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.

What is claimed is: 
     
         1 . A T cell activating bispecific antigen binding molecule comprising
 (a) a first antigen binding moiety which specifically binds to a first antigen;   (b) a second antigen binding moiety which specifically binds to a second antigen;   wherein the first antigen is an activating T cell antigen and the second antigen is CEA, or the first antigen is CEA and the second antigen is an activating T cell antigen; and   wherein the antigen binding moiety which specifically binds to CEA comprises a heavy chain variable region, particularly a humanized heavy chain variable region, comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, and a light chain variable region, particularly a humanized light chain variable region, comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.   
     
     
         2 . The T cell activating bispecific antigen binding molecule according to  claim 1 , wherein the antigen binding moiety which specifically binds to CEA 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: 22 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: 23. 
     
     
         3 . The T cell activating bispecific antigen binding molecule according to  claim 1  or  2 , wherein the first and/or the second antigen binding moiety is a Fab molecule. 
     
     
         4 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1 - 3 , wherein the second antigen binding moiety is a Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are replaced by each other. 
     
     
         5 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1 - 4 , wherein the first antigen is CEA and the second antigen is an activating T cell antigen. 
     
     
         6 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1 - 5 , wherein the activating T cell antigen is CD3, particularly CD3 epsilon. 
     
     
         7 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1 - 6 , wherein the antigen binding moiety which specifically binds to the activating T cell antigen comprises 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. 
     
     
         8 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1 - 7 , wherein the antigen binding moiety which specifically binds to the activating T cell antigen 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. 
     
     
         9 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1 - 8 , wherein the first antigen binding moiety under (a) is a first Fab molecule which specifically binds to a first antigen, the second antigen binding moiety under (b) is a second Fab molecule which specifically binds to a second antigen wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other;
 and   i) in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index); or   ii) in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).   
     
     
         10 . The T cell activating bispecific antigen binding molecule according to  claim 9 , wherein in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). 
     
     
         11 . The T cell activating bispecific antigen binding molecule according to  claim 9  or  10 , wherein in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). 
     
     
         12 . The T cell activating bispecific antigen binding molecule according to any one of  claims 9 - 11 , wherein in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). 
     
     
         13 . The T cell activating bispecific antigen binding molecule according to any one of  claims 9 - 12 , wherein in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). 
     
     
         14 . The T cell activating bispecific antigen binding molecule according to any one of  claims 9 - 12 , wherein in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and wherein in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). 
     
     
         15 . The T cell activating bispecific antigen binding molecule according to  claim 9 , wherein in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). 
     
     
         16 . The T cell activating bispecific antigen binding molecule according to  claim 9  or  15 , wherein in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). 
     
     
         17 . The T cell activating bispecific antigen binding molecule according to any one of  claim 9 , and  16 , wherein in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). 
     
     
         18 . The T cell activating bispecific antigen binding molecule according to any one of  claims 9  and  15 - 17 , wherein in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). 
     
     
         19 . The T cell activating bispecific antigen binding molecule according to any one of  claims 9  and  15 - 17 , wherein in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and wherein in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). 
     
     
         20 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1 - 19 , further comprising
 c) a third antigen binding moiety which specifically binds to the first antigen.   
     
     
         21 . The T cell activating bispecific antigen binding molecule according to  claim 20 , wherein the third antigen binding moiety is a Fab molecule. 
     
     
         22 . The T cell activating bispecific antigen binding molecule according to  claim 20  or  21 , wherein the third antigen binding moiety is identical to the first antigen binding moiety. 
     
     
         23 . The T cell activating bispecific antigen binding molecule according to any one of  claims 20 - 22 , wherein the first and the third antigen binding moiety specifically bind to a target cell antigen, and the second antigen binding moiety specifically binds to an activating T cell antigen, particularly CD3, more particularly CD3 epsilon. 
     
     
         24 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1  to  23 , additionally comprising
 d) an Fc domain composed of a first and a second subunit capable of stable association. 
 
     
     
         25 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1  to  24 , wherein the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker. 
     
     
         26 . The T cell activating bispecific antigen binding molecule according to any one of  claims 1  to  25 , wherein the first and the second antigen binding moieties are Fab molecules 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. 
     
     
         27 . The T cell activating bispecific antigen binding molecule of any one of  claims 1  to  25 , wherein the first and the second antigen binding moieties are Fab molecules 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. 
     
     
         28 . The T cell activating bispecific antigen binding molecule of  claim 26  or  27 , wherein the first and the second antigen binding moieties are Fab molecules and 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. 
     
     
         29 . The T cell activating bispecific antigen binding molecule according to  claim 24 , wherein the first and the second antigen binding moieties are Fab molecules and 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. 
     
     
         30 . The T cell activating bispecific antigen binding molecule according to  claim 24 , wherein the first and the second antigen binding moieties are Fab molecules and the first 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. 
     
     
         31 . The T cell activating bispecific antigen binding molecule according to  claim 24 , wherein the first and the second antigen binding moieties are Fab molecules and 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. 
     
     
         32 . The T cell activating bispecific antigen binding molecule according to any one of  claims 24 ,  29  or  30 , wherein the third antigen binding moiety is a Fab molecule and 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. 
     
     
         33 . The T cell activating bispecific antigen binding molecule of  claim 24 , wherein the first, second and third antigen binding moieties are Fab molecules and 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. 
     
     
         34 . The T cell activating bispecific antigen binding molecule according to  claim 24 , wherein the first, second and third antigen binding moieties are Fab molecules and 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. 
     
     
         35 . The T cell activating bispecific antigen binding molecule according to  claim 34 , wherein the first and the third antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, particularly an IgG class immunoglobulin. 
     
     
         36 . The T cell activating bispecific antigen binding molecule according to any one of  claims 24 - 35 , wherein the Fc domain is an IgG, specifically an IgG or IgG 4 , Fc domain. 
     
     
         37 . The T cell activating bispecific antigen binding molecule according to any one of  claims 24 - 36 , wherein the Fc domain is a human Fc domain. 
     
     
         38 . The T cell activating bispecific antigen binding molecule according to any one of  claims 24 - 37 , 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 39 , wherein said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W), and said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). 
     
     
         41 . The T cell activating bispecific antigen binding molecule of  claim 39  or  40 , wherein in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index). 
     
     
         42 . The T cell activating bispecific antigen binding molecule of any one of  claims 39 - 41 , wherein in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). 
     
     
         43 . The T cell activating bispecific antigen binding molecule of any one of  claims 39 - 42 , wherein the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index). 
     
     
         44 . The T cell activating bispecific antigen binding molecule according to any one of  claims 24 - 43 , 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. 
     
     
         45 . The T cell activating bispecific antigen binding molecule according to any one of  claims 24 - 44 , wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. 
     
     
         46 . The T cell activating bispecific antigen binding molecule according to  claim 45 , wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (Kabat EU index numbering). 
     
     
         47 . The T cell activating bispecific antigen binding molecule according to any one of  claims 24 - 46 , 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 (Kabat EU index numbering). 
     
     
         48 . The T cell activating bispecific antigen binding molecule of any one of  claims 44  to  47 , wherein the Fc receptor is an Fcγ receptor. 
     
     
         49 . The T cell activating bispecific antigen binding molecule of any one of  claims 44  to  48 , wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). 
     
     
         50 . One or more isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of any one of  claims 1  to  49 . 
     
     
         51 . One or more vector, particularly expression vector, comprising the polynucleotide(s) of  claim 50 . 
     
     
         52 . A host cell comprising the polynucleotide(s) of  claim 50  or the vector(s) of  claim 51 . 
     
     
         53 . A method of producing a T cell activating bispecific antigen binding molecule capable of specific binding to CEA and an activating T cell antigen, comprising the steps of a) culturing the host cell of  claim 52  under conditions suitable for the expression of the T cell activating bispecific antigen binding molecule and b) optionally recovering the T cell activating bispecific antigen binding molecule. 
     
     
         54 . A T cell activating bispecific antigen binding molecule produced by the method of  claim 53 . 
     
     
         55 . A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of any one of  claims 1  to  49  or  54  and a pharmaceutically acceptable carrier. 
     
     
         56 . 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 any one of  claims 1  to  49  or  54  in a pharmaceutically acceptable form. 
     
     
         57 . The method of  claim 56 , wherein said disease is cancer. 
     
     
         58 . A method for inducing lysis of a target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of any one of  claims 1 - 49  or  54  in the presence of a T cell.