Novel bispecific antigen binding molecules capable of specific binding to cd40 and to fap

Abstract

The invention relates to novel bispecific antigen binding molecules, comprising (a) at least one antigen binding domain capable of specific binding to CD40, and (b) at least one antigen binding domain capable of specific binding to a target cell antigen, in particular Fibroblast Activation Protein (FAP), and to methods of producing these molecules and to methods of using the same.

1 . A bispecific antigen binding molecule, comprising
 (a) at least one antigen binding domain capable of specific binding to CD40, and   (b) at least one antigen binding domain capable of specific binding to a target cell antigen.   
     
     
         2 . The bispecific antigen binding molecule of  claim 1 , additionally comprising
 (c) a Fc region composed of a first and a second subunit capable of stable association.   
     
     
         3 . The bispecific antigen binding molecule of  claim 1  or  claim 2 , wherein the antigen binding domain capable of specific binding to CD40 binds to a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:1. 
     
     
         4 . The bispecific antigen binding molecule of any one of  claims 1  to  3 , wherein the antigen binding domain capable of specific binding to a target cell antigen is an antigen binding domain capable of specific binding to Fibroblast Activation Protein (FAP). 
     
     
         5 . The bispecific antigen binding molecule of any one of  claims 1  to  4 , wherein the antigen binding domain capable of specific binding to FAP comprises
 (a) a heavy chain variable region (V H FAP) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:5, and a light chain variable region (V L FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:6, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, or 
 (b) a heavy chain variable region (V H FAP) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:11, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:12, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:13, and a a light chain variable region (V L FAP) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:15, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:16. 
 
     
     
         6 . The bispecific antigen binding molecule of any one of  claims 1  to  5 , wherein the antigen binding domain capable of specific binding to FAP comprises
 (a) a heavy chain variable region (V H FAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:9, and a light chain variable region (V L FAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:10, or 
 (b) a heavy chain variable region (V H FAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:17, and a light chain variable region (V L FAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:18. 
 
     
     
         7 . The bispecific antigen binding molecule of any one of  claims 1  to  6 , wherein the antigen binding domain capable of specific binding to CD40 comprises a heavy chain variable region (V H CD40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:19, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:20, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:21, and a light chain variable region (V L CD40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:24. 
     
     
         8 . The bispecific antigen binding molecule of any one of  claims 1  to  6 , wherein the antigen binding domain capable of specific binding to CD40 comprises a heavy chain variable region (V H CD40) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:29, and a light chain variable region (V L CD40) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:30, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:32. 
     
     
         9 . The bispecific antigen binding molecule of any one of  claims 1  to  8 , wherein the antigen binding domain capable of specific binding to CD40 comprises
 (a) a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL comprising the amino acid sequence of SEQ ID NO:26, or 
 (b) a VH comprising the amino acid sequence of SEQ ID NO:33 and a VL comprising the amino acid sequence of SEQ ID NO:34. 
 
     
     
         10 . The bispecific antigen binding molecule of any one of  claims 1  to  7 , wherein the antigen binding domain capable of specific binding to CD40 comprises
 (i) a heavy chain variable region (V H CD40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173 and SEQ ID NO:174, and 
 (ii) a light chain variable region (V L CD40) comprising the amino acid sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178. 
 
     
     
         11 . The bispecific antigen binding molecule of any one of  claims 1  to  7 , wherein the antigen binding domain capable of specific binding to CD40 comprises
 (i) a heavy chain variable region (V H CD40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184, and 
 (ii) a light chain variable region (V L CD40) comprising the amino acid sequence selected from the group consisting of SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, and SEQ ID NO:188. 
 
     
     
         12 . The bispecific antigen binding molecule of any one of  claim 1  to  7  or  10 , wherein the antigen binding domain capable of specific binding to CD40 comprises
 (a) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VL comprising the amino acid sequence of SEQ ID NO:175, or 
 (b) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VL comprising the amino acid sequence of SEQ ID NO:177, or 
 (c) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VL comprising the amino acid sequence of SEQ ID NO:178, or 
 (d) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VL comprising the amino acid sequence of SEQ ID NO:177, or 
 (e) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VL comprising the amino acid sequence of SEQ ID NO:178, or 
 (f) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VL comprising the amino acid sequence of SEQ ID NO:175, or 
 (g) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VL comprising the amino acid sequence of SEQ ID NO:178, or 
 (h) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VL comprising the amino acid sequence of SEQ ID NO:175, or 
 (i) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VL comprising the amino acid sequence of SEQ ID NO:177, or 
 (j) a VH comprising the amino acid sequence of SEQ ID NO:171 and a VL comprising the amino acid sequence of SEQ ID NO:176, or 
 (k) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VL comprising the amino acid sequence of SEQ ID NO:175, or 
 (l) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VL comprising the amino acid sequence of SEQ ID NO:176, or 
 (m) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VL comprising the amino acid sequence of SEQ ID NO:177, or 
 (n) a VH comprising the amino acid sequence of SEQ ID NO:172 and a VL comprising the amino acid sequence of SEQ ID NO:178, or 
 (o) a VH comprising the amino acid sequence of SEQ ID NO:173 and a VL comprising the amino acid sequence of SEQ ID NO:176, or 
 (p) a VH comprising the amino acid sequence of SEQ ID NO:174 and a VL comprising the amino acid sequence of SEQ ID NO:176. 
 
     
     
         13 . The bispecific antigen binding molecule of any one of  claim 1  to  7  or  10  or  12 , wherein the antigen binding domain capable of specific binding to CD40 comprises a VH comprising the amino acid sequence of SEQ ID NO:171 and a VL comprising the amino acid sequence of SEQ ID NO:175. 
     
     
         14 . The bispecific antigen binding molecule of any one of  claim 1  to  7  or  11 , wherein the antigen binding domain capable of specific binding to CD40 comprises
 (a) a VH comprising the amino acid sequence of SEQ ID NO:179 and a VL comprising the amino acid sequence of SEQ ID NO:185, or 
 (b) a VH comprising the amino acid sequence of SEQ ID NO:180 and a VL comprising the amino acid sequence of SEQ ID NO:185, or 
 (c) a VH comprising the amino acid sequence of SEQ ID NO:181 and a VL comprising the amino acid sequence of SEQ ID NO:185, or 
 (d) a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:185, or 
 (e) a VH comprising the amino acid sequence of SEQ ID NO:179 and a VL comprising the amino acid sequence of SEQ ID NO:186, or 
 (f) a VH comprising the amino acid sequence of SEQ ID NO:180 and a VL comprising the amino acid sequence of SEQ ID NO:186, or 
 (g) a VH comprising the amino acid sequence of SEQ ID NO:181 and a VL comprising the amino acid sequence of SEQ ID NO:186, or 
 (h) a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:186, or 
 (i) a VH comprising the amino acid sequence of SEQ ID NO:183 and a VL comprising the amino acid sequence of SEQ ID NO:187, or 
 (j) a VH comprising the amino acid sequence of SEQ ID NO:183 and a VL comprising the amino acid sequence of SEQ ID NO:188, or 
 (k) a VH comprising the amino acid sequence of SEQ ID NO:184 and a VL comprising the amino acid sequence of SEQ ID NO:187, or 
 (l) a VH comprising the amino acid sequence of SEQ ID NO:184 and a VL comprising the amino acid sequence of SEQ ID NO:188. 
 
     
     
         15 . The bispecific antigen binding molecule of any one of  claim 1  to  7  or  11  or  14 , wherein the antigen binding domain capable of specific binding to CD40 comprises a VH comprising the amino acid sequence of SEQ ID NO:179 and a VL comprising the amino acid sequence of SEQ ID NO:185 or wherein the antigen binding domain capable of specific binding to CD40 comprises a VH comprising the amino acid sequence of SEQ ID NO:182 and a VL comprising the amino acid sequence of SEQ ID NO:185. 
     
     
         16 . The bispecific antigen binding molecule of any one of  claims 1  to  7 , comprising
 (i) at least one antigen binding domain capable of specific binding to CD40, comprising a heavy chain variable region (V H CD40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184, and a light chain variable region (V L CD40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:188, and 
 (ii) at least one antigen binding domain capable of specific binding to FAP, comprising a heavy chain variable region (V H FAP) comprising an amino acid sequence of SEQ ID NO:9 and a light chain variable region (V L FAP) comprising an amino acid sequence of SEQ ID NO:10, or a heavy chain variable region (V H FAP) comprising an amino acid sequence of SEQ ID NO:17 and a light chain variable region (V L FAP) comprising an amino acid sequence of SEQ ID NO:18. 
 
     
     
         17 . The bispecific antigen binding molecule of any one of  claims 2  to  16 , wherein the Fc region is an IgG, particularly an IgG1 Fc region or an IgG4 Fc region and wherein the Fc region comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor and/or effector function. 
     
     
         18 . The bispecific antigen binding molecule of any one of  claims 2  to  17 , wherein the Fc region is (i) of human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index), or (ii) of mouse IgG1 subclass with the amino acid mutations D265A and P329G (numbering according to Kabat EU index). 
     
     
         19 . The bispecific antigen binding molecule of any one of  claims 1  to  18 , wherein the bispecific antigen binding molecule comprises
 (a) at least two Fab fragments capable of specific binding to CD40 connected to a Fc region, and 
 (b) one antigen binding domain capable of specific binding to FAP connected to the C-terminus of the Fc region. 
 
     
     
         20 . The bispecific antigen binding molecule of any one of  claims 1  to  19 , wherein the bispecific antigen binding molecule comprises
 (a) at least two Fab fragments capable of specific binding to CD40 connected to a Fc region, and 
 (b) a cross-fab fragment capable of specific binding to FAP connected to the C-terminus of the Fc region. 
 
     
     
         21 . The bispecific antigen binding molecule of any one of  claims 1  to  19 , wherein the bispecific antigen binding molecule comprises four Fab fragments capable of specific binding to CD40. 
     
     
         22 . Polynucleotide encoding the bispecific antigen binding molecule of any one of  claims 1  to  21 . 
     
     
         23 . An expression vector comprising the polynucleotide of  claim 22 . 
     
     
         24 . A host cell comprising polynucleotide of  claim 22  or the expression vector of  claim 23 . 
     
     
         25 . A method of producing a bispecific antigen binding molecule, comprising culturing the host cell of  claim 24  under conditions suitable for the expression of the bispecific antigen binding molecule, and isolating the bispecific antigen binding molecule. 
     
     
         26 . A pharmaceutical composition comprising the bispecific antigen binding molecule of any one of  claims 1  to  21  and at least one pharmaceutically acceptable excipient. 
     
     
         27 . The bispecific antigen binding molecule of any one of  claims 1  to  21 , or the pharmaceutical composition of  claim 26 , for use as a medicament. 
     
     
         28 . The bispecific antigen binding molecule of any one of  claims 1  to  21 , or the pharmaceutical composition of  claim 26 , for use
 (i) in inducing immune stimulation by CD40 expressing antigen-presenting cells (APCs), 
 (ii) in stimulating tumor-specific T cell response, 
 (iii) in causing apoptosis of tumor cells, 
 (iv) in the treatment of cancer, 
 (v) in delaying progression of cancer, 
 (vi) in prolonging the survival of a patient suffering from cancer, 
 (vii) in the treatment of infections. 
 
     
     
         29 . The bispecific antigen binding molecule of any one of  claims 1  to  21 , or the pharmaceutical composition of  claim 26 , for use in the treatment of cancer. 
     
     
         30 . Use of the bispecific antigen binding molecule of any one of  claims 1  to  21 , or the pharmaceutical composition of  claim 26 , in the manufacture of a medicament for the treatment of cancer. 
     
     
         31 . A method of treating an individual having cancer comprising administering to the individual an effective amount of the bispecific antigen binding molecule of any one of  claims 1  to  21 , or the pharmaceutical composition of  claim 26 . 
     
     
         32 . The bispecific antigen binding molecule according to any one of  claims 1  to  21  or the pharmaceutical composition according to  claim 26  for use in the treatment of cancer, wherein the bispecific antigen binding molecule is administered in combination with a chemotherapeutic agent, radiation and/or other agents for use in cancer immunotherapy.