present invention 1047] The modified human IgM antibody of the invention 1046, wherein said heterologous polypeptide is fused to said J chain or a fragment thereof via a peptide linker. [The present invention 1048] The modified human IgM antibody of the invention 1047, wherein said peptide linker comprises at least 5 amino acids but not more than 25 amino acids. [The present invention 1049] The peptide linker A modified human IgM antibody of the present invention 1048, consisting of TIFF2024177479000002.tif4128. [The present invention 1050] The modified human IgM antibody of any of claims 1046 to 1049, wherein the heterologous polypeptide is fused to the N-terminus of the J chain, or a fragment or variant thereof, to the C-terminus of the J chain, or a fragment or variant thereof, or to both the N-terminus and C-terminus of the J chain, or a fragment or variant thereof. [The present invention 1051] The modified human IgM antibody of any of claims 1046 to 1050, wherein the heterologous polypeptide comprises a binding domain. [The present invention 1052] The modified human IgM antibody of the invention 1051, wherein said binding domain of said heterologous polypeptide is an antibody or an antigen-binding fragment thereof. [The present invention 1053] The modified human IgM antibody of the present invention 1052, wherein the antigen-binding fragment comprises a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment, or any combination thereof. [The present invention 1054] The modified human IgM antibody of the present invention, wherein said antigen-binding fragment is an scFv fragment. [The present invention 1055] The modified human IgM antibody of any of claims 1051 to 1054, wherein said heterologous polypeptide is capable of specifically binding to CD3ε. [The present invention 1056] The modified human IgM antibody of the present invention, wherein the modified J chain comprises the amino acid sequence of SEQ ID NO:9 (V15J) or SEQ ID NO:11 (J15V). [The present invention 1057] The modified human IgM antibody of the present invention, wherein the modified J chain further comprises a signal peptide. [The present invention 1058] 1057. A modified human IgM antibody of the invention, wherein the modified J chain comprises the amino acid sequence of SEQ ID NO:8 (V15J) or SEQ ID NO:10 (J15V). [The present invention 1059] The modified human IgM antibody of any of claims 1055 to 1058, which is capable of directing T cell-mediated killing of cells expressing said target antigen, and said T cell-mediated killing is equivalent to that of a corresponding IgM antibody that is identical to said modified human IgM antibody except for the modified IgM constant region. [The present invention 1060] The modified human IgM antibody of any of claims 1042 to 1059, wherein the cell expressing the target antigen is a eukaryotic cell. [The present invention 1061] A polynucleotide comprising a nucleic acid sequence encoding a modified human IgM constant region of any of the present inventions 1001 to 1040 or a nucleic acid sequence encoding a heavy chain polypeptide subunit of a modified human IgM antibody of any of the present inventions 1042 to 1059. [The present invention 1062] A composition comprising a polynucleotide of the present invention. [The present invention 1063] The composition of claim 1062, further comprising a nucleic acid sequence encoding a light chain polypeptide subunit. [The present invention 1064] The composition of the present invention 1063, wherein said light chain polypeptide subunit comprises a human antibody light chain constant region or a fragment thereof fused to the C-terminus of the VL. [The present invention 1065] The composition of claim 1063 or 1064, wherein the nucleic acid sequence encoding the heavy chain polypeptide subunit and the nucleic acid sequence encoding the light chain polypeptide subunit are present on separate vectors. [The present invention 1066] The composition of claim 1063 or 1064, wherein the nucleic acid sequence encoding the heavy chain polypeptide subunit and the nucleic acid sequence encoding the light chain polypeptide subunit are present on a single vector. [The present invention 1067] The composition of any of claims 1062 to 1066, further comprising a nucleic acid sequence encoding a J chain, or a functional fragment or a functional variant thereof. [The present invention 1068] The composition of claim 1067, wherein the J chain or fragment thereof is a modified J chain further comprising a heterologous polypeptide, wherein the heterologous polypeptide is fused directly or indirectly to the J chain or fragment thereof. [The present invention 1069] The composition of invention 1067 or invention 1068, wherein the nucleic acid sequence encoding the heavy chain polypeptide subunit, the nucleic acid sequence encoding the light chain polypeptide subunit, and the nucleic acid sequence encoding the J chain are present on a single vector. [The present invention 1070] The composition of invention 1067 or invention 1068, wherein the nucleic acid sequence encoding the heavy chain polypeptide subunit, the nucleic acid sequence encoding the light chain polypeptide subunit, and the nucleic acid sequence encoding the J chain are each present on a separate vector. [The present invention 1071] The vector of the present invention 1066 or 1069. [The present invention 1072] The vector of the present invention 1065 or 1070. [The present invention 1073] A host cell comprising a polynucleotide of the present invention 1061, a composition of any of the present inventions 1062 to 1070, or a vector of the present invention 1071 or 1072, and capable of expressing a modified human IgM constant region of any of the present inventions 1001 to 1040, or a modified human IgM antibody of any of the present inventions 1042 to 1059, or a functional fragment thereof. [The present invention 1074] A method for producing a modified human IgM constant region or an antibody, comprising culturing a host cell of the present invention 1073, and recovering the modified human IgM constant region or the antibody of any of the present inventions 1001 to 1040 or any of the modified human IgM antibodies of the present inventions 1042 to 1060. [Brief description of the drawings]

[0023] [Figure 1] Figure 1 shows SDS-PAGE gel run under non-reducing conditions to demonstrate pentameric and hexameric assembly.Gel patterns are shown for control, IgM (pure), IgM+J (pure), and supernatant preparations of IgM and IgM variants S283N, P313S, P311A, and D294G (all without J chain). [Diagram 2] FIG. 1 shows the percent complement dependent cytolysis (CDC) activity for (1) control anti-CD20 antibodies (1.5.3 IgG and 1.5.3 IgM×V15J) tested in the absence of complement, (2) control IgM (1.5.3 IgM, which has no J chain) tested in the presence of complement, and (3) the corresponding 1.5.3 IgM mutants, P311A, P313S, D294G, and S283N, which all have no J chain, tested in the presence of complement. [Diagram 3] FIG. 13 shows the percent CDC activity for anti-CD20×anti-CD3 bispecific IgM antibodies with anti-CD3 modified J chains, including the control 1.5.3 IgM×V15J antibody and the corresponding 1.5.3 IgM×V15J mutants P311A, P313S, D294G, and S283N. [Figure 4]FIG. 1 shows T cell activation assays for control anti-CD20 IgM lacking an anti-CD3 modified J chain, and bispecific IgM antibodies containing an anti-CD3 modified J chain (V15J), including control antibody 1.5.3 IgM×V15J, and the corresponding 1.5.3×V15J IgM mutants, D294G, S283N, P313S, and P311A. [Diagram 5] Figure 1 shows hybrid and reduced gels for 1.5.3 IgMxV15J, and the corresponding 1.5.3xV15J IgM mutants, P311A, P313S, and the double mutant P311A / P313S. The double mutant is expressed and assembles similarly to each of the single mutants. [Figure 6] Figure 1 shows CDC activity for 1.5.3 IgMxV15J and the corresponding 1.5.3xV15J IgM mutants, P311A, P313S and the double mutant P311A / P313S, the double mutant showing complete loss of CDC activity. [Figure 7] Figure 14. T cell activation assay for 1.5.3 IgMxV15J, and the corresponding 1.5.3xV15J IgM mutants, P311A, P313S, and the double mutant P311A / P313S. The double mutant is as effective at activating T cells as each of the single mutants. [Figure 8] Figure 1 shows CDC activity for 1.5.3 IgM without J chain, 1.5.3 IgMxV15J, the corresponding 1.5.3xV15J IgM mutant, P311A, P313S, L310A, K315A and the double mutant P311A / P313S, the double mutant showing complete abolition of CDC activity. [Figure 9] Figure 9A shows CDC activity for 1.5.3 IgM without J chain, 1.5.3 IgMxV15J, 1.5.3xV15J IgM mutants L310D and L310A and the double mutant P311A / P313S, and Figure 9B shows CDC activity for 1.5.3 IgM without J chain, 1.5.3 IgMxV15J, 1.5.3xV15J IgM mutants K315D, K315Q and K315A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description definition The term "a" or "an" entity refers to one or more of that entity, for example, "binding molecule" is understood to refer to one or more binding molecules. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0025] Furthermore, "and / or", when used herein, should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or", when used herein in phrases such as "A and / or B", is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or", when used in phrases such as "A, B, and / or C", is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0026] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0027] Units, prefixes, and symbols are shown in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the present disclosure, which may be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0028] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to a chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains" or other terms used to refer to a chain of amino acids or chains of two or more amino acids are included within the definition of "polypeptide" and the term "polypeptide" can be used in place of or interchangeably with these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, and derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant methods, but is not necessarily translated from a specified nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0029] Polypeptides as disclosed herein can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids in size. A polypeptide does not necessarily have a defined three-dimensional structure, but can have such a structure. A polypeptide with a defined three-dimensional structure is called folded, and a polypeptide that does not have a defined three-dimensional structure, but rather can adopt a number of different conformations, is called unfolded. As used herein, the term glycoprotein refers to a protein bound to at least one carbohydrate moiety that is linked to the protein through an oxygen- or nitrogen-containing side chain of an amino acid, such as serine or asparagine.

[0030] By "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0031] As used herein, the term "non-naturally occurring polypeptide" or any grammatical variation thereof expressly excludes, but does not exclude, forms of the polypeptide that have been or may be determined or construed as "naturally occurring" by a judicial or administrative agency or judicial body. No more than This is a conditional definition.

[0032] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the aforementioned polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative," and "analog" as disclosed herein include any polypeptide that retains at least some of the properties of the corresponding native antibody or polypeptide, e.g., specifically binding to an antigen. Fragments of polypeptides include, for example, specific antibody fragments as discussed elsewhere herein, as well as proteolytic fragments as well as deletion fragments. For example, variants of polypeptides include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain aspects, variants may not be naturally occurring. Non-naturally occurring variants can be made using mutagenesis methods known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been altered to exhibit additional characteristics not found in the original polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein, a "derivative" of a polypeptide can refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side chain. Also included as a "derivative" is a peptide containing one or more derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be used in place of proline, 5-hydroxylysine can be used in place of lysine, 3-methylhistidine can be used in place of histidine, homoserine can be used in place of serine, and ornithine can be used in place of lysine.

[0033] A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids with similar side chains are defined in the art to include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abolish binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the binding molecule binds. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell, et al., Biochem. 32:1180-1, 187 (1993); Kobayashi, et al., Protein Eng. 12(10):879-884 (1999); and Burks, et al., Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0034] The term "polynucleotide" is intended to encompass the singular form of nucleic acid as well as the plural form of nucleic acid, and refers to an isolated nucleic acid molecule or a nucleic acid construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acid (PNA)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more nucleic acid segments, e.g., fragments of DNA or RNA, present in a polynucleotide.

[0035] Any form of nucleic acid or polynucleotide that is separated from its native environment by an "isolated" nucleic acid or polynucleotide is intended. For example, a gel-purified or recombinant polynucleotide encoding a polypeptide contained in a vector would be considered "isolated". Also, a segment of a polynucleotide that has been engineered to have a restriction site for cloning, e.g., a PCR product, would be considered "isolated". Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or polynucleotides that have been purified (partially or substantially) in a non-native solution, such as in a buffer or saline solution. Isolated RNA molecules include in vitro or in vivo RNA transcripts of a polynucleotide, which transcripts are not found in nature. Isolated polynucleotides or nucleic acids further include such molecules that are produced synthetically. In addition, a polynucleotide or nucleic acid can be or include regulatory elements, such as, for example, a promoter, a ribosome binding site, or a transcription terminator.

[0036] As used herein, the term "non-naturally occurring polynucleotide" or any grammatical variation thereof expressly excludes, but does not exclude, forms of nucleic acids or polynucleotides that have been or may be determined or construed as "naturally occurring" by a judicial or administrative agency or judicial body. No more than This is a conditional definition.

[0037] As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered part of the coding region, but adjacent sequences, e.g., promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding region or can include two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can include a heterologous coding region, fused or unfused to another coding region. Heterologous coding regions include, but are not limited to, those that encode specialized elements or motifs, e.g., secretory signal peptides or heterologous functional domains.

[0038] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide can usually include a promoter and / or other transcriptional or translational control elements operably linked to one or more coding regions. Operable linkage is when a gene product, e.g., a coding region for a polypeptide, is linked to one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequences. Two DNA fragments (a coding region for a polypeptide and its associated promoter) are "operably linked" if induction of promoter function results in transcription of an mRNA encoding the desired gene product, and if the nature of the link between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA in a given cell. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably linked to the polynucleotide to direct cell-specific transcription.

[0039] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, for example, promoter and enhancer segments derived from cytomegalovirus (immediate early promoter combined with intron-A), simian virus 40 (early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences that can control the expression of genes in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0040] Likewise, various translation control elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (in particular, internal ribosome entry sites, also called CITE sequences, or IRES).

[0041] In other embodiments, the polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0042] The coding regions of the polynucleotides and nucleic acids can be associated with additional coding regions that encode secretory or signal peptides that direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain from the rough endoplasmic reticulum has been initiated. Those skilled in the art are aware that polypeptides secreted by vertebrate cells can have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the complete or "full-length" polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide is used, for example, a signal peptide of an immunoglobulin heavy or light chain, or a functional derivative of that sequence that retains the ability to direct the secretion of an operably linked polypeptide. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0043] As used herein, the term "CD20" refers to a membrane protein expressed on the surface of B-lymphocytes. CD20 protein is referred to in the literature by other names, such as B-lymphocyte antigen CD20, B-lymphocyte cell surface antigen B1, Bp35, CVID5, LEU-16, transmembrane 4-domain subfamily A member 1, or MS4A2. Certain multivalent anti-CD20 binding molecules, such as IgM antibodies or fragments thereof as disclosed herein, are further described in PCT Publication No. WO / 2016 / 141303, the entirety of which is incorporated herein by reference.

[0044] Disclosed herein are specific binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless a full-sized antibody is specifically referred to, the term "binding molecule" includes full-sized antibodies as well as antigen-binding subunits, fragments, variants, analogs, or derivatives of such antibodies, for example, engineered antibody molecules or fragments that bind to antigens in a manner similar to antibody molecules but use different scaffolds.

[0045] As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to a target or molecular determinant, such as an epitope or antigenic determinant. As further described herein, a binding molecule can include one or more "antigen binding domains" as described herein. A non-limiting example of a binding molecule is an antibody or a fragment thereof that retains antigen-specific binding.

[0046] As used herein, a "binding domain" or "antigen-binding domain" refers to a region of a binding molecule that is sufficient to specifically bind to an epitope. For example, an "Fv", e.g., the variable heavy and variable light chains of an antibody, are considered to be a "binding domain" either as two separate polypeptide subunits or as a single chain. Other antigen-binding domains include, but are not limited to, the variable heavy chain (VHH) of an antibody from a camelid species, or the six immunoglobulin complementarity determining regions (CDRs) expressed on a fibronectin scaffold. A "binding molecule" as described herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more "antigen-binding domains".

[0047] The terms "antibody" and "immunoglobulin" may be used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof as disclosed herein) includes at least a heavy chain variable domain (for camelids) or at least heavy and light chain variable domains. Basic immunoglobulin structure in vertebrate systems is relatively well understood (see, e.g., Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise stated, the term "antibody" encompasses anything ranging from a small antigen-binding fragment of an antibody to a full-sized antibody, e.g., an IgG antibody, which includes two complete heavy chains and two complete light chains, an IgA antibody, which includes four complete heavy chains and four complete light chains and may include a J chain and / or a secretory component, or an IgM antibody, which includes 10 or 12 complete heavy chains and 10 or 12 complete light chains and may include a J chain.

[0048] As discussed in more detail below, the term "immunoglobulin" includes various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4 or α1-α2) among them. It is the nature of this chain that determines the "isotype" of an antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (subtypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, are well characterized and are known to confer functional properties. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in light of the present disclosure and are therefore within the scope of the present disclosure. In certain embodiments, the present disclosure provides modified human IgM antibodies.

[0049] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is expressed, for example, by a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked ends of the Y-shape to the C-terminus at the bottom of each chain. The basic structure of a particular antibody, for example an IgG antibody, includes two heavy chain subunits and two light chain subunits covalently linked via disulfide bonds to form a "Y" structure, also referred to herein as an "H2L2" structure or "binding unit".

[0050] The term "binding unit" is used herein to refer to a portion of a binding molecule, e.g., an antibody or antigen-binding fragment thereof, that corresponds to a standard immunoglobulin structure, e.g., two heavy chains or fragments thereof and two light chains or fragments thereof, or two heavy chains or fragments thereof, e.g., from a camelid or condricthoid antibody. In certain embodiments, e.g., when the binding molecule is a bivalent IgG antibody or antigen-binding fragment thereof, the terms "binding molecule" and "binding unit" are synonymous. In other embodiments, when the binding molecule is an IgM pentamer or IgM hexamer, the binding molecule comprises two or more "binding units", five or six "binding units" in the case of an IgM pentamer or hexamer, respectively. A binding unit need not comprise full-length antibody heavy and light chains, but will usually be bivalent, i.e., comprise two "antigen-binding domains" as defined below. Certain IgM-derived binding molecules provided in this disclosure are pentamers or hexamers and contain five or six bivalent binding units that contain an IgM constant region, e.g., a modified human IgM constant region or a fragment thereof. As used herein, a binding molecule that contains more than one binding unit, e.g., five or six binding units, can be referred to as a "multimer."

[0051] The terms "J chain," "native sequence J chain," or "native J chain," as used herein, refer to the J chain of a native sequence IgM or IgA antibody of any animal species, including the mature human J chain whose amino acid sequence is presented as SEQ ID NO:7.

[0052] The term "modified J chain" is used herein to refer to a variant of a native sequence J chain polypeptide that includes a heterologous moiety, e.g., a heterologous polypeptide, e.g., an exogenous binding domain, introduced into the native sequence. Introduction can be accomplished by any means including direct or indirect fusion of the heterologous polypeptide or other moiety, or by linkage via a peptide or chemical linker. The term "modified human J chain" includes, but is not limited to, a native sequence human J chain of the amino acid sequence of SEQ ID NO:7, or a functional fragment thereof, that has been modified by the introduction of a heterologous moiety, e.g., a heterologous polypeptide, e.g., an exogenous binding domain. In certain embodiments, the heterologous moiety does not interfere with efficient polymerization of IgM into pentamers and binding of such polymers to targets. Exemplary modified J chains can be found, for example, in PCT Publication No. WO2015 / 153912, PCT Publication No. WO / 2017 / 059387, and PCT Publication No. WO / 2017 / 059380, each of which is incorporated by reference herein in its entirety.

[0053] The terms "valency", "bivalent", "multivalent" and grammatical equivalents refer to the number of antigen-binding domains in a given binding molecule or binding unit. As such, the terms "bivalent", "tetravalent" and "hexavalent" in reference to a given binding molecule, e.g., an IgM antibody or fragment thereof, refer to the presence of two antigen-binding domains, four antigen-binding domains and six antigen-binding domains, respectively. In a typical IgM-derived binding molecule in which each binding unit is bivalent, the binding molecule itself can have a valency of 10 or 12. A bivalent or multivalent binding molecule can be monospecific, i.e., all of the antigen-binding domains are identical, or can be bispecific or multispecific, e.g., where two or more antigen-binding domains are different, e.g., bind different epitopes on the same antigen or bind completely different antigens.

[0054] The term "epitope" includes any molecular determinant capable of specific binding to an antibody. In certain aspects, an epitope can include, for example, chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain aspects can have three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of a target to which an antibody binds.

[0055] The term "target" is used in the broadest sense to include any substance to which a binding molecule can bind. A target can be, for example, a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule. Furthermore, a "target" can be, for example, a cell, an organ, or an organism that contains a bound epitope to which a binding molecule can bind.

[0056] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the variable light chain (VL) and the variable heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant region (CL) of the light chain and the constant region (e.g., CH1, CH2, CH3, or CH4) of the heavy chain confer biological properties such as, for example, secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 (CH4 for IgM) and CL domains are at the carboxy termini of the heavy and light chains, respectively.

[0057] A "full-length IgM antibody heavy chain" is a set of antibody heavy chain variable domains (V H ), antibody heavy chain constant domain 1 (CM1 or Cμ1), antibody heavy chain constant domain 2 (CM2 or Cμ2), antibody heavy chain constant domain 3 (CM3 or Cμ3), and antibody heavy chain constant domain 4 (CM4 or Cμ4), which may include a tail.

[0058] As indicated above, the variable regions (i.e., "antigen-binding domains") enable a binding molecule to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains (or simply the VH domain (designated VHH) for camelid or chondrictoid antibodies), or a subset of the complementarity determining regions (CDRs) of a binding molecule, e.g., an antibody, can combine to form an antigen-binding domain. More specifically, an antigen-binding domain can be defined by three CDRs in each of the VH and VL chains (or three CDRs in a VHH). Certain antibodies form larger structures. For example, IgM can form dimeric, pentameric, or hexameric molecules comprising two, five, or six H2L2 binding units and, optionally, a J chain operably covalently linked via disulfide bonds.

[0059] The six "complementarity determining regions" or "CDRs" present in the antigen-binding domain of an antibody are short, non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain such that the antibody assumes its three-dimensional structure in an aqueous environment. The remainder of the amino acids in the antigen-binding domain, called the "framework" regions, show little intermolecular variability. The framework regions largely adopt a β-sheet conformation, while the CDRs form loops that connect, and in some cases, form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that provides for positioning the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to the epitope in the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. The amino acids that constitute the CDR and framework regions, respectively, have been defined in a variety of different ways and can be readily identified by one of skill in the art for any given heavy or light chain variable region (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).

[0060] Where there are more than one definition of a term used and / or accepted within the art, the definition of a term as used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region ("CDR")" to describe the non-contiguous antigen combining sites found within the variable regions of both heavy and light chain polypeptides. These regions are described, for example, by Kabat, et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia, et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference. The Kabat and Chothia definitions include overlapping or subsets of amino acids compared to each other. Nevertheless, application of either definition (or other definitions known to those of skill in the art) to refer to the CDRs of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein, unless otherwise indicated. The appropriate amino acids which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 for comparison. The exact number of amino acids which encompass a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine which amino acids constitute a particular CDR given the amino acid sequence of the variable region of an antibody.

[0061] (Table 1) CDR definition * TIFF2024177479000003.tif34128 * All numbering of the CDR definitions in Table 1 follows the numbering convention set forth by Kabat, et al. (see below).

[0062] Immunoglobulin variable domains can also be analyzed to identify segments of the variable regions that contain the CDRs, for example using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V-Quest). See, e.g., Brochet, X. et al., Nucl. Acids Res. 36:W503-508 (2008).

[0063] Kabat, et al. also defined a numbering system for variable and constant region sequences that is applicable to any antibody. One skilled in the art can clearly assign this system of "Kabat numbering" to any variable domain sequence without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat, et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). However, consecutive numbering is used for amino acid sequences in this disclosure unless the use of the Kabat numbering system is explicitly mentioned. Another numbering scheme is the Eu numbering system for IgG (Edelman, GM, et al., Proc. Natl. Acad. Sci. USA, 63:78-85 (1969)), which has been adapted to other immunoglobulins, including IgM (see, e.g., Arya, S., et al., J. Immunol. 152:1206-1212 (1994)).

[0064] Binding molecules, such as antibodies or antigen-binding fragments, variants, or derivatives thereof, include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, such as Fab, Fab', and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either the VL or VH domains, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0065] By "specifically binds", it is generally meant that a binding molecule, e.g., an antibody or a fragment, variant, or derivative thereof, binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to the epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular binding molecule binds to a particular epitope. For example, binding molecule "A" can be considered to have a higher specificity for a given epitope than binding molecule "B", or binding molecule "A" can be said to bind epitope "C" with a higher specificity than it has for related epitope "D".

[0066] The binding molecules disclosed herein, such as antibodies or fragments, variants, or derivatives thereof, can be used to bind to 5×10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , 10 -3 seconds -1 , 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10-5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 Or 10 -7 seconds -1 Lower off-rate (k(off)) or 5×10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , 10 -3 seconds -1 , 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 or 10 -7 seconds -1 The antibody can be said to bind to the target antigen with an off rate (k(off)) of

[0067] The binding molecules disclosed herein, e.g., antibodies or antigen-binding fragments, variants or derivatives thereof, can be used in a variety of applications, including the following: 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , 5×10 4 M -1 seconds -1 , 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5 × 10 6 M-1 seconds -1 Or 10 7 M -1 seconds -1 On-rate (k(on)) exceeding 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , 5×10 4 M -1 seconds -1 , 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5 × 10 6 M -1 seconds -1 Or 10 7 M -1 seconds -1 It can be said that an antibody binds to a target antigen with an on-rate (k(on)) of

[0068] A binding molecule, such as an antibody or a fragment, variant, or derivative thereof, may be said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope if it preferentially binds to that epitope to such an extent that it blocks, to some extent, the binding of the reference antibody or antigen-binding fragment to that epitope. Competitive inhibition may be measured by any method known in the art, such as a competitive ELISA assay. A binding molecule may be said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0069] As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope with one or more antigen-binding domains, e.g., those of an immunoglobulin molecule. See, e.g., pages 27-28 of Harlow, et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). As used herein, the term "avidity" refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, e.g., pages 29-34 of Harlow. Avidity is related to both the affinity of an individual antigen-binding domain in the population with a particular epitope and the valency of the immunoglobulin and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, e.g., a polymer, is of high avidity. The interaction between a bivalent monoclonal antibody and a receptor present at high density on the cell surface is also of high avidity.

[0070] A binding molecule or its antigen-binding fragment, variant or derivative as disclosed herein can also be described or specified in terms of its cross-reactivity.As used herein, the term "cross-reactivity" refers to the ability of a binding molecule, such as an antibody or its fragment, variant or derivative, specific for one antigen to react with a second antigen; a measure of the homology between two different antigenic substances.Thus, a binding molecule is cross-reactive if it binds to an epitope other than the one that induced its formation.A cross-reactive epitope generally contains many of the same complementary structural characteristics as the induced epitope, and in some cases can actually fit better than the original.

[0071] A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, may also be described or specified in terms of its binding affinity to an antigen. For example, a binding molecule may have a binding affinity of 5×10 -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or 10 -15 Dissociation constant or K not exceeding M D can bind to the antigen.

[0072] Single chain antibodies or other antibody fragments containing antigen-binding domains can be present alone or in combination with one or more of the following: hinge region, CH1, CH2, CH3, or CH4 domains, J chain, or secretory component. Also included are antigen-binding fragments that can include any combination of the variable region with one or more of the hinge region, CH1, CH2, CH3, CH4 domains, J chain, or secretory component. The binding molecules, e.g., antibodies or antigen-binding fragments thereof, can be from any animal origin, including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be chondricthoid in origin (e.g., from sharks). As used herein, a "human" antibody includes antibodies having a human immunoglobulin amino acid sequence, and includes antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins, which may or may not express endogenous immunoglobulins, as described above and, for example, in U.S. Patent No. 5,939,598 by Kucherlapati, et al.

[0073] As used herein, the term "heavy chain subunit" includes an amino acid sequence derived from an immunoglobulin heavy chain, and a binding molecule, e.g., an antibody, comprising a heavy chain subunit can include at least one of a VH domain, a CH1 domain, a hinge (e.g., upper, middle and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof. For example, a binding molecule, e.g., an antibody, or a fragment, variant or derivative thereof, can include, but is not limited to, a CH1 domain, in addition to a VH domain; a CH1 domain and a hinge and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain and a hinge and a CH3 domain; or a CH1 domain and a hinge domain and a CH2 domain and a CH3 domain. In certain aspects, a binding molecule, e.g., an antibody, or a fragment, variant or derivative thereof, can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain and a CH4 domain and a J chain. Additionally, a binding molecule for use in the present disclosure can lack portions of certain constant regions, e.g., all or part of the CH2 domain. It will be understood by those of skill in the art that these domains (e.g., heavy chain subunits) can be modified such that they differ in amino acid sequence from the native immunoglobulin molecule.

[0074] As used herein, the term "light chain subunit" includes amino acid sequences derived from an immunoglobulin light chain. A light chain subunit can include at least a VL, and can further include a CL (e.g., a Cκ or Cλ) domain.

[0075] Binding molecules, such as antibodies or antigen-binding fragments, variants, or derivatives thereof, can be described or specified in terms of the epitope or portion of the antigen that they recognize or specifically bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant." A target antigen can contain a single epitope, or at least two epitopes, and can contain any number of epitopes depending on the size, conformation, and type of antigen.

[0076] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0077] As used herein, the term "chimeric antibody" refers to an antibody in which the immunoreactive regions or sites are obtained or derived from a first species and the constant regions (which may be intact, partial or modified) are obtained from a second species. In some embodiments, the target binding regions or sites will be derived from a non-human source (e.g., mouse or primate) and the constant regions are human.

[0078] The term "multispecific antibody", e.g., "bispecific antibody", refers to an antibody that has antigen-binding domains for two or more different epitopes within a single antibody molecule. In addition to standard antibody structures, other binding molecules can be constructed with two binding specificities. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means (Strohlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). Bispecific antibodies can also be diabodies.

[0079] As used herein, the term "engineered antibody" refers to an antibody in which the variable domains and / or constant regions in either or both of the heavy and light chains have been altered by at least partial replacement of one or more amino acids in the CDRs, framework, and / or constant regions. In certain embodiments, the entire CDRs from an antibody of known specificity can be grafted into the framework regions of a heterologous antibody. The replacement CDRs can be from an antibody of the same class or equivalent subclass as the antibody from which the framework region is derived, but the CDRs can also be from an antibody of a different class, e.g., an antibody of a different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity are grafted into the framework regions of a human heavy or light chain is referred to herein as a "humanized antibody." In certain embodiments, not all of the CDRs are replaced with complete CDRs from the variable regions of the donor, and furthermore, the antigen-binding capacity of the donor can be transferred to the variable domains of the recipient. Given the descriptions set forth in, for example, U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, obtaining functional engineered or humanized antibodies would be well within the capabilities of one of ordinary skill in the art by routine experimentation or trial and error testing.

[0080] As used herein, the term "engineered" includes the manipulation of a nucleic acid molecule or a polypeptide molecule by synthetic means (e.g., by recombinant techniques, by in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or any combination of these techniques).

[0081] As used herein, the terms "linked," "fused," or "fusion," or other grammatical equivalents, can be used interchangeably. These terms refer to the joining of two or more elements or components by any means, including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous, longer ORF in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein that contains two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature). The reading frames are thus made continuous throughout the fused segments, but the segments can be physically or spatially separated, for example, by in-frame linker sequences. For example, polynucleotides encoding the CDRs of immunoglobulin variable regions can be fused in frame, but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0082] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide, in the amino-terminal to carboxy-terminal direction, in which amino acids adjacent to each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is "amino-terminal" or "N-terminal" to another portion of the polypeptide is that portion that comes earlier in the contiguous polypeptide chain. Similarly, a portion of a polypeptide that is "carboxy-terminal" or "C-terminal" to another portion of the polypeptide is that portion that comes later in the contiguous polypeptide chain. For example, in a typical antibody, the variable domain is "N-terminal" to the constant region, and the constant region is "C-terminal" to the variable domain.

[0083] The term "expression" as used herein refers to the process by which a gene produces a biochemical, e.g., a polypeptide. The process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, knockdown of the gene and both transient and stable expression. It includes, but is not limited to, transcription of the gene into RNA, e.g., messenger RNA (mRNA), and translation of such mRNA into a polypeptide. If the final desired product is a biochemical, expression includes producing that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product is either a nucleic acid produced by transcription of the gene, e.g., messenger RNA, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0084] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to a therapeutic approach that cures, alleviates, reduces the symptoms of an existing diagnosed condition or disease, and / or halts or slows its progression. Terms such as "prevent", "prevention", "avoid", "prevention", and the like refer to a prophylactic or preventative approach that prevents the onset of an undiagnosed targeted condition or disease. Thus, "those in need of treatment" can include those already with the disease, those prone to having the disease, and those in whom the disease is to be prevented.

[0085] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, farm animals, industrial animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0086] As used herein, phrases such as "subject benefiting from treatment" and "animal in need of treatment" include subjects, e.g., mammalian subjects, who will benefit from the administration of a binding molecule, such as an antibody, that comprises one or more antigen-binding domains. Such binding molecules, e.g., antibodies, can be used, for example, in the diagnosis and / or treatment or prevention of disease.

[0087] IgM binding molecule IgM is the first immunoglobulin produced by B cells in response to stimulation by an antigen and is present in serum at around 1.5 mg / ml with a half-life of 5 days. IgM is usually a pentameric or hexameric molecule. An IgM binding unit usually comprises two light chains and two heavy chains. While IgG contains three heavy chain constant domains (CH1, CH2 and CH3), the IgM heavy chain (μ) further contains a fourth constant domain (CH4) that comprises the C-terminal "tail". The constant region of human IgM usually comprises the amino acid sequence SEQ ID NO:1 (see Table 3). The human Cμ1 region spans from about amino acid 5 to about amino acid 102 of SEQ ID NO:1; the human Cμ2 region spans from about amino acid 114 to about amino acid 205 of SEQ ID NO:1; the human Cμ3 region spans from about amino acid 224 to about amino acid 319 of SEQ ID NO:1; the human Cμ4 region spans from about amino acid 329 to about amino acid 430 of SEQ ID NO:1; and the tail spans from about amino acid 431 to about amino acid 453 of SEQ ID NO:1.

[0088] Five IgM binding units can complex with an additional small polypeptide chain (J chain) to form an IgM antibody. The precursor human J chain comprises the amino acid sequence SEQ ID NO:7 (Table 3). The first 22 amino acids of SEQ ID NO:7 are a secretory signal peptide, and the mature human J chain begins at amino acid 23 of SEQ ID NO:7. In the absence of a J chain, the IgM binding unit is typically a hexamer. Without wishing to be bound by theory, it is believed that the assembly of the IgM binding unit into a hexameric or pentameric binding molecule involves the Cμ3, Cμ4, and tail domains. Thus, the hexameric or pentameric binding molecules provided in this disclosure typically comprise an IgM constant region that includes at least the Cμ3, Cμ4, and tail domains.

[0089] The IgM heavy chain constant region can further comprise a Cμ2 domain or a fragment thereof, a Cμ1 domain or a fragment thereof, and / or other IgM heavy chain domains. In certain aspects, a binding molecule as provided herein can comprise the complete IgM heavy chain (μ) constant region (SEQ ID NO:1) or a variant, derivative, or analog thereof.

[0090] Complement System and Complement-Dependent Cytotoxicity The complement system contains more than 30 glycoproteins, of which 20 are present in plasma and 10 are cell-associated regulators or receptors. Activation of the complement cascade induces diverse immune effector functions, including cell lysis, phagocytosis, chemotaxis, and activation of immune cells. Complement can be activated by three different pathways: the classical pathway, the alternative pathway, and the mannose-binding lectin pathway, all of which converge at the level of the C3 protein and result in complement-dependent cytotoxicity of target cells by the formation of the membrane attack complex (MAC). IgM and certain subclasses of IgG antibodies in immune complexes activate the classical complement pathway. The formation of antigen-antibody complexes induces a conformational change in the Fc portion of the IgM or IgG molecules that exposes the binding site for the C1 component of the complement system. C1 in serum is a macromolecular complex consisting of C1q and two molecules each of C1r and C1s. Binding of C1q to the Fc binding site induces a conformational change in C1r and C1s. Subsequently, proteolytically activated C1r cleaves C1s, resulting in activation of C1s. C1s then cleaves C2 and C4 to generate C2a, C2b, C4a, and C4b. C2a and C4b together form C3 convertase. Cleavage of the central component C3 by C3 convertase leads to the formation of C3b, a portion of which binds to the plasma membrane of target cells. Membrane-bound C3b interacts with C3 convertase, resulting in the formation of C5 convertase. As a result, C5 is cleaved into C5a and C5b. Activation of the final pathway leads to deposition of C5b-C9 components on the membrane of opsonized target cells to form the membrane attack complex, ultimately resulting in complement-dependent cytotoxicity (CDC).

[0091] The end result of complement-dependent cytolysis is the formation of holes in the lipid bilayer membrane of cells, disrupting the membrane's integrity. Complement-dependent cytotoxicity assays (CDC assays) examine the effectiveness of antibodies to activate the complement immune pathway to initiate the membrane attack complex and lysis of targeted cells. One method for CDC assays is to mix target cells to which the antibody being evaluated has bound with an additive, e.g., serum, containing components of the complement system, and then measure cell death. Cell death can be measured, for example, by preloading the target cells with a radioactive compound that is released from the cells upon cell death, and the effectiveness of the antibody to mediate cell death is measured by the level of radioactivity. Non-radioactive CDC assays, e.g., the CELLTITER-GLO® assay available from Promega, measure the release of abundant cellular components such as ATP by measuring fluorescence or luminescence. In a CDC event, mixing results in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture.

[0092] Modified human IgM constant regions with reduced CDC activity The present disclosure provides modified human IgM constant regions that, when expressed as part of a modified target-specific human IgM antibody directed against a target antigen expressed on a cell, exhibit reduced CDC activity of the cell in the presence of complement compared to the corresponding wild-type human IgM antibody. A "corresponding wild-type human IgM antibody" refers to a wild-type IgM antibody that is identical to the antibody comprising the modified human IgM constant region except for a modification or modifications in the constant region that affect CDC activity. For example, a "corresponding wild-type human IgM antibody" would include the same VH and VL regions, and any other modifications or truncations that the modified human IgM antibody may have other than the modifications that affect CDC activity. In certain aspects, the modified human IgM constant region comprises one or more amino acid substitutions, e.g., in or near the Cμ3 domain, e.g., in the region extending from about amino acid T302 of SEQ ID NO:1 to about amino acid K322 of SEQ ID NO:1, e.g., at least one, at least two, or at least three or more amino acid substitutions relative to a wild-type human IgM constant region at positions T302, C303, T304, V305, T306, H307, T308, D309, L310, P311, S312, P313, L314, K315, Q316, T317, I318, S319, R320, P321, and / or K322 of SEQ ID NO:1. Without wishing to be bound by theory, it is believed that the C1q component of complement binds to the human IgM constant region through at least certain amino acid residues present in the Cμ3 domain. Assays for measuring CDC are well known to those of skill in the art, and exemplary assays are described herein.

[0093] In certain embodiments, the modified human IgM constant region as provided herein comprises a substitution at position P311 of SEQ ID NO:1 compared to the wild-type human IgM constant region. In other embodiments, the modified IgM constant region as provided herein comprises a substitution at position P313 of SEQ ID NO:1 compared to the wild-type human IgM constant region. In other embodiments, the modified IgM constant region as provided herein comprises a substitution at position L310 of SEQ ID NO:1 compared to the wild-type human IgM constant region. In other embodiments, the modified IgM constant region as provided herein comprises a substitution at position K315 of SEQ ID NO:1 compared to the wild-type human IgM constant region. In other aspects, the modified IgM constant region as provided herein contains a combination of substitutions compared to the wild-type human IgM constant region at two or more of positions L310 of SEQ ID NO:1, P311 of SEQ ID NO:1, P313 of SEQ ID NO:1, and / or K315 of SEQ ID NO:1. The modified IgM constant region as provided herein can be substituted at amino acid position P311 of SEQ ID NO:1, for example, with alanine (P311A) (SEQ ID NO:2), serine (P311S), or glycine (P311G). The modified IgM constant region as provided herein can be substituted at amino acid position P313 of SEQ ID NO:1, for example, with alanine (P313A), serine (P313S) (SEQ ID NO:3), or glycine (P313G). The modified IgM constant regions provided herein can have an amino acid substitution at position L310 of SEQ ID NO:1, for example, with alanine (L310A) (SEQ ID NO:15), serine (L310S), glycine (L310G) or aspartic acid (L310D) (SEQ ID NO:23).Modified IgM constant regions as provided herein can have amino acid substitutions at amino acid positions K315 of SEQ ID NO:1, for example, with alanine (K315A) (SEQ ID NO:16), serine (K315S), glycine (K315G), aspartic acid (K315D) (SEQ ID NO:24), or glutamine (K315Q) (SEQ ID NO:25). Modified IgM constant regions as provided herein can have amino acid substitutions at amino acid positions P311 and P313 of SEQ ID NO:1, for example, with alanine (P311A) and serine (P313S), respectively (SEQ ID NO:4), or any combination of alanine, serine, and / or glycine. Modified IgM constant regions as provided herein can be substituted at amino acid positions L310 and K315 of SEQ ID NO:1, for example, with alanine (L310A, K315A) (SEQ ID NO:17), or serine (L310S, K315S) (SEQ ID NO:18), or a combination of alanine, serine, aspartic acid, glutamine, and / or glycine. Modified IgM constant regions as provided herein can be substituted at amino acid positions L310 and P311 of SEQ ID NO:1, for example, with alanine (L310A, P311A) (SEQ ID NO:19), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine. Modified IgM constant regions as provided herein can have amino acids at positions L310 and P313 of SEQ ID NO:1 substituted, for example, with alanine and serine, respectively (L310A, P313S) (SEQ ID NO:20), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine.Modified IgM constant regions as provided herein can have amino acids at positions P311 and K315 of SEQ ID NO:1 substituted, for example, by alanine (P311A, K315A) (SEQ ID NO:21), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine. Modified IgM constant regions as provided herein can have amino acids at positions P313 and K315 of SEQ ID NO:1 substituted, for example, by serine and alanine (P313S, K315A), respectively (SEQ ID NO:22), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine.

[0094] In certain aspects, a polypeptide as provided herein may be used, e.g., comprising an amino acid substitution at L310, P311, P313, and / or K315, e.g., L310A, L310S, L310G, L310D, P311A, P311S, P311G, P313A, P313S, P313G, K315A, K315S, K315G, K315D, and / or K315Q, or any combination thereof. The complement-dependent cytotoxicity (CDC) activity of a target-specific IgM antibody comprising such a modified human IgM constant region can be reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a corresponding wild-type IgM antibody, i.e., compared to a wild-type target-specific IgM antibody that is identical except for the modified human IgM constant region.

[0095] In certain aspects, the antibody concentration resulting in 50% CDC activity (EC) of a target-specific IgM antibody comprising a modified human IgM constant region as provided herein, e.g., comprising amino acid substitutions at L310, P311, P313, and / or K315, e.g., L310A, L310S, L310G, L310D, P311A, P311S, P311G, P313A, P313S, P313G, K315A, K315S, K315G, K315D, and / or K315Q, or any combination thereof.50 ) can be increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to a corresponding wild-type IgM antibody, i.e., compared to a wild-type target-specific IgM antibody that is identical except for the modified human IgM constant region.

[0096] Modified human IgM constant regions as provided herein can be constructed by standard mutagenesis techniques well known to those skilled in the art or can be obtained from various suppliers. Methods of mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983), Techniques in Molecular Biology. (MacMillan Publishing Company, New York); Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492 (1985); Kunkel, et al., Methods Enzymol. 154:367-382 (1987); Sambrook, et al. (1989), Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor, NY); U.S. Patent No. 4,873,192; and references cited therein, all of which are incorporated herein by reference. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found in the model of Dayhoff, et al. (1978), in Atlas of Protein Sequence and Structure, (Natl. Biomed. Res. Found., Washington, DC), pp. 345-352, which is incorporated herein by reference in its entirety. The Dayhoff et al. model uses an acceptable point mutation (PAM) amino acid similarity matrix (PAM250 matrix) to determine suitable conservative amino acid substitutions. In certain embodiments, conservative substitutions, e.g., replacing one amino acid with another amino acid with similar properties, are used. Examples of conservative amino acid substitutions as taught by the PAM250 matrix of the Dayhoff et al. model include, but are not limited to, Gly Ala, Val Ile Leu, Asp Glu, Lys Arg, Asn Gln, and Phe Trp Tyr.

[0097] Pentameric or hexameric modified human IgM antibodies In another aspect, the disclosure provides modified human IgM antibodies, the modified human IgM antibodies comprising a modified human IgM constant region as provided herein and further comprising a heavy chain variable region (VH) located amino-terminal to the modified human IgM constant region, the heavy chain variable region (VH) specifically binds to a target antigen (alone or in combination with a light chain variable region (VL)) and exhibits reduced complement dependent cytotoxicity (CDC) of cells expressing the target antigen and / or an antibody concentration that results in 50% CDC activity (EC 50 In certain embodiments, the modified human IgM antibody comprises one or more amino acid substitutions, e.g., in or near the Cμ3 domain, e.g., in the region extending from about amino acid T302 of SEQ ID NO:1 to about amino acid K322 of SEQ ID NO:1, e.g., at least one, at least two, or at least three or more amino acid substitutions at positions T302, C303, T304, V305, T306, H307, T308, D309, L310, P311, S312, P313, L314, K315, Q316, T317, I318, S319, R320, P321, and / or K322 of SEQ ID NO:1 relative to a wild-type human IgM constant region. Without wishing to be bound by theory, it is believed that the C1q component of complement binds to the human IgM constant region at least through specific amino acid residues present in the Cμ3 domain. Assays for measuring CDC are well known to those of skill in the art, and exemplary assays are described herein.

[0098] In certain embodiments, modified human IgM antibodies as provided herein contain a substitution at position P311 of SEQ ID NO:1 compared to a wild-type human IgM antibody. In other embodiments, modified human IgM antibodies as provided herein contain a substitution at position P313 of SEQ ID NO:1 compared to a wild-type human IgM antibody. In other embodiments, modified human IgM antibodies as provided herein contain a substitution at position L310 of SEQ ID NO:1 compared to a wild-type human IgM antibody. In other embodiments, modified human IgM antibodies as provided herein contain a substitution at position K315 of SEQ ID NO:1 compared to a wild-type human IgM antibody. In other aspects, modified IgM antibodies as provided herein contain a combination of substitutions compared to a wild-type human IgM antibody at two or more of position L310 of SEQ ID NO:1, position P311 of SEQ ID NO:1, position P313 of SEQ ID NO:1, and / or position K315 of SEQ ID NO:1. Modified IgM antibodies as provided herein can have a substitution at amino acid position P311 of SEQ ID NO:1, for example, with alanine (P311A) (SEQ ID NO:2), serine (P311S) or glycine (P311G). Modified IgM antibodies as provided herein can have a substitution at amino acid position P313 of SEQ ID NO:1, for example, with alanine (P313A), serine (P313S) (SEQ ID NO:3) or glycine (P313G). Modified IgM antibodies as provided herein can have a substitution at amino acid position L310 of SEQ ID NO:1, for example, with alanine (L310A) (SEQ ID NO:15), serine (L310S), glycine (L310G) or aspartic acid (L310D) (SEQ ID NO:23).Modified IgM antibodies as provided herein can have amino acid substitutions at position K315 of SEQ ID NO:1, for example, with alanine (K315A) (SEQ ID NO:16), serine (K315S), glycine (K315G), aspartic acid (K315D) (SEQ ID NO:24), or glutamine (K315Q) (SEQ ID NO:25). Modified IgM antibodies as provided herein can have amino acid substitutions at positions P311 and P313 of SEQ ID NO:1, for example, with alanine (P311A) and serine (P313S), respectively (SEQ ID NO:4), or any combination of alanine, serine, and / or glycine. Modified IgM antibodies as provided herein can have amino acids at positions L310 and K315 of SEQ ID NO:1 substituted, for example, by alanine (L310A, K315A) (SEQ ID NO:17), or serine (L310S, K315S) (SEQ ID NO:18), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine. Modified IgM antibodies as provided herein can have amino acids at positions L310 and P311 of SEQ ID NO:1 substituted, for example, by alanine (L310A, P311A) (SEQ ID NO:19), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine. Modified IgM antibodies as provided herein can have amino acids at positions L310 and P313 of SEQ ID NO:1 substituted, for example, with alanine and serine (L310A, P313S) (SEQ ID NO:20), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine. Modified IgM antibodies as provided herein can have amino acids at positions P311 and K315 of SEQ ID NO:1 substituted, for example, with alanine (P311A, K315A) (SEQ ID NO:21), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine.Modified IgM antibodies as provided herein can have amino acids at positions P313 and K315 of SEQ ID NO:1 substituted, for example, with serine and alanine, respectively (P313S, K315A) (SEQ ID NO:22), or any combination of alanine, serine, aspartic acid, glutamine, and / or glycine.

[0099] In certain aspects, the complement dependent cytotoxicity (CDC) activity of a modified IgM antibody as provided herein, comprising, for example, amino acid substitutions at L310, P311, P313, and / or K315, e.g., L310A, L310S, L310G, L310D, P311A, P311S, P311G, P313A, P313S, P313G, K315A, K315S, K315G, K315D, and / or K315Q, or any combination thereof, can be reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a corresponding wild-type IgM antibody, i.e., compared to a wild-type target-specific IgM antibody that is identical except for the modified human IgM constant region.

[0100] In certain aspects, the antibody concentration resulting in 50% CDC activity (EC) of an IgM antibody as provided herein includes, for example, amino acid substitutions at L310, P311, P313, and / or K315, e.g., L310A, L310S, L310G, L310D, P311A, P311S, P311G, P313A, P313S, P313G, K315A, K315S, K315G, K315D, and / or K315Q, or any combination thereof. 50 ) can be increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to a corresponding wild-type IgM antibody, i.e., compared to a wild-type target-specific IgM antibody that is identical except for the modified human IgM constant region.

[0101] The modified human IgM antibodies as provided herein can be hexamers or pentamers comprising five or six IgM "binding units" as defined herein, one or more of which can specifically bind to a target antigen of interest. The present disclosure encompasses modified human IgM antibodies that specifically bind to any target antigen. In certain embodiments, the target antigen can be expressed on the surface of a cell, e.g., a eukaryotic cell. Target antigens can include, but are not limited to, tumor antigens, other oncological targets, e.g., immune-oncological targets such as immune checkpoint inhibitors, infectious disease antigens, e.g., viral antigens expressed on the surface of infected cells, target antigens involved in blood-brain barrier trafficking, target antigens involved in neurodegenerative and neuroinflammatory diseases, and any combination thereof. Non-limiting examples of target antigens, as well as non-limiting examples of antibody binding domains that bind to such target antigens, can be found, for example, in PCT Publication Nos. WO2016 / 141303, WO2016 / 168758, WO2016 / 154593, WO2016 / 118641, WO2015 / 153912, WO2015 / 053887, WO2013 / 120012, WO / 2017 / 059387, WO / 2017 / 059380, WO / 2018 / 017888, WO / 2018 / 017889, WO / 2018 / 017761, and WO / 2018 / 017763, the disclosures of which are incorporated herein by reference in their entireties.

[0102] In certain aspects, the present disclosure provides pentameric or hexameric modified human IgM antibodies comprising five or six bivalent binding units, respectively, each binding unit comprising two modified human IgM heavy chain constant regions or fragments thereof as provided herein.

[0103] When the modified human IgM antibody provided herein is a pentamer, the antibody can further comprise a J chain or a functional fragment or variant thereof. In certain embodiments, the J chain is a modified J chain comprising a heterologous moiety or one or more heterologous moieties, e.g., a heterologous polypeptide sequence, e.g., an exogenous binding domain introduced into the native sequence. In certain embodiments, the exogenous binding domain specifically binds to CD3, e.g., CD3ε. In certain embodiments, the modified J chain comprises V15J (SEQ ID NO:9) or J15V (SEQ ID NO:11).

[0104] The IgM heavy chain constant region can comprise one or more of a Cμ1 domain, a Cμ2 domain, a Cμ3 domain (as provided herein, Cμ3 can comprise one or more amino acid substitutions), and / or a Cμ4 domain, provided that the constant region is capable of serving a desired function in the modified human IgM antibody, e.g., capable of combining with a second IgM constant region to form a binding unit, or of combining with other binding units to form a hexamer or pentamer. In certain embodiments, the two modified human IgM heavy chain constant regions or fragments thereof within an individual binding unit each comprise a modified Cμ3 domain (e.g., with substitution at one or more of L310 of SEQ ID NO:1, P311 of SEQ ID NO:1, P313 of SEQ ID NO:1, K315 of SEQ ID NO:1, and / or any combination thereof) or fragment thereof, a Cμ4 domain or fragment thereof, a tail (TP) or fragment thereof, or any combination of a modified Cμ3 domain, a Cμ4 domain, and a TP or fragment thereof. In certain embodiments, the two modified human IgM heavy chain constant regions or fragments thereof within an individual binding unit each further comprise a Cμ2 domain or fragment thereof, a Cμ1 domain or fragment thereof, or a Cμ1 domain or fragment thereof and a Cμ2 domain or fragment thereof.

[0105] In a particular embodiment, each of the two modified human IgM heavy chain constant regions in a given binding unit is associated with an antigen binding domain, e.g., a heavy chain variable region (VH) or Fv portion of an antibody, e.g., the VH and VL of the antibody.

[0106] Modified J chain In certain aspects, modified human IgM antibodies as provided herein can be bispecific by incorporating a modified J chain. As provided herein and in PCT Publication Nos. WO2015 / 153912, WO / 2017 / 059387, and WO / 2017 / 059380, the modified J chain can include a heterologous moiety, e.g., a heterologous polypeptide, e.g., a polypeptide binding domain capable of specifically binding to a target, e.g., a foreign binding domain. The binding domain can be, e.g., an antibody or antigen-binding fragment thereof, an antibody-drug conjugate or antigen-binding fragment thereof, or an antibody-like molecule. The polypeptide binding domain can be introduced into the J chain by appropriately selecting the position and type of attachment (e.g., direct or indirect fusion, chemical linkage, etc.).

[0107] In certain embodiments, the binding domain can be an antibody or an antigen-binding fragment of an antibody, including monospecific, bispecific, and multispecific antibodies and antibody fragments. The antibody fragment can be, but is not limited to, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv, an (scFv)2 fragment, a single-chain antibody molecule, a minibody, or a multispecific antibody formed from antibody fragments. In certain embodiments, the antibody fragment is an scFv.

[0108] In other aspects, the binding domain can be an antibody-like molecule, such as a human domain antibody (dAb), a dual affinity retargeting (DART) molecule, a diabody, a di-diabody, a dual variable domain antibody, a stacked variable domain antibody, a small modular immunopharmaceutical (SMIP), a Surrobody, a strand exchange engineered domain (SEED)-body, or a TandAb.

[0109] The binding domain can be introduced into the native J chain sequence at any position that allows the binding domain to bind to its binding target without interfering with the binding of the recipient IgM molecule to its binding target or targets or the ability of the J chain to efficiently incorporate into an IgM pentamer. In certain embodiments, the binding domain can be inserted at or near the C-terminus, at or near the mature N-terminus (e.g., amino acid number 23 of SEQ ID NO:7 after cleavage of the signal peptide), or at an internal position that is accessible based on the three-dimensional structure of the J chain. In certain embodiments, the binding domain can be introduced into the native sequence J chain about 10 residues removed from the C-terminus or about 10 residues removed from the mature N-terminus (amino acid 23) of the human J chain of SEQ ID NO:7. In another embodiment, the binding domain can be introduced into the native sequence human J chain of SEQ ID NO:7 between cysteine ​​residues 113 and 122 of SEQ ID NO:7, or at the equivalent position of another native sequence J chain. In further embodiments, a binding domain can be introduced into a native sequence J chain at or near a glycosylation site, e.g., the J chain of SEQ ID NO:7. In particular embodiments, a binding domain can be introduced into a native sequence human J chain of SEQ ID NO:7 within about 10 residues of the C-terminus.

[0110] The introduction can be achieved by direct or indirect fusion, i.e., by combining the J chain and the binding domain in one polypeptide chain by in-frame combination of the coding nucleotide sequences with or without a peptide linker. The peptide linker (indirect fusion), if used, can be about 1-50, or about 1-40, or about 1-30, or about 1-20, or about 1-10, or about 10-20 amino acids in length and can be present at either or both ends of the binding domain introduced into the J chain sequence. In certain embodiments, the peptide linker is about 10-20 or 10-15 amino acids in length. In certain embodiments, the peptide linker is 15 amino acids in length. In certain embodiments, the peptide linker is (GGGGS)3 (SEQ ID NO:12).

[0111] It is also possible to introduce multiple heterologous polypeptides into the J chain, eg, multiple binding domains or other moieties.

[0112] The modified J chain can be produced by well known techniques of recombinant DNA technology by expressing a nucleic acid encoding the modified J chain in a suitable prokaryotic or eukaryotic host organism.

[0113] The modified J chain can also be co-expressed with the heavy and light chains of a recipient modified human IgM antibody as described elsewhere herein. The recipient antibody can be monospecific, bispecific or multispecific prior to incorporation of the modified J chain, e.g., a monospecific, bispecific or multispecific modified human IgM antibody. Bispecific or multispecific IgM antibodies, including antibodies, are described, e.g., in PCT Publication Nos. WO2015 / 053887 and WO2015 / 120474, the contents of which are expressly incorporated herein by reference in their entireties.

[0114] In certain embodiments, modified human IgM antibodies as described herein can include a modified J chain with binding specificity for immune effector cells, such as, for example, T cells, NK cells, macrophages, or neutrophils. In certain embodiments, the effector cells are T cells and the binding target is CD3 (discussed below). By activating and redirecting effector cells, e.g., effector T cells, such as cytotoxic T cells (CTLs), to cells expressing a target antigen of interest, modified human IgM antibodies as provided herein can generate enhanced effector responses against the target antigen, thereby further increasing potency and efficacy. In certain embodiments, the ability of bispecific modified human IgM antibodies as provided herein to elicit T cell-mediated cytotoxicity by binding to CD3 via a modified J chain is not affected by modifications in the IgM constant region that reduce CDC compared to the corresponding wild-type IgM antibody.

[0115] In T cells, the cluster of differentiation 3 (CD3) is a multimeric protein complex historically known as the T3 complex, which is composed of four distinct polypeptide chains (ε, γ, δ, ζ) that assemble and function as three pairs of dimers (εγ, εδ, ζζ). The CD3 complex serves as a T cell coreceptor that binds non-covalently to the T cell receptor (TCR). Components of this CD3 complex, particularly CD3ε, can be targets for the modified J chains of the bispecific IgM or IgA binding molecules provided herein.

[0116] In a particular embodiment, where the J chain is modified to bind to CD3ε, a bispecific modified human IgM antibody is provided.

[0117] In certain embodiments, the anti-CD3ε binding domain of the modified J chain provided herein is an scFv. The anti-CD3ε scFv can be fused directly or indirectly to or near the N-terminus of the J chain or to or near the C-terminus of the J chain by a synthetic linker, such as a (GGGGS)3 linker (SEQ ID NO:12), introduced between the scFv and the J chain sequence. In certain embodiments, the scFv comprises the VH and VL regions of visilizumab (Nuvion). In certain embodiments, the modified J chain comprises an scFv comprising the VH of visilizumab, a (GGGGS)3 linker, and the VL of visilizumab.

[0118] In certain embodiments, the modified J chain comprises a scFv of visilizumab fused to the N-terminus of a human J chain via a 15 amino acid (GGGGS)3 linker, a modified J chain referred to herein as V15J. V15J can further comprise a signal peptide to facilitate transport and assembly into modified human IgM antibodies as provided herein. The mature V15J protein is presented as SEQ ID NO:9, and the precursor form comprising a 19 amino acid immunoglobulin heavy chain signal peptide is presented as SEQ ID NO:8. In certain embodiments, the modified J chain comprises a scFv of visilizumab fused to the C-terminus of a human J chain via a 15 amino acid (GGGGS)3 linker, a modified J chain referred to herein as J15V. J15V can further comprise a signal peptide to facilitate transport and assembly into modified human IgM antibodies as provided herein. The mature J15V protein is presented as SEQ ID NO:11, and the precursor form containing the 22 amino acid human J chain signal peptide is presented as SEQ ID NO:10. In certain embodiments, other signal peptides can be used. The selection and inclusion of a suitable signal peptide to facilitate expression, secretion and incorporation of the modified J chain into modified human IgM antibodies as provided herein is well within the capabilities of one of skill in the art.

[0119] The present disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art and are fully explained in the literature. For example, Sambrook, et al., ed. (1989), Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook, et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DNGlover, ed., (1985) DNA Cloning, Volumes I and II; Synthesis; Mullis, et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press)(1986);Perbal(1984)A Practical Guide To Molecular Cloning;the treatise,Methods In Enzymology(Academic Press,Inc.,NY);Miller and Calos,eds.(1987)Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory);Wu,et al.,eds.,Methods In Enzymology,Vols.154 and 155;Mayer and Walker,eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and Ausubel, et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). See also.

[0120] The general principles of antibody engineering are described in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). The general principles of protein engineering are described in Rickwood, et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). The general principles of antibodies and antibody-hapten binding are described in Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, NY). Additionally, routine methods in immunology known in the art and not specifically described can be followed, such as those found in Current Protocols in Immunology, John Wiley & Sons, New York; Stites, et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (WH Freeman and Co., NY).

[0121] Standard references describing the general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett, et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) “Monoclonal Antibody Technology” in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden, et al., (Elsevier, Amsterdam); Goldsby, et al., eds. (2000) Kuby Immunology (4th ed.; W. H. Freeman and Co., NY); Roitt, et al. (2001) Immunology (6th ed. ed.;London:Mosby);Abbas,et al.(2005)Cellular and Molecular Immunology(5th ed.;Elsevier Health Sciences Division);Kontermann and Dubel(2001)Antibody Engineering(Springer Verlag);Sambrook and Russell(2001)Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Press);Lewin(2003)Genes VIII(Prentice Hall,2003);Harlow and Lane(1988)Antibodies:A Laboratory Manual(Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer, (Cold Spring Harbor Press). .

[0122] All references cited above, as well as all references cited herein, are hereby incorporated by reference in their entirety.

[0123] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0124] Example 1: Effect of Mutations in the Cμ3 Domain on IgM Association pFUSEss-CHIg-hM encodes a modified human IgM constant region with single site mutations in the Cμ3 domain: P311A (SEQ ID NO:2), P313S (SEQ ID NO:3), both P311A and P313S (SEQ ID NO:4), D294G (SEQ ID NO:5), and S283N (SEQ ID NO:6). * Plasmid variants of 03 were designed and submitted to a supplier for synthesis. Exemplary plasmid constructs capable of expressing wild-type or modified human pentameric or hexameric IgM antibodies containing wild-type or modified IgM constant regions and capable of specifically binding to CD20 were generated by the following method.

[0125] DNA fragments encoding the VH and VL regions of 1.5.3 (SEQ ID NOs: 13 and 14, respectively) with EcoRV and XbaI restriction sites at the 5' and 3' ends for subcloning into heavy and light chain expression vectors were synthesized by a supplier (Genescript). The synthesized DNA constructs were resuspended in Tris-EDTA buffer at 1 μg / ml. DNA samples (1 μg) were digested with EcoRV and XbaI and the synthesized VH and VL were separated from the carrier plasmid DNA by electrophoresis. The digested DNA was ligated to predigested plasmid DNA (pFUSEss-CHIg-hM available from Invivogen) by standard molecular biology techniques. * The constructs were ligated into pFUSE2ss-CLIg-hk for the kappa chain, a modified construct as described above for the μ chain available from Invivogen (pFUSE2ss-CLIg-hk for the kappa chain). The ligated DNA was transformed into competent bacteria and plated on LB plates with multiple selective antibiotics. Several bacterial colonies were picked and DNA preparations made by standard molecular biology techniques. The constructs encoding the heavy and light chains were verified by sequencing.

[0126] Plasmid constructs encoding IgM heavy and light chains or heavy, light and J chains were co-transfected into CHO cells and cells expressing wild-type or modified CD20 IgM antibodies with or without the J chain were selected, all according to standard procedures.

[0127] Antibodies present in cell supernatants were recovered using Capture Select IgM (Catalog 2890.05, BAC, Thermo Fisher) according to the manufacturer's protocol. Antibodies were assessed on SDS PAGE under non-reducing conditions (Figures 1 and 5) or, for certain bispecific antibodies, under reducing conditions (Figure 5) to demonstrate association as previously described, for example, in PCT Publication No. WO2016 / 141303.

[0128] Control antibodies (IgM(pure), IgM+J(pure) (IgM antibody with wild-type J chain) and IgM(IgM lacking J chain)), and IgM Cμ3 domain mutants, S283N, P313S, P311A, and D294G, were evaluated in SDS-PAGE under non-reducing conditions to demonstrate antibody association. NuPage LDS sample buffer (Life Technologies) was added to the samples before loading onto a NativePage Novex 3-12% Bis-Tris gel (Life Technologies catalog no. BN1003). Novex Tris-acetate SDS running buffer (Life Technologies catalog no. LA0041) was used for gel electrophoresis. The gel was run until the dye front reached the bottom of the gel. Following electrophoresis, the gel was stained with colloidal blue stain (Life Technologies catalog no. LC6025).

[0129] The results shown in Figure 1 demonstrate that the IgM Cμ3 domain mutant antibodies associate similarly to the control antibody.

[0130] A control bispecific antibody (1.5.3 IgM V15J) and the 1.5.3 IgM V15J Cμ3 domain mutants P313S, P311A, and the P311A / P313S double mutant were evaluated in SDS PAGE under non-reducing ("hybrid") conditions and under reducing conditions to demonstrate antibody association. NuPage LDS sample buffer (Life Technologies) was added to the samples before loading onto a NativePage Novex 3-12% Bis-Tris gel (Life Technologies Catalog No. BN1003). Novex Tris-Acetate SDS Running Buffer (Life Technologies Catalog No. LA0041) was used for gel electrophoresis. The gel was run until the dye front reached the bottom of the gel. Following electrophoresis, the gel was stained with colloidal blue stain (Life Technologies Catalog No. LC6025).

[0131] The results shown in Figure 5 demonstrate that the bispecific IgM Cμ3 domain mutant antibodies associate similarly to the control antibody.

[0132] Example 2: Complement-dependent cytotoxicity of IgM Cμ3 mutants The CD20 expressing Ramos (ATCC Catalog No. CRL-1596) cell line was obtained from ATCC and DSMZ. 50,000 cells were seeded in 96-well plates. Cells were treated with wild-type control antibody, 1.5.3 IgM, 1.5.3 IgG, and 1.5.3 IgM×V15J, and Cμ3 domain single mutants of IgM (P311A, P313S, D294G, and S283N). Human serum complement (Quidel Catalog No. A113) was added to the antibody-treated cells at a final concentration of 10%. The reaction mixture was incubated at 37° C. for 4 hours. CELLTITER-GLO® Reagent (Promega Catalog No. G7572) was added in a volume equal to the volume of culture medium present in each well. The plate was shaken for 2 minutes, incubated at room temperature for 10 minutes, and luminescence was measured in a luminometer.

[0133] The results are shown in Figure 2. The two IgM Cμ3 single mutants P311A and P313S showed nearly 50% less cell killing in the presence of complement than the control 1.5.3 IgM antibody. The IgM Cμ3 mutants S283N and D294G showed similar complement-mediated cell killing as the control wild-type IgM. In the absence of complement, minimal cell killing was observed for the 1.5.3 IgG and 1.5.3 IgMxV15J antibodies.

[0134] Example 3: Complement-dependent cytotoxicity of bispecific anti-CD20 x anti-CD3 IgM Cμ3 mutants Ramos (ATCC Catalog No. CRL-1596) cell line expressing CD20 was obtained from ATCC and DSMZ. 50,000 cells were seeded in 96-well plates. Cells were treated with control antibody, 1.5.3 IgM×V15J, and bispecific IgM Cμ3 domain mutants (P311A×V15J, P313S×V15J, D294G×V15J, and S283N×V15J). All antibodies contained bispecific J chains for CD20 and CD3 generated and expressed as previously described. See PCT Publication No. WO2016 / 141303. Human serum complement (Quidel Catalog No. A113) was added to the antibody-treated cells at a final concentration of 10%. The reaction mixture was incubated at 37° C. for 4 hours. CELLTITER-GLO® reagent (Promega catalog number G7572) was added in a volume equal to the volume of culture medium present in each well. The plates were shaken for 2 minutes, incubated at room temperature for 10 minutes, and luminescence was measured in a luminometer.

[0135] The results are shown in Figures 3 and 6. Bispecific IgM Cμ3 mutants P311A and P313S exhibited half-maximal killing of Ramos cells by complement, while control 1.5.3 IgM x V15J and bispecific IgM Cμ3 mutants D294G and S283N achieved near-maximal complement-dependent cytotoxicity (90-100% effective) in the presence of complement. In the assay shown in Figure 6, bispecific IgM Cμ3 mutant P311A exhibited approximately 46% maximal killing by complement (EC 50 The EC 50 (The IL-10 binding site was 243 pM.) Complement-mediated killing was essentially abolished in the double mutant P311A / P313S (Fig. 6; see also Table 2).

[0136] Example 4: T cell activation potential of bispecific anti-CD20 x anti-CD3 IgM Cμ3 mutants Engineered Jurkat T cells (Promega CS176403) and RPMI8226 cells (ATCC CCL-155) were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Serially diluted bispecific 1.5.3 IgMxV15J antibodies and Cμ3 mutants of bispecific IgM, D294GxV15J, S283NxV15J, P313SxV15J, P311AxV15J, P311A / P313SxV15J, and 1.5.3 IgM (no J chain) were incubated with 7500 RPMI8226 cells in 20 μL in white 384-well assay plates at 37° C., 5% CO2 for 2 hours. Engineered Jurkat cells (25,000) were added to the mixture to a final volume of 40 μL. The mixture was incubated at 37° C., 5% CO2 for 5 hours. The cell mixture was then mixed with 20 μL of lysis buffer containing luciferin (Promega, CELLTITER-GLO®) to measure luciferase reporter activity. Light output was measured by an EnVision plate reader. EC 50 was determined by four-parameter curve fitting using Prism software.

[0137] The results are shown in Figures 4 and 7. All bispecific IgM Cμ3 mutants activated T cells as effectively as the bispecific control antibody (1.5.3 IgM×V15J). No T cell activation was observed with the IgM control lacking the bispecific J chain (1.5.3 IgM).

[0138] Example 5: Complement-dependent cytotoxicity of additional bispecific anti-CD20 x anti-CD3 IgM Cμ3 mutants To further evaluate the effect of the Cμ3 mutations on CDC activity, deliberate single amino acid Ala substitutions were made at each position from T302 of SEQ ID NO:1 to K322 of SEQ ID NO:1. Each of these mutant IgM constant regions was constructed as a Cμ3 domain mutant of 1.5.3 IgM V15J as described in Example 1 and examined for CDC activity as described in Example 3 and T cell activation as described in Example 4. In addition to the previously evaluated mutations at P311 and P313, Ala substitutions in the Cμ3 domain of 1.5.3 IgM V15J at L310 (SEQ ID NO:15) and K315 (SEQ ID NO:16) increased CDC activity as shown in Figure 8 and Table 2 without significantly affecting T cell activation (data not shown). 50 The CDC activity decreased moderately with increasing concentration.

[0139] Various combinations of double substitutions at two of the positions L310, P311, P313, and K315 were also constructed as described in Example 1 and tested for CDC activity as described in Example 3. These included the double mutants L310A, K315A (SEQ ID NO:17), L310S, K315S (SEQ ID NO:18), L310A, P311A (SEQ ID NO:19), L310A, P313S (SEQ ID NO:20), P311A, K315A (SEQ ID NO:21), and P313S, K315A (SEQ ID NO:22). The CDC activity of these double mutants compared to wild-type human IgM is summarized in Table 2. The L310A, K315A double mutant showed a large reduction in CDC activity compared to either of the single mutants.

[0140] Additional amino acid substitutions at positions L310 and K315 were evaluated to determine the effect of charged or polar amino acids on CDC activity. L310 was substituted with a negatively charged aspartic acid (D, SEQ ID NO:23) and K315 was substituted with an aspartic acid (D, SEQ ID NO:24) or the polar amino acid glutamine (Q, SEQ ID NO:25). These mutants were tested for CDC activity as described in Example 3. The results are shown in Figures 9A and 9B and in Table 2. Introduction of a negatively charged amino acid at positions 310 or 315 abolished CDC activity to a greater extent than substitution with the neutral Ala.

[0141] Table 2 CDC activity of human IgM Cμ3 mutants TIFF2024177479000004.tif145138

[0142] (Table 3) Sequence TIFF2024177479000005.tif216156TIFF2024177479000006.tif221156TIFF2024177479000007.tif216156 TIFF2024177479000008.tif226156TIFF2024177479000009.tif226156TIFF2024177479000010.tif199156

[0143] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0144] Sequence information SEQUENCE LISTING <110> IGM BIOSCIENCES, INC. <120> MODIFIED HUMAN IGM CONSTANT REGIONS FOR MODULATION OF COMPLEMENT-DEPENDENT CYTOLYSIS EFFECTOR FUNCTION <150> US 62 / 483,087 <151> 2017-04-07 <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 453 <212> PRT <213> Homo sapiens <400> 1 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 2 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Ala Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 3 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Ser Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 4 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 4 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Ala Ser Ser Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 5 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Gly Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 6 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Asn Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 7 <211> 158 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Met Lys Asn His Leu Leu Phe Trp Gly Val Leu Ala Val Phe Ile Lys 1 5 10 15 Ala Val His Val Lys Ala Gln Glu Asp Glu Arg Ile Val Leu Val Asp 20 25 30 Asn Lys Cys Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser 35 40 45 Glu Asp Pro Asn Glu Asp Ile Val Glu Arg Asn Ile Ile Ile Val Pro 50 55 60 Leu Asn Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr 65 70 75 80 Arg Phe Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr 85 90 95 Glu Val Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile 100 105 110 Cys Asp Glu Asp Ser Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn 115 120 125 Lys Cys Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys 130 135 140 Met Val Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp 145 150 155 <210> 8 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Ile Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn 65 70 75 80 Gln Lys Leu Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 165 170 175 Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys 180 185 190 Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala 195 200 205 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 210 215 220 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 225 230 235 240 Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys 245 250 255 Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 260 265 270 Gly Gly Ser Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys 275 280 285 Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro 290 295 300 Asn Glu Asp Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn 305 310 315 320 Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe 325 330 335 Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val 340 345 350 Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp 355 360 365 Glu Asp Ser Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn Lys Cys 370 375 380 Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val 385 390 395 400 Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp 405 410 <210> 9 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Trp Ser Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 245 250 255 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 260 265 270 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 275 280 285 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 290 295 300 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 305 310 315 320 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 325 330 335 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 340 345 350 Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 355 360 365 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 370 375 380 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 385 390 <210> 10 <211> 415 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Met Lys Asn His Leu Leu Phe Trp Gly Val Leu Ala Val Phe Ile Lys 1 5 10 15 Ala Val His Val Lys Ala Gln Glu Asp Glu Arg Ile Val Leu Val Asp 20 25 30 Asn Lys Cys Lys Cys Ala Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser 35 40 45 Glu Asp Pro Asn Glu Asp Ile Val Glu Arg Asn Ile Arg Ile Ile Val 50 55 60 Pro Leu Asn Asn Arg Glu Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg 65 70 75 80 Thr Arg Phe Val Tyr His Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro 85 90 95 Thr Glu Val Glu Leu Asp Asn Gln Ile Val Thr Ala Thr Gln Ser Asn 100 105 110 Ile Cys Asp Glu Asp Ser Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg 115 120 125 Asn Lys Cys Tyr Thr Ala Val Val Pro Leu Val Tyr Gly Gly Glu Thr 130 135 140 Lys Met Val Glu Thr Ala Leu Thr Pro Asp Ala Cys Tyr Pro Asp Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 165 170 175 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 180 185 190 Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr Thr Met 195 200 205 His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Tyr 210 215 220 Ile Asn Pro Arg Ser Gly Tyr Thr His Tyr Asn Gln Lys Leu Lys Asp 225 230 235 240 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ala Ser Thr Ala Tyr Met Glu 245 250 255 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 260 265 270 Ser Ala Tyr Tyr Asp Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr 275 280 285 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 290 295 300 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 305 310 315 320 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser 325 330 335 Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 340 345 350 Lys Arg Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser 355 360 365 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 370 375 380 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser 385 390 395 400 Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 405 410 415 <210> 11 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn Lys Cys Lys Cys Ala 1 5 10 15 Arg Ile Thr Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn Glu Asp 20 25 30 Ile Val Glu Arg Asn Ile Arg Ile Ile Val Pro Leu Asn Asn Arg Glu 35 40 45 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 50 55 60 Leu Ser Asp Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 65 70 75 80 Asn Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 85 90 95 Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala 100 105 110 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu Thr Ala 115 120 125 Leu Thr Pro Asp Ala Cys Tyr Pro Asp Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly 145 150 155 160 Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala 165 170 175 Ser Gly Tyr Thr Phe Ile Ser Tyr Thr Met His Trp Val Arg Gln Ala 180 185 190 Pro Gly Gln Gly Leu Glu Trp Met Gly Tyr Ile Asn Pro Arg Ser Gly 195 200 205 Tyr Thr His Tyr Asn Gln Lys Leu Lys Asp Lys Ala Thr Leu Thr Ala 210 215 220 Asp Lys Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser 225 230 235 240 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Ala Tyr Tyr Asp Tyr 245 250 255 Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 275 280 285 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 290 295 300 Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn 305 310 315 320 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr Asp 325 330 335 Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 340 345 350 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 355 360 365 Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Thr Phe 370 375 380 Gly Gly Gly Thr Lys Leu Glu Ile Lys 385 390 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 13 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Pro Ser Tyr Gly Ser Gly Ser Pro Asn Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Ala 85 90 95 Thr Gln Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 15 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Ala Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 16 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 16 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Ala Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 17 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 17 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Ala Pro Ser Pro Leu Ala Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 18 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 18 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Ser Pro Ser Pro Leu Ser Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 19 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Ala Ala Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 20 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Ala Pro Ser Ser Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 21 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Ala Ser Pro Leu Ala Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 22 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 22 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Ser Leu Ala Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 23 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 23 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Asp Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 24 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 24 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Asp Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 25 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn 1 5 10 15 Ser Pro Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp 20 25 30 Phe Leu Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser 35 40 45 Asp Ile Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys 50 55 60 Tyr Ala Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln 65 70 75 80 Gly Thr Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95 Lys Glu Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110 Val Ser Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120 125 Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile 130 135 140 Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr 145 150 155 160 Thr Asp Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr 165 170 175 Lys Val Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln 180 185 190 Ser Met Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln 195 200 205 Asn Ala Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val 210 215 220 Phe Ala Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr 225 230 235 240 Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr 245 250 255 Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn 260 265 270 Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala 275 280 285 Ser Ile Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr 290 295 300 Val Thr His Thr Asp Leu Pro Ser Pro Leu Gln Gln Thr Ile Ser Arg 305 310 315 320 Pro Lys Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro 325 330 335 Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu 340 345 350 Val Thr Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg 355 360 365 Gly Gln Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro 370 375 380 Glu Pro Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val 385 390 395 400 Ser Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala 405 410 415 His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser 420 425 430 Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr 435 440 445 Ala Gly Thr Cys Tyr 450 <210> 26 <211> 8 <212> PRT <213> Homo sapiens <400> 26 Asp Leu Pro Ser Pro Leu Lys Gln 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 27 Asp Ala Pro Ser Pro Leu Lys Gln 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Asp Leu Ala Ser Pro Leu Lys Gln 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Asp Leu Pro Ser Ser Leu Lys Gln 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Asp Leu Pro Ser Pro Leu Ala Gln 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Asp Asp Pro Ser Pro Leu Lys Gln 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 32 Asp Leu Pro Ser Pro Leu Asp Gln 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Asp Leu Pro Ser Pro Leu Gln Gln 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 Asp Ala Pro Ser Pro Leu Ala Gln 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 Asp Ser Pro Ser Pro Leu Ser Gln 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 Asp Ala Ala Ser Pro Leu Lys Gln 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 Asp Ala Pro Ser Ser Leu Lys Gln 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 Asp Leu Ala Ser Pro Leu Ala Gln 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 Asp Leu Pro Ser Ser Leu Ala Gln 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Asp Leu Ala Ser Ser Leu Lys Gln 1 5

Claims

1. A modified human IgM constant region comprising one or more amino acid substitutions compared to a wild-type human IgM constant region, The modified human IgM constant region, wherein at least one amino acid substitution is present at a position in the Cμ3 domain spanning from T302 of SEQ ID NO:1 to K322 of SEQ ID NO:1, and a modified IgM antibody comprising the modified IgM constant region and a heavy chain variable region specific for a target antigen exhibits reduced complement dependent cytotoxicity (CDC) of cells expressing the target antigen compared to a corresponding wild-type human IgM antibody.

2. 2. The modified human IgM constant region of claim 1, wherein the at least one amino acid substitution is at amino acid T302, C303, T304, V305, T306, H307, T308, D309, L310, P311, S312, P313, L314, K315, Q316, T317, I318, S319, R320, P321, and / or K322 of SEQ ID NO:

1.

3. 3. The modified human IgM constant region of claim 2, wherein the at least one amino acid substitution is at position L310 of SEQ ID NO:1, position P311 of SEQ ID NO:1, position P313 of SEQ ID NO:1, position K315 of SEQ ID NO:1, or any combination thereof.

4. 4. The modified human IgM constant region of claim 3, wherein the amino acid substitution is at position L310 of SEQ ID NO:

1.

5. 5. The modified human IgM constant region of claim 4, wherein L310 of SEQ ID NO:1 is substituted with alanine (L310A), serine (L310S), aspartic acid (L310D), or glycine (L310G).

6. 6. The modified human IgM constant region of claim 5, wherein L310 of SEQ ID NO:1 is replaced with alanine (L310A).

7. The modified human IgM constant region of claim 6, comprising SEQ ID NO:

15.

8. 6. The modified human IgM constant region of claim 5, wherein L310 of SEQ ID NO:1 is replaced with aspartic acid (L310D).

9. The modified human IgM constant region of claim 8 comprising SEQ ID NO:

23.

10. 4. The modified human IgM constant region of claim 3, wherein the amino acid substitution is at position P311 of SEQ ID NO:

1.

11. 11. The modified human IgM constant region of claim 10, wherein P311 of SEQ ID NO: 1 is replaced with alanine (P311A), serine (P311S), or glycine (P311G).

12. 12. The modified human IgM constant region of claim 11, wherein P311 of SEQ ID NO: 1 is replaced with alanine (P311A).

13. The modified human IgM constant region of claim 12, comprising SEQ ID NO:

2.

14. 4. The modified human IgM constant region of claim 3, wherein the amino acid substitution is at position P313 of SEQ ID NO:

1.

15. 15. The modified human IgM constant region of claim 14, wherein P313 of SEQ ID NO: 1 is substituted with alanine (P313A), serine (P313S), or glycine (P313G).

16. 16. The modified human IgM constant region of claim 15, wherein P313 of SEQ ID NO: 1 is replaced with serine (P313S).

17. The modified human IgM constant region of claim 16, comprising SEQ ID NO:

3.

18. 4. The modified human IgM constant region of claim 3, wherein the amino acid substitution is at position K315 of SEQ ID NO:

1.

19. 19. The modified human IgM constant region of claim 18, wherein K315 of SEQ ID NO: 1 is substituted with alanine (K315A), serine (K315S), aspartic acid (K315D), glutamine (K315Q), or glycine (P313G).

20. 20. The modified human IgM constant region of claim 19, wherein K315 of SEQ ID NO: 1 is replaced with alanine (K315A).

21. The modified human IgM constant region of claim 20 comprising SEQ ID NO:

16.

22. 20. The modified human IgM constant region of claim 19, wherein K315 of SEQ ID NO: 1 is replaced with aspartic acid (K315D).

23. The modified human IgM constant region of claim 22, comprising SEQ ID NO:

24.

24. 20. The modified human IgM constant region of claim 19, wherein K315 of SEQ ID NO: 1 is replaced with glutamine (K315Q).

25. The modified human IgM constant region of claim 24, comprising SEQ ID NO:

25.

26. 4. The modified human IgM constant region of claim 3, comprising amino acid substitutions at positions P311 and P313 of SEQ ID NO:

1.

27. 27. The modified human IgM constant region of claim 26, wherein P311 of SEQ ID NO: 1 is replaced with alanine (P311A), serine (P311S), or glycine (P311G), and P313 of SEQ ID NO: 1 is replaced with alanine (P313A), serine (P313S), or glycine (P313G).

28. 28. The modified human IgM constant region of claim 27, wherein P311 of SEQ ID NO: 1 is replaced with alanine (P311A) and P313 of SEQ ID NO: 1 is replaced with serine (P313S).

29. 29. The modified human IgM constant region of claim 28 comprising SEQ ID NO:

4.

30. 4. The modified human IgM constant region of claim 3, comprising amino acid substitutions at positions L310 and K315 of SEQ ID NO: 1, wherein L310 is substituted with alanine (L310A) or serine (L310S) and K315 is substituted with alanine (K315A) or serine (K315S).

31. 31. The modified human IgM constant region of claim 30, comprising SEQ ID NO: 17 or SEQ ID NO:

18.

32. 4. The modified human IgM constant region of claim 3, comprising amino acid substitutions at positions L310 and P311 of SEQ ID NO: 1, wherein L310 is replaced with alanine (L310A) and P311 is replaced with alanine (P311A).

33. 33. The modified human IgM constant region of claim 32, comprising SEQ ID NO:

19.

34. 4. The modified human IgM constant region of claim 3, comprising amino acid substitutions at positions L310 and P313 of SEQ ID NO: 1, wherein L310 is replaced with alanine (L310A) and P313 is replaced with serine (P313S).

35. 35. The modified human IgM constant region of claim 34, comprising SEQ ID NO:

20.

36. 4. The modified human IgM constant region of claim 3, comprising amino acid substitutions at positions P311 and K315 of SEQ ID NO: 1, wherein P311 is replaced with alanine (P311A) and K315 is replaced with alanine (K315A).

37. 37. The modified human IgM constant region of claim 36, comprising SEQ ID NO:

21.

38. 4. The modified human IgM constant region of claim 3, comprising amino acid substitutions at positions P313 and K315 of SEQ ID NO: 1, wherein P313 is replaced with serine (P313S) and K315 is replaced with alanine (K315A).

39. 39. The modified human IgM constant region of claim 38, comprising SEQ ID NO:

22.

40. 40. The modified human IgM constant region of any one of claims 1 to 39, wherein the maximum CDC activity achieved in a dose response assay by a target-specific IgM antibody comprising the modified human IgM constant region is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a corresponding wild-type IgM antibody that is identical except for the modified human IgM constant region.

41. 40. The modified human IgM constant region of any one of claims 1 to 39, wherein the antibody concentration of a target-specific IgM antibody comprising the modified human IgM constant region that results in 50% CDC activity (EC50) is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, and at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to a corresponding wild-type IgM antibody that is identical except for the modified human IgM constant region.

42. 42. A modified human IgM antibody comprising a modified human IgM constant region according to any one of claims 1 to 41 and a heavy chain variable region (VH) located amino-terminal to the modified human IgM constant region, which specifically binds to a target antigen and exhibits reduced complement-dependent cytotoxicity (CDC) of cells expressing the target antigen compared to a corresponding wild-type human IgM antibody.

43. 43. The modified human IgM antibody of claim 42, which is a pentameric or hexameric antibody comprising five or six bivalent IgM binding units, respectively, each binding unit comprising two IgM heavy chains, each comprising a VH positioned amino terminal to the modified human IgM constant region, and two immunoglobulin light chains, each comprising a light chain variable domain (VL) positioned amino terminal to a human immunoglobulin light chain constant region.

44. 44. The modified human IgM antibody of claim 43, which is a pentamer and further comprises a J chain or a functional fragment or a functional variant thereof.

45. 45. The modified human IgM antibody of claim 44, wherein the J chain or a fragment thereof comprises amino acids 23 to 158 of SEQ ID NO: 7 or a functional fragment or a functional variant thereof.

46. 46. ​​The modified human IgM antibody of claim 44 or claim 45, wherein the J chain, or a fragment or variant thereof, is a modified J chain further comprising a heterologous polypeptide, wherein the heterologous polypeptide is fused directly or indirectly to the J chain, or a fragment or variant thereof.

47. 47. The modified human IgM antibody of claim 46, wherein the heterologous polypeptide is fused to the J chain or fragment thereof via a peptide linker.

48. 48. The modified human IgM antibody of claim 47, wherein the peptide linker comprises at least 5 amino acids but not more than 25 amino acids.

49. The peptide linker 49. The modified human IgM antibody of claim 48, consisting of:

50. 50. The modified human IgM antibody of any one of claims 46 to 49, wherein the heterologous polypeptide is fused to the N-terminus of the J chain, or fragment or variant thereof, to the C-terminus of the J chain, or fragment or variant thereof, or to both the N-terminus and C-terminus of the J chain, or fragment or variant thereof.

51. The modified human IgM antibody of any one of claims 46 to 50, wherein the heterologous polypeptide comprises a binding domain.

52. 52. The modified human IgM antibody of claim 51 , wherein the binding domain of the heterologous polypeptide is an antibody or an antigen-binding fragment thereof.

53. 53. The modified human IgM antibody of claim 52, wherein the antigen-binding fragment comprises a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment, or any combination thereof.

54. 54. The modified human IgM antibody of claim 53, wherein the antigen-binding fragment is an scFv fragment.

55. The modified human IgM antibody of any one of claims 51 to 54, wherein said heterologous polypeptide is capable of specifically binding to CD3ε.

56. 56. The modified human IgM antibody of claim 55, wherein the modified J chain comprises the amino acid sequence SEQ ID NO:9 (V15J) or SEQ ID NO:11 (J15V).

57. 57. The modified human IgM antibody of claim 56, wherein the modified J chain further comprises a signal peptide.

58. 58. The modified human IgM antibody of claim 57, wherein the modified J chain comprises the amino acid sequence SEQ ID NO:8 (V15J) or SEQ ID NO:10 (J15V).

59. 59. The modified human IgM antibody of any one of claims 55 to 58, capable of directing T cell mediated killing of cells expressing the target antigen, wherein the T cell mediated killing is equivalent to that of a corresponding IgM antibody that is identical to the modified human IgM antibody except for the modified IgM constant region.

60. The modified human IgM antibody of any one of claims 42 to 59, wherein the cell expressing the target antigen is a eukaryotic cell.

61. A polynucleotide comprising a nucleic acid sequence encoding a modified human IgM constant region according to any one of claims 1 to 40 or a nucleic acid sequence encoding a heavy chain polypeptide subunit of a modified human IgM antibody according to any one of claims 42 to 59.

62. A composition comprising the polynucleotide of claim 61.

63. 63. The composition of claim 62, further comprising a nucleic acid sequence encoding a light chain polypeptide subunit.

64. 64. The composition of claim 63, wherein the light chain polypeptide subunit comprises a human antibody light chain constant region, or a fragment thereof, fused to the C-terminus of the VL.

65. 65. The composition of claim 63 or claim 64, wherein the nucleic acid sequences encoding the heavy chain polypeptide subunits and the nucleic acid sequences encoding the light chain polypeptide subunits are present on separate vectors.

66. 65. The composition of claim 63 or claim 64, wherein the nucleic acid sequence encoding the heavy chain polypeptide subunit and the nucleic acid sequence encoding the light chain polypeptide subunit are present on a single vector.

67. 67. The composition of any one of claims 62 to 66, further comprising a nucleic acid sequence encoding a J chain, or a functional fragment or functional variant thereof.

68. The composition of claim 67, wherein the J chain or fragment thereof is a modified J chain further comprising a heterologous polypeptide, wherein the heterologous polypeptide is fused directly or indirectly to the J chain or fragment thereof.

69. 69. The composition of claim 67 or claim 68, wherein the nucleic acid sequence encoding the heavy chain polypeptide subunit, the nucleic acid sequence encoding the light chain polypeptide subunit, and the nucleic acid sequence encoding the J chain are present on a single vector.

70. 69. The composition of claim 67 or claim 68, wherein the nucleic acid sequence encoding the heavy chain polypeptide subunit, the nucleic acid sequence encoding the light chain polypeptide subunit, and the nucleic acid sequence encoding the J chain are each present on a separate vector.

71. 70. The vector of claim 66 or claim 69.

72. 71. The vector of claim 65 or claim 70.

73. A host cell comprising the polynucleotide of claim 61, the composition of any one of claims 62 to 70, or the vector of claim 71 or claim 72, and capable of expressing the modified human IgM constant region of any one of claims 1 to 40, or the modified human IgM antibody of any one of claims 42 to 59, or a functional fragment thereof.

74. 74. A method of producing a modified human IgM constant region of any one of claims 1 to 40 or a modified human IgM antibody of any one of claims 42 to 60, comprising culturing a host cell of claim 73 and recovering said constant region or said antibody.

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