Cd20 binding molecules and uses thereof

Abstract

This disclosure provides pentameric and hexameric CD20 binding molecules and methods of using such molecules to direct complement-mediated, T-cell-mediated, or both complement-mediated and T-cell-mediated killing of CD20-expressing cells.

1 . A multimeric binding molecule comprising at least two bivalent binding units, or variants or fragments thereof, wherein each binding unit comprises at least two heavy chain constant regions or fragments thereof, each associated with an antigen-binding domain, wherein at least one antigen binding domain of the binding molecule is a CD20 antigen binding domain comprising six immunoglobulin complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 39 with one or two single amino acid substitutions; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 40 with one or two single amino acid substitutions; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 41 with one or two single amino acid substitutions; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 43, or SEQ ID NO: 43 with one or two single amino acid substitutions; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 44 with one or two single amino acid substitutions; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 45 with one or two single amino acid substitutions. 
     
     
         2 . A multimeric binding molecule comprising at least two bivalent binding units, or variants or fragments thereof, wherein each binding unit comprises at least two heavy chain constant regions or fragments thereof, each associated with an antigen-binding domain, wherein at least one antigen binding domain of the binding molecule is a CD20 antigen binding domain comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 38, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 42. 
     
     
         3 . The binding molecule of  claim 1 , which is a dimeric binding molecule comprising two bivalent IgA binding units or fragments thereof and a J-chain or fragment or variant thereof, wherein each binding unit comprises two IgA heavy chain constant regions or fragments thereof each associated with an antigen-binding domain, wherein the IgA heavy chain constant regions or fragments thereof each comprise a Cα3-tp domain. 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The binding molecule of  claim 3 , wherein one or more IgA heavy chain constant regions or fragments thereof further comprise a Cα1 domain, a Cα2, or a Cα1 domain and a Cα2 domain. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The binding molecule of  claim 3 , wherein each binding unit comprises two IgA heavy chains each comprising a VH situated amino terminal to the IgA constant region or fragment thereof, and two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region. 
     
     
         10 . The binding molecule of  claim 1 , which is a pentameric or a hexameric binding molecule comprising five or six bivalent IgM binding units, respectively, wherein each binding unit comprises two IgM heavy chain constant regions or fragments thereof each associated with an antigen-binding domain, wherein the IgM heavy chain constant regions or fragments thereof each comprise a Cα4-tp domain. 
     
     
         11 . (canceled) 
     
     
         12 . The binding molecule of  claim 10 , wherein one or more IgM heavy chain constant regions or fragments thereof further comprise a Cμ3 domain, a Cμ2 domain, a Cμ1 domain, or any combination thereof. 
     
     
         13 . The binding molecule of  claim 10 , wherein the binding molecule is pentameric, and further comprises a J-chain, or functional fragment thereof, or a functional variant thereof. 
     
     
         14 . The binding molecule of  claim 13 , wherein J-chain or fragment thereof comprises the amino acid sequence SEQ ID NO: 49 or a functional fragment thereof. 
     
     
         15 . The binding molecule of  claim 13 , wherein the J-chain or fragment thereof is a modified J-chain further comprising a heterologous polypeptide, wherein the heterologous polypeptide is directly or indirectly fused via a peptide linker to the J-chain or fragment thereof, wherein the peptide linker comprises at least 5 amino acids, but no more than 25 amino acids. 
     
     
         16 - 17 . (canceled) 
     
     
         18 . The binding molecule of  claim 15 , wherein the peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 67). 
     
     
         19 . The binding molecule of  claim 15 , wherein the heterologous polypeptide is fused to the N-terminus of the J-chain or fragment thereof, the C-terminus of the J-chain or fragment thereof, or to both the N-terminus and C-terminus of the J-chain or fragment thereof. 
     
     
         20 . The binding molecule of  claim 15 , wherein the heterologous polypeptide comprises a binding domain. 
     
     
         21 . The binding molecule of  claim 20 , wherein the binding domain of the heterologous polypeptide is an antibody or antigen-binding fragment thereof. 
     
     
         22 . The binding molecule of  claim 21 , 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. 
     
     
         23 . The binding molecule of  claim 22 , wherein the antigen-binding fragment is a scFv fragment. 
     
     
         24 . The binding molecule of  claim 23 , wherein the heterologous polypeptide can specifically bind to CD3. 
     
     
         25 - 27 . (canceled) 
     
     
         28 . The binding molecule of  claim 10 , wherein the IgM heavy chain constant region is a human IgM constant region. 
     
     
         29 . The binding molecule of  claim 10 , wherein each binding unit comprises two identical IgM heavy chains each comprising a VH situated amino terminal to the IgM constant region or fragment thereof, and two identical immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region. 
     
     
         30 . (canceled) 
     
     
         31 . The binding molecule of  claim 29 , wherein the two IgM heavy chains within at least one binding unit comprise the amino acid sequence SEQ ID NO: 56, and wherein each light chain comprises the amino acid sequence SEQ ID NO: 58. 
     
     
         32 - 35 . (canceled) 
     
     
         36 . The binding molecule of  claim 1 , which can direct complement-mediated killing of a CD-20-expressing cell at higher potency than an equivalent amount of a monospecific, bivalent IgG1 antibody comprising a VH having the amino acid sequence SEQ ID NO: 38 and a VL having the amino acid sequence SEQ ID NO: 42. 
     
     
         37 . (canceled) 
     
     
         38 . The binding molecule of  claim 36 , wherein the CD-20-expressing cell is a lymphoma cell line or a malignant B cell in a subject with cancer. 
     
     
         39 - 43 . (canceled) 
     
     
         44 . The binding molecule of  claim 38 , wherein the cancer is minimally responsive or non-responsive to rituximab therapy. 
     
     
         45 . The binding molecule of  claim 38 , wherein the subject is human. 
     
     
         46 . A composition comprising the binding molecule of  claim 1  any and a carrier. 
     
     
         47 - 49 . (canceled) 
     
     
         50 . A composition comprising
 (a) a first polynucleotide encoding a heavy chain polypeptide subunit of the binding molecule of  claim 1 , wherein the heavy chain polypeptide subunit comprises an IgM heavy chain constant region or fragment thereof or an IgA heavy chain constant region or fragment thereof, and at least the antibody VH portion of the CD20 antigen binding domain; and   (b) a second polynucleotide encoding a light chain polypeptide subunit, wherein the light chain polypeptide subunit comprises the antibody VL portion of the CD20 antigen binding domain.   
     
     
         51 - 68 . (canceled) 
     
     
         69 . A host cell comprising the composition of  claim 50 , wherein the host cell can express the binding molecule. 
     
     
         70 . A method of producing the binding molecule comprising culturing the host cell of  claim 69 , and recovering the binding molecule. 
     
     
         71 . A method for directing complement-mediated, T-cell-mediated, or both complement-mediated and T-cell-mediated killing of a CD20-expressing cell comprising contacting a CD20-expressing cell with the binding molecule of  claim 1 , wherein the binding molecule can direct complement-mediated killing of a CD-20-expressing cell at higher potency than an equivalent amount of a monospecific, bivalent IgG1 antibody or fragment thereof that specifically binds to the same CD20 epitope as the CD20 antigen binding domain. 
     
     
         72 - 79 . (canceled) 
     
     
         80 . A method for directing complement-mediated, T-cell-mediated, or both complement-mediated and T-cell-mediated killing of a CD20-expressing cell comprising contacting a CD20-expressing cell with a dimeric, pentameric, or hexameric binding molecule comprising two, five, or six bivalent binding units, respectively, wherein each binding unit comprises two IgA or IgM heavy chain constant regions or fragments thereof and two antigen binding domains, wherein at least one antigen binding domain of the binding molecule is a CD20 antigen binding domain, and wherein the binding molecule can direct complement-mediated killing of a CD-20-expressing cell at higher potency than an equivalent amount of a monospecific, bivalent IgG1 antibody or fragment thereof that specifically binds to the same CD20 epitope as the CD20 antigen binding domain. 
     
     
         81 - 121 . (canceled)