Bispecific t cell activating antigen binding molecules

Abstract

The present invention generally relates to novel bispecific antigen binding molecules for T cell activation and re-direction to specific target cells. In addition, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.

1 . A T cell activating bispecific antigen binding molecule comprising
 (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, comprising at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at least one light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10;   (ii) a second antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen.   
     
     
         2 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the first antigen binding moiety comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO: 31. 
     
     
         3 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the first antigen binding moiety comprises a heavy chain variable region 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: 3 and a light chain variable region 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: 7. 
     
     
         4 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the second antigen binding moiety is capable of specific binding to Carcinoembryonic Antigen (CEA) and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDR selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30. 
     
     
         5 . The T cell activating bispecific antigen binding molecule of  claim 4 , wherein the second antigen binding moiety comprises a heavy chain variable region 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: 23 and a light chain variable region 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: 27. 
     
     
         6 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the second antigen binding moiety is capable of specific binding to Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP) and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one light chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50. 
     
     
         7 . The T cell activating bispecific antigen binding molecule of  claim 6 , wherein the second antigen binding moiety comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. 
     
     
         8 . The T cell activating bispecific antigen binding molecule of  claim 6 , wherein the second antigen binding moiety is capable of specific binding to MCSP and comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51. 
     
     
         9 . The T cell activating bispecific antigen binding molecule of  claim 8 , wherein the second antigen binding moiety comprises a heavy chain variable region 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: 13 and a light chain variable region 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. 
     
     
         10 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged. 
     
     
         11 . The T cell activating bispecific antigen binding molecule of  claim 10 , wherein the first antigen binding moiety is a crossover Fab molecule wherein the constant regions of the Fab light chain and the Fab heavy chain are exchanged. 
     
     
         12 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the second antigen binding moiety is a conventional Fab molecule. 
     
     
         13 . The T cell activating bispecific antigen binding molecule of  claim 1 , comprising not more than one antigen binding moiety capable of specific binding to CD3. 
     
     
         14 . The T cell activating bispecific antigen binding molecule of  claim 1 , comprising a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen. 
     
     
         15 . The T cell activating bispecific antigen binding molecule of  claim 14 , wherein the third antigen binding moiety is a conventional Fab molecule. 
     
     
         16 . The T cell activating bispecific antigen binding molecule of  claim 14 , wherein the third antigen binding moiety is identical to the second antigen binding moiety. 
     
     
         17 . The T cell activating bispecific antigen binding molecule of  claim 16 , wherein the third antigen binding moiety is an antigen binding moiety capable of specific binding to CEA and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDR selected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30. 
     
     
         18 . The T cell activating bispecific antigen binding molecule of  claim 16 , wherein the third antigen binding moiety is an antigen binding moiety capable of specific binding to MCSP and comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least one light chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50. 
     
     
         19 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker. 
     
     
         20 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. 
     
     
         21 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. 
     
     
         22 . The T cell activating bispecific antigen binding molecule of  claim 20 , wherein the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a peptide linker. 
     
     
         23 . The T cell activating bispecific antigen binding molecule of  claim 1 , additionally comprising (iii) an Fc domain composed of a first and a second subunit capable of stable association. 
     
     
         24 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. 
     
     
         25 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. 
     
     
         26 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the first and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. 
     
     
         27 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein a third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. 
     
     
         28 . The T cell activating bispecific antigen binding molecule of  claim 27 , wherein the second and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. 
     
     
         29 . The T cell activating bispecific antigen binding molecule of  claim 27 , wherein the first and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. 
     
     
         30 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the first and the third antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, particularly an IgG class immunoglobulin. 
     
     
         31 . A T cell activating bispecific antigen binding molecule comprising
 (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, comprising the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;   (ii) a second and a third antigen binding moiety each of which is a Fab molecule capable of specific binding to CEA comprising the heavy chain CDR 1 of SEQ ID NO: 24, the heavy chain CDR 2 of SEQ ID NO: 25, the heavy chain CDR 3 of SEQ ID NO: 26, the light chain CDR 1 of SEQ ID NO: 28, the light chain CDR 2 of SEQ ID NO: 29 and the light chain CDR3 of SEQ ID NO: 30.   
     
     
         32 . The T cell activating bispecific antigen binding molecule of  claim 31 , comprising
 (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a heavy chain variable region 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: 3 and a light chain variable region 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: 7, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;   (ii) a second and a third antigen binding moiety each of which is a Fab molecule capable of specific binding to CEA comprising heavy chain variable region 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: 23 and a light chain variable region 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: 27.   
     
     
         33 . A T cell activating bispecific antigen binding molecule comprising
 (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3, comprising the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;   (ii) a second and a third antigen binding moiety each of which is a Fab molecule capable of specific binding to MCSP comprising the heavy chain CDR 1 of SEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15, the heavy chain CDR 3 of SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO: 18, the light chain CDR 2 of SEQ ID NO: 19 and the light chain CDR3 of SEQ ID NO: 20.   
     
     
         34 . The T cell activating bispecific antigen binding molecule of  claim 33 , comprising
 (i) a first antigen binding moiety which is a Fab molecule capable of specific binding to CD3 comprising a heavy chain variable region 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: 3 and a light chain variable region 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: 7, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;   (ii) a second and a third antigen binding moiety each of which is a Fab molecule capable of specific binding to MCSP comprising a heavy chain variable region 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: 13 and a light chain variable region 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.   
     
     
         35 . The T cell activating bispecific antigen binding molecule of  claim 31 , further comprising
 (iii) an Fc domain composed of a first and a second subunit capable of stable association,   wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.   
     
     
         36 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the Fc domain is an IgG, specifically an IgG 1  or IgG 4 , Fc domain. 
     
     
         37 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the Fc domain is a human Fc domain. 
     
     
         38 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. 
     
     
         39 . The T cell activating bispecific antigen binding molecule of  claim 38 , wherein in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. 
     
     
         40 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG 1  Fc domain. 
     
     
         41 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. 
     
     
         42 . The T cell activating bispecific antigen binding molecule of  claim 41 , wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (Kabat numbering). 
     
     
         43 . The T cell activating bispecific antigen binding molecule of  claim 42 , wherein each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function wherein said amino acid substitutions are L234A, L235A and P329G. 
     
     
         44 . The T cell activating bispecific antigen binding molecule of  claim 40 , wherein the Fc receptor is an Fey receptor. 
     
     
         45 . The T cell activating bispecific antigen binding molecule of  claim 40 , wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). 
     
     
         46 . An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of  claim 1  or a fragment thereof. 
     
     
         47 . An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of  claim 31  or a fragment thereof. 
     
     
         48 . An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of  claim 33  or a fragment thereof. 
     
     
         49 . A polypeptide encoded by the polynucleotide of  claim 46 . 
     
     
         50 . A vector, particularly an expression vector, comprising the polynucleotide of  claim 46 . 
     
     
         51 . A host cell comprising the vector of  claim 50 . 
     
     
         52 . A method of producing the T cell activating bispecific antigen binding molecule capable of specific binding to CD3 and a target cell antigen, comprising the steps of a) culturing the host cell of  claim 51  under conditions suitable for the expression of the T cell activating bispecific antigen binding molecule and b) recovering the T cell activating bispecific antigen binding molecule. 
     
     
         53 . A T cell activating bispecific antigen binding molecule produced by the method of  claim 52 . 
     
     
         54 . A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         55 . A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of  claim 31  and a pharmaceutically acceptable carrier. 
     
     
         56 . A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of  claim 33  and a pharmaceutically acceptable carrier. 
     
     
         57 . A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of  claim 53  and a pharmaceutically acceptable carrier. 
     
     
         58 . The T cell activating bispecific antigen binding molecule of  claim 1  for use as a medicament. 
     
     
         59 . The T cell activating bispecific antigen binding molecule of  claim 31  for use as a medicament. 
     
     
         60 . The T cell activating bispecific antigen binding molecule of  claim 33  for use as a medicament. 
     
     
         61 . The T cell activating bispecific antigen binding molecule of  claim 53  for use as a medicament. 
     
     
         62 . The T cell activating bispecific antigen binding molecule of  claim 1  for use in the treatment of a disease in an individual in need thereof. 
     
     
         63 . The T cell activating bispecific antigen binding molecule of  claim 62 , wherein the disease is cancer. 
     
     
         64 . The T cell activating bispecific antigen binding molecule of  claim 31  for use in the treatment of a disease in an individual in need thereof. 
     
     
         65 . The T cell activating bispecific antigen binding molecule of  claim 33  for use in the treatment of a disease in an individual in need thereof. 
     
     
         66 . The T cell activating bispecific antigen binding molecule of  claim 53  for use in the treatment of a disease in an individual in need thereof. 
     
     
         67 . A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of  claim 1  in a pharmaceutically acceptable form. 
     
     
         68 . The method of  claim 67 , wherein said disease is cancer. 
     
     
         69 . A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of  claim 31  in a pharmaceutically acceptable form. 
     
     
         70 . A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of  claim 33  in a pharmaceutically acceptable form. 
     
     
         71 . A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the T cell activating bispecific antigen binding molecule of  claim 53  in a pharmaceutically acceptable form. 
     
     
         72 . A method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of  claim 1  in the presence of a T cell. 
     
     
         73 . A method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of  claim 31  in the presence of a T cell. 
     
     
         74 . A method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of  claim 33  in the presence of a T cell. 
     
     
         75 . A method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of  claim 53  in the presence of a T cell. 
     
     
         76 . The T cell activating bispecific antigen binding molecule of  claim 16 , wherein the third antigen binding moiety is an antigen binding moiety capable of specific binding to CEA and comprises a heavy chain variable region 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: 23 and a light chain variable region 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: 27. 
     
     
         77 . The T cell activating bispecific antigen binding molecule of  claim 16 , wherein the third antigen binding moiety is an antigen binding moiety capable of specific binding to MCSP and comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51. 
     
     
         78 . The T cell activating bispecific antigen binding molecule of  claim 21 , wherein the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a peptide linker. 
     
     
         79 . A host cell comprising the polynucleotide of  claim 46 .