Antigen binding molecules comprising a trimeric tnf family ligand

Abstract

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising (a) at least one moiety capable of specific binding to a target cell antigen, (b) a polypeptide comprising three ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by peptide linkers and (c) a Fc domain composed of a first and a second subunit capable of stable association, and to methods of producing these molecules and to methods of using the same.

1 . A TNF family ligand trimer-containing antigen binding molecule comprising
 (a) at least one moiety capable of specific binding to a target cell antigen,   (b) a polypeptide comprising three ectodomains of a TNF ligand family member or fragments thereof that are connected to each other, optionally by peptide linkers, and   (c) a Fc domain composed of a first and a second subunit capable of stable association.   
     
     
         2 . The TNF family ligand trimer-containing antigen binding molecule of  claim 1  comprising
 (a) at least one moiety capable of specific binding to a target cell antigen, 
 (b) a polypeptide comprising three ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by peptide linkers and 
 (c) a Fc domain composed of a first and a second subunit capable of stable association. 
 
     
     
         3 . The TNF family ligand trimer containing antigen binding molecule of  claim 1  or  2  comprising
 (a) at least one moiety capable of specific binding to a target cell antigen, 
 (b) a polypeptide comprising three ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by peptide linkers and 
 (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the polypeptide comprising the three ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by peptide linkers is fused to the N- or C-terminal amino acid of one of the two subunits of the Fc domain, optionally through a peptide linker. 
 
     
     
         4 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  3 , wherein the TNF ligand family member costimulates human T-cell activation. 
     
     
         5 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  4 , wherein the TNF ligand family member is selected from 4-1BBL and OX40L. 
     
     
         6 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  5 , wherein the TNF ligand family member is 4-1BBL. 
     
     
         7 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  6 , wherein the ectodomain of a TNF ligand family member comprises the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO:101 and SEQ ID NO:102, particularly the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:99. 
     
     
         8 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  6 , comprising
 (a) at least one moiety capable of specific binding to a target cell antigen and 
 (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103, and 
 (c) a Fc domain composed of a first and a second subunit capable of stable association. 
 
     
     
         9 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  5 , wherein the TNF ligand family member is OX40L. 
     
     
         10 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claim 1  to  5  or  9 , wherein the ectodomain of a TNF ligand family member comprises the amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7, particularly the amino acid sequence of SEQ ID NO:6. 
     
     
         11 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claim 1  to  5  or  9  or  10 , comprising
 (a) at least one moiety capable of specific binding to a target cell antigen and 
 (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:8, and 
 (c) a Fc domain composed of a first and a second subunit capable of stable association. 
 
     
     
         12 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  11 , wherein the polypeptide comprising three ectodomains of a TNF ligand family member or fragments thereof is fused at the C-terminal amino acid to a N-terminal amino acid of one of the subunits of the Fc domain. 
     
     
         13 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  11 , wherein the polypeptide comprising three ectodomains of a TNF ligand family member or fragments thereof is fused at the N-terminal amino acid to a C-terminal amino acid of one of the subunits of the Fc domain. 
     
     
         14 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  13 , wherein the moiety capable of specific binding to a target cell antigen is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, an aVH and a scaffold antigen binding protein. 
     
     
         15 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  14 , wherein the moiety capable of specific binding to a target cell antigen is a Fab molecule capable of specific binding to a target cell antigen. 
     
     
         16 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  15 , wherein the target cell antigen is selected from the group consisting of Fibroblast Activation Protein (FAP), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33. 
     
     
         17 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  16 , wherein the target cell antigen is Fibroblast Activation Protein (FAP). 
     
     
         18 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  17 , wherein the moiety capable of specific binding to FAP comprises
 (a) a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:13 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:14, or 
 (b) a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:104, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:105 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:106, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:107, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:108 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:109. 
 
     
     
         19 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  18 , wherein the moiety capable of specific binding to FAP comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO:16 and a variable light chain comprising an amino acid sequence of SEQ ID NO:17 or wherein the moiety capable of specific binding to FAP comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO:110 and a variable light chain comprising an amino acid sequence of SEQ ID NO:111. 
     
     
         20 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  16 , wherein the target cell antigen is CEA. 
     
     
         21 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claim 1  to  16  or  20 , wherein the moiety capable of specific binding to CEA comprises a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:112, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:113 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:114, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:115, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:116 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:117. 
     
     
         22 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claim 1  to  16  or  20  or  21 , wherein the moiety capable of specific binding to CEA comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO:118 and a variable light chain comprising an amino acid sequence of SEQ ID NO:119. 
     
     
         23 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  16 , wherein the target cell antigen is CD19. 
     
     
         24 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claim 1  to  16  or  23 , wherein the moiety capable of specific binding to CD19 comprises
 (a) a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:120, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:121 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:122, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:123, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:124 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:125, or 
 (b) a VH domain comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:128, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:129 and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:130, and a VL domain comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:131, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:132 and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:133. 
 
     
     
         25 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claim 1  to  16  or  23  or  24 , wherein the moiety capable of specific binding to CD19 comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO:126 and a variable light chain comprising an amino acid sequence of SEQ ID NO:127 or wherein the moiety capable of specific binding to FAP comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO:134 and a variable light chain comprising an amino acid sequence of SEQ ID NO:135. 
     
     
         26 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  25 , wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain. 
     
     
         27 . The TNF family ligand trimer-containing antigen binding molecule of  claim 26 , wherein the Fc domain is an IgG1 Fc domain comprising the amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering). 
     
     
         28 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  27 , comprising one moiety capable of specific binding to a target cell antigen. 
     
     
         29 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  28 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising the VH domain of a Fab molecule capable of specific binding to a target cell antigen, 
 (ii) a light chain comprising the VL domain of a Fab molecule capable of specific binding to a target cell antigen, and 
 (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8. 
 
     
     
         30 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to to  29 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:16 or a VH domain comprising an amino acid sequence of SEQ ID NO:110, 
 (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO:17 or a VL domain comprising an amino acid sequence of SEQ ID NO:111, and 
 (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8. 
 
     
     
         31 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  29 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:118, 
 (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO:119, and 
 (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8. 
 
     
     
         32 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  29 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:126 or a VH domain comprising an amino acid sequence of SEQ ID NO:134, 
 (ii) a light chain comprising a VL domain comprising an amino acid sequence of SEQ ID NO:127 or a VL domain comprising an amino acid sequence of SEQ ID NO:135, and 
 (iii) a second heavy chain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8. 
 
     
     
         33 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  27 , comprising two moieties capable of specific binding to a target cell antigen. 
     
     
         34 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claim 1  to  27  or  33 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising a VH domain of a Fab molecule capable of specific binding to a target cell antigen, 
 (ii) two light chains comprising each the VL domain of a Fab molecule capable of specific binding to a target cell antigen, and 
 (iii) a second heavy chain comprising a VH domain of a Fab molecule capable of specific binding to a target cell antigen and the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8. 
 
     
     
         35 . The TNF family ligand trimer-containing antigen binding molecule of  claim 34 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:16 or a VH domain comprising an amino acid sequence of SEQ ID NO:110,   (ii) two light chains comprising each a VL domain comprising an amino acid sequence of SEQ ID NO:17 or a VL domain comprising an amino acid sequence of SEQ ID NO:111, and   (iii) a second heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:16 or a VH domain comprising an amino acid sequence of SEQ ID NO:110 and the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.   
     
     
         36 . The TNF family ligand trimer-containing antigen binding molecule of  claim 34 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:118,   (ii) two light chains comprising each a VL domain comprising an amino acid sequence of SEQ ID NO:119, and   (iii) a second heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:118 and the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.   
     
     
         37 . The TNF family ligand trimer-containing antigen binding molecule of  claim 34 , wherein the antigen binding molecule comprises
 (i) a first heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:126 or a VH domain comprising an amino acid sequence of SEQ ID NO:134,   (ii) two light chains comprising a VL domain comprising an amino acid sequence of SEQ ID NO:127 or a VL domain comprising an amino acid sequence of SEQ ID NO:135, and   (iii) a second heavy chain comprising a VH domain comprising an amino acid sequence of SEQ ID NO:126 or a VH domain comprising an amino acid sequence of SEQ ID NO:134 and the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.   
     
     
         38 . An isolated polynucleotide encoding the TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  37 . 
     
     
         39 . A vector, particularly an expression vector, comprising the isolated polynucleotide of  claim 38 . 
     
     
         40 . A host cell comprising the isolated polynucleotide of  claim 38  or the vector of  claim 39 . 
     
     
         41 . A method for producing the TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  37 , comprising the steps of
 (i) culturing the host cell of  claim 40  under conditions suitable for expression of the antigen binding molecule, and 
 (ii) recovering the antigen binding molecule. 
 
     
     
         42 . A pharmaceutical composition comprising the TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  37  and at least one pharmaceutically acceptable excipient. 
     
     
         43 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  37 , or the pharmaceutical composition of  claim 38 , for use as a medicament. 
     
     
         44 . The TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  37 , or the pharmaceutical composition of  claim 42 , for use in the treatment of cancer. 
     
     
         45 . Use of the TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  37  for the manufacture of a medicament for the treatment of cancer. 
     
     
         46 . A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen binding molecule of any one of  claims 1  to  37  in a pharmaceutically acceptable form. 
     
     
         47 . The method of  claim 46 , wherein said disease is cancer.