US2022340676A1PendingUtilityA1

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

67
Assignee: IGM BIOSCIENCES INCPriority: Apr 7, 2017Filed: Jun 10, 2022Published: Oct 27, 2022
Est. expiryApr 7, 2037(~10.7 yrs left)· nominal 20-yr term from priority
Inventors:Ramesh BaligaDean Ng
C07K 16/2887C07K 2317/526C07K 16/00C07K 16/2809C07K 2317/71C07K 2317/31C07K 2317/622C07K 2317/734C12Q 1/68A61P 35/00
67
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Claims

Abstract

The disclosure provides modified human IgM heavy chain constant regions that include one or more amino acid substitutions, e.g., in the Cμ3 domain, where a modified human 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.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . 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 of SEQ ID NO: 1 to K322 of SEQ ID NO: 1, 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. 
     
     
         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 . 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 . The modified human IgM constant region of  claim 3 , wherein the amino acid substitution is at position L310 of SEQ ID NO: 1. 
     
     
         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 . The modified human IgM constant region of  claim 5 , wherein L310 of SEQ ID NO: 1 is substituted with alanine (L310A). 
     
     
         7 . The modified human IgM constant region of  claim 6 , comprising SEQ ID NO: 15. 
     
     
         8 . The modified human IgM constant region of  claim 5 , wherein L310 of SEQ ID NO: 1 is substituted with aspartic acid (L310D). 
     
     
         9 . The modified human IgM constant region of  claim 8 , comprising SEQ ID NO: 23. 
     
     
         10 . The modified human IgM constant region of  claim 3 , wherein the amino acid substitution is at position P311 of SEQ ID NO: 1. 
     
     
         11 . The modified human IgM constant region of  claim 10 , wherein P311 of SEQ ID NO: 1 is substituted with alanine (P311A), serine (P311S), or glycine (P311G). 
     
     
         12 . The modified human IgM constant region of  claim 11 , wherein P311 of SEQ ID NO: 1 is substituted with alanine (P311A). 
     
     
         13 . The modified human IgM constant region of  claim 12 , comprising SEQ ID NO: 2. 
     
     
         14 . The modified human IgM constant region of  claim 3 , wherein the amino acid substitution is at position P313 of SEQ ID NO: 1. 
     
     
         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 . The modified human IgM constant region of  claim 15 , wherein P313 of SEQ ID NO: 1 is substituted with serine (P313S). 
     
     
         17 . The modified human IgM constant region of  claim 16 , comprising SEQ ID NO: 3. 
     
     
         18 . The modified human IgM constant region of  claim 3 , wherein the amino acid substitution is at position K315 of SEQ ID NO: 1. 
     
     
         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 . The modified human IgM constant region of  claim 19 , wherein K315 of SEQ ID NO: 1 is substituted with alanine (K315A). 
     
     
         21 . The modified human IgM constant region of  claim 20 , comprising SEQ ID NO: 16. 
     
     
         22 . The modified human IgM constant region of  claim 19 , wherein K315 of SEQ ID NO: 1 is substituted with aspartic acid (K315D). 
     
     
         23 . The modified human IgM constant region of  claim 22 , comprising SEQ ID NO: 24. 
     
     
         24 . The modified human IgM constant region of  claim 19 , wherein K315 of SEQ ID NO: 1 is substituted with glutamine (K315Q). 
     
     
         25 . The modified human IgM constant region of  claim 24 , comprising SEQ ID NO: 25. 
     
     
         26 . The modified human IgM constant region of  claim 3 , comprising amino acid substitutions at positions P311 and P313 of SEQ ID NO: 1. 
     
     
         27 . The modified human IgM constant region of  claim 26 , wherein P311 of SEQ ID NO: 1 is substituted with alanine (P311A), serine (P311S), or glycine (P311G), and wherein P313 of SEQ ID NO: 1 is substituted with alanine (P313A), serine (P313S), or glycine (P313G). 
     
     
         28 . The modified human IgM constant region of  claim 27 , wherein P311 of SEQ ID NO: 1 is substituted with alanine (P311A) and P313 of SEQ ID NO: 1 is substituted with serine (P313S). 
     
     
         29 . The modified human IgM constant region of  claim 28 , comprising SEQ ID NO: 4. 
     
     
         30 . 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 . The modified human IgM constant region of  claim 30 , comprising SEQ ID NO: 17 or SEQ ID NO: 18. 
     
     
         32 . 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 substituted with alanine (L310A) and P311 is substituted with alanine (P311A). 
     
     
         33 . The modified human IgM constant region of  claim 32 , comprising SEQ ID NO: 19. 
     
     
         34 . 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 substituted with alanine (L310A) and P313 is substituted with serine (P313S). 
     
     
         35 . The modified human IgM constant region of  claim 34 , comprising SEQ ID NO: 20. 
     
     
         36 . 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 substituted with alanine (P311A) and K315 is substituted with alanine (K315A). 
     
     
         37 . The modified human IgM constant region of  claim 36 , comprising SEQ ID NO: 21. 
     
     
         38 . 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 substituted with serine (P313S) and K315 is substituted with alanine (K315A). 
     
     
         39 . The modified human IgM constant region of  claim 38 , comprising SEQ ID NO: 22. 
     
     
         40 . The modified human IgM constant region of any one of  claims 1  to  39 , wherein the maximum CDC activity achieved by a target-specific IgM antibody comprising the modified human IgM constant region in a dose-response assay is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to a corresponding wild-type IgM antibody identical except for the modified human IgM constant region. 
     
     
         41 . 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 effecting 50% CDC activity (EC50) is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, and 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 relative to a corresponding wild-type IgM antibody identical except for the modified human IgM constant region. 
     
     
         42 . A modified human IgM antibody comprising the modified human IgM constant region of any one of  claims 1  to  41 , and a heavy chain variable region (VH) situated amino terminal to the modified human IgM constant region, wherein the modified human IgM antibody specifically binds to a target antigen and exhibits reduced complement-dependent cytotoxicity (CDC) of cells expressing the target antigen relative to a corresponding wild-type human IgM antibody. 
     
     
         43 . The modified human IgM antibody of  claim 42 , which is a pentameric or a hexameric antibody comprising five or six bivalent IgM binding units, respectively, wherein each binding unit comprises two IgM heavy chains each comprising a VH situated amino terminal to the modified human IgM constant region, and two immunoglobulin light chains each comprising a light chain variable domain (VL) situated amino terminal to a human immunoglobulin light chain constant region. 
     
     
         44 . The modified human IgM antibody of  claim 43 , which is a pentameric, and further comprises a J-chain, or functional fragment thereof, or a functional variant thereof. 
     
     
         45 . The modified human IgM antibody of  claim 44 , wherein J-chain or fragment thereof comprises amino acids 23 to 158 of SEQ ID NO: 7 or a functional fragment thereof, or a functional variant thereof. 
     
     
         46 . The modified human IgM antibody of  claim 44  or  claim 45 , wherein the J-chain or fragment or variant thereof is a modified J-chain further comprising a heterologous polypeptide, wherein the heterologous polypeptide is directly or indirectly fused to the J-chain or fragment or variant thereof. 
     
     
         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 . The modified human IgM antibody of  claim 47 , wherein the peptide linker comprises at least 5 amino acids, but no more than 25 amino acids. 
     
     
         49 . The modified human IgM antibody of  claim 48 , wherein the peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 12). 
     
     
         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, 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 . The modified human IgM antibody of  claim 51 , wherein the binding domain of the heterologous polypeptide is an antibody or antigen-binding fragment thereof. 
     
     
         53 . The modified human IgM antibody of  claim 52 , wherein the antigen-binding fragment comprises an Fab fragment, an 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 . The modified human IgM antibody of  claim 53 , wherein the antigen-binding fragment is a scFv fragment. 
     
     
         55 . The modified human IgM antibody of any one of  claims 51  to  54  wherein the heterologous polypeptide can specifically bind to CD3ε. 
     
     
         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 . The modified human IgM antibody of  claim 56 , wherein the modified J-chain further comprises a signal peptide. 
     
     
         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 . The modified human IgM antibody of any one of  claims 55  to  58 , which can direct T-cell-mediated killing of a cell expressing the target antigen equivalent to that of a corresponding IgM antibody that is identical to the modified 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 that encodes the modified human IgM constant region of any one of  claims 1  to  40 , or a heavy chain polypeptide subunit of the modified human IgM antibody of any one of  claims 42  to  59 . 
     
     
         62 . A composition comprising the polynucleotide of  claim 61 . 
     
     
         63 . The composition of  claim 62 , further comprising a nucleic acid sequence that encodes a light chain polypeptide subunit. 
     
     
         64 . The composition of  claim 63 , wherein the light chain polypeptide subunit comprises a human antibody light chain constant region or fragment thereof fused to the C-terminal end of a VL. 
     
     
         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 on separate vectors. 
     
     
         66 . 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 on a single vector. 
     
     
         67 . The composition of any one of  claims 62  to  66 , further comprising a nucleic acid sequence that encodes a J-chain, or functional fragment thereof, or a functional variant thereof. 
     
     
         68 . The composition of  claim 67 , wherein the J-chain or fragment thereof is a modified J-chain that further comprises a heterologous polypeptide, wherein the heterologous polypeptide is directly or indirectly fused to the J-chain or fragment thereof. 
     
     
         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 on a single vector. 
     
     
         70 . 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 on separate vectors. 
     
     
         71 . A vector of  claim 66  or  claim 69 . 
     
     
         72 . The vectors 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 or vectors of  claim 71  or  claim 72 , wherein the host cell can express 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 . A method of producing 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  60 , comprising culturing the host cell of  claim 73 , and recovering the constant region or antibody.

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