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 a first antigen binding moiety capable of specific binding to an activating T cell antigen, and a second antigen binding moiety capable of specific binding to a target cell antigen, wherein said one antigen binding moiety is a Fab molecule or a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged and wherein the other antigen binding moiety comprises a single domain antigen binding molecule or is a binding protein comprising at least one ankyrin repeat motif. 
     
     
         2 . The T cell activating bispecific antigen binding molecule according to  claim 1  wherein said a single domain antigen binding molecule is a single domain variable heavy chain. 
     
     
         3 . The T cell activating bispecific antigen binding molecule according to  claim 2 , wherein the first antigen binding moiety capable of specific binding to an activating T cell antigen 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 and wherein the second antigen binding moiety capable of specific binding to a target cell antigen consists of a single domain variable heavy chain. 
     
     
         4 . (canceled) 
     
     
         5 . The T cell activating bispecific antigen binding molecule according to  claim 1 , wherein the first antigen binding moiety capable of specific binding to an activating T cell antigen 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 and wherein the second antigen binding moiety is a binding protein comprising at least one ankyrin repeat motif. 
     
     
         6 . The T cell activating bispecific antigen binding molecule of  claim 5 , wherein the second antigen moiety comprises a binding protein comprising two, three, four or five ankyrin repeat motifs. 
     
     
         7 . The T cell activating bispecific antigen binding molecule of  claim 1 , further comprising an Fc domain composed of a first and a second subunit capable of stable association. 
     
     
         8 . The T cell activating bispecific antigen binding molecule of  claim 1 , comprising not more than one antigen binding moiety capable of specific binding to an activating T cell antigen. 
     
     
         9 . 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. 
     
     
         10 . 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. 
     
     
         11 . The T cell activating bispecific antigen binding molecule of  claim 1 , comprising a third antigen binding moiety capable of specific binding to a target cell antigen. 
     
     
         12 . The T cell activating bispecific antigen binding molecule of  claim 11 , wherein the third antigen binding moiety is a single domain antigen binding molecule. 
     
     
         13 . The T cell activating bispecific antigen binding molecule of  claim 12 , wherein the single domain antigen binding molecule is a single domain variable heavy chain. 
     
     
         14 . The T cell activating bispecific antigen binding molecule of  claim 13 , comprising
 a) an Fc domain composed of a first and a second subunit capable of stable association,   b) a first antigen binding moiety comprising a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged, wherein said crossover Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;   c) a second antigen binding moiety comprising a single domain variable heavy chain, wherein said single domain variable heavy chain is fused to the N-terminus of one of the subunits of the Fc domain, and   d) a third antigen binding moiety comprising a single domain variable heavy chain wherein said single domain variable heavy chain is fused to the N-terminus of the Fab heavy chain of the first antigen binding moiety.   
     
     
         15 . The T cell activating bispecific antigen binding molecule of  claim 11 , wherein the third antigen binding moiety is a binding protein comprising at least one ankyrin repeat motiv. 
     
     
         16 . The T cell activating bispecific antigen binding molecule of  claim 15 , wherein the third antigen binding moiety is a binding protein comprising two, three, four or five ankyrin repeat motifs. 
     
     
         17 . The T cell activating bispecific antigen binding molecule of  claim 15 , comprising
 a) an Fc domain composed of a first and a second subunit capable of stable association,   b) a first antigen binding moiety comprising a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged, wherein said crossover Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain;   c) a second antigen binding moiety comprising a binding protein comprising at least one ankyrin repeat motiv, wherein said binding protein comprising at least one ankyrin repeat motiv is fused to the N-terminus of one of the subunits of the Fc domain, and   d) a third antigen binding moiety comprising binding protein comprising at least one ankyrin repeat motiv wherein binding protein comprising at least one ankyrin repeat motiv is fused to the N-terminus of the Fab heavy chain of the first antigen binding moiety.   
     
     
         18 . The T cell activating bispecific antigen binding molecule of  claim 11 , wherein the first antigen binding moiety binds to an activating T cell antigen and the second and third antigen binding moiety bind to the same target cell antigen. 
     
     
         19 . The T cell activating bispecific antigen binding molecule of  claim 7 , wherein the Fc domain is an IgG, specifically an IgG 1  or IgG 4 , Fc domain. 
     
     
         20 . The T cell activating bispecific antigen binding molecule of  claim 19 , wherein the Fc domain is a human Fc domain. 
     
     
         21 . The T cell activating bispecific antigen binding molecule of  claim 7 , wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. 
     
     
         22 . The T cell activating bispecific antigen binding molecule of  claim 21 , 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. 
     
     
         23 . The T cell activating bispecific antigen binding molecule of  claim 7 , wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG1 Fc domain. 
     
     
         24 . The T cell activating bispecific antigen binding molecule of  claim 7 , wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. 
     
     
         25 . The T cell activating bispecific antigen binding molecule of  claim 24 , wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329. 
     
     
         26 . The T cell activating bispecific antigen binding molecule of  claim 25 , 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. 
     
     
         27 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the Fc receptor is an Fcγ receptor. 
     
     
         28 . The T cell activating bispecific antigen binding molecule of  claim 23 , wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). 
     
     
         29 . A T cell activating bispecific antigen binding molecule comprising a first and a second antigen binding moiety, one of which is a Fab molecule capable of specific binding to an activating T cell antigen and the other one of which is a Fab molecule capable of specific binding to a target cell antigen;
 wherein the first antigen binding moiety is
 (a) a single chain Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker, or 
 (b) a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged; 
   and an Fc domain composed of a first and a second subunit capable of stable association, wherein said first subunit and said second subunit have been modified to comprise one or more charged amino acids electrostatically favorable to heterodimer formation.   
     
     
         30 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein said first subunit comprises amino acid mutations E356K, E357K and D399K and said second subunit comprises amino acid mutations K370E, K409E and K439E. 
     
     
         31 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein said first subunit comprises amino acid mutations K392D, K409D and said second subunit comprises amino acid mutations E356K, D399K (DDKK). 
     
     
         32 . The T cell activating bispecific antigen binding molecule of  claim 29 , comprising not more than one antigen binding moiety capable of specific binding to an activating T cell antigen. 
     
     
         33 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker. 
     
     
         34 . The T cell activating bispecific antigen binding molecule of  claim 29 , 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 or 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. 
     
     
         35 . (canceled) 
     
     
         36 . The T cell activating bispecific antigen binding molecule of  claim 34 , wherein the first antigen binding moiety is a crossover Fab molecule 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. 
     
     
         37 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the second antigen binding moiety of the T cell activating bispecific antigen binding molecule is fused at the C-terminus of the Fab light chain to the N-terminus of the Fab light chain of the first antigen binding moiety. 
     
     
         38 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the second antigen binding moiety of the T cell activating bispecific antigen binding molecule 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. 
     
     
         39 . The T cell activating bispecific antigen binding molecule of  claim 29 , 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. 
     
     
         40 . The T cell activating bispecific antigen binding molecule of  claim 29 , 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. 
     
     
         41 . The T cell activating bispecific antigen binding molecule of  claim 29 , comprising a third antigen binding moiety which is a Fab molecule capable of specific binding to a target cell antigen. 
     
     
         42 . The T cell activating bispecific antigen binding molecule of  claim 41 , wherein the 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. 
     
     
         43 . The T cell activating bispecific antigen binding molecule of  claim 41 , 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. 
     
     
         44 . The T cell activating bispecific antigen binding molecule of  claim 41 , 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. 
     
     
         45 . The T cell activating bispecific antigen binding molecule of  claim 43 , wherein the second and the third antigen binding moiety and the Fc domain are part of an immunoglobulin molecule, particularly an IgG class immunoglobulin. 
     
     
         46 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the Fc domain is an IgG, specifically an IgG 1  or IgG 4 , Fc domain. 
     
     
         47 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the Fc domain is a human Fc domain. 
     
     
         48 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG1 Fc domain. 
     
     
         49 . The T cell activating bispecific antigen binding molecule of  claim 29 , wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function. 
     
     
         50 . The T cell activating bispecific antigen binding molecule of  claim 49 , wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329. 
     
     
         51 . The T cell activating bispecific antigen binding molecule of  claim 50 , 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. 
     
     
         52 . The T cell activating bispecific antigen binding molecule of  claim 1  or  claim 29 , wherein the activating T cell antigen is CD3. 
     
     
         53 . The T cell activating bispecific antigen binding molecule of  claim 52 , wherein the target cell antigen is selected from the group consisting of: Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), CD19, CD20, CD33, Carcinoembryonic Antigen (CEA) and Fibroblast Activation Protein (FAP). 
     
     
         54 . An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of  claim 1  or  29  or a fragment thereof. 
     
     
         55 . A polypeptide encoded by the isolated polynucleotide of  claim 54 . 
     
     
         56 . A vector, particularly an expression vector, comprising the isolated polynucleotide of  claim 54 . 
     
     
         57 . A host cell comprising the isolated polynucleotide of  claim 54 . 
     
     
         58 . A method of producing the T cell activating bispecific antigen binding molecule, comprising the steps of a) culturing the host cell of  claim 57  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. 
     
     
         59 . A T cell activating bispecific antigen binding molecule produced by the method of  claim 58 . 
     
     
         60 . A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of  claim 1  or  29  and a pharmaceutically acceptable carrier. 
     
     
         61 . (canceled) 
     
     
         62 . (canceled) 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . 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  or  29  in a pharmaceutically acceptable form. 
     
     
         66 . The method of  claim 65 , wherein said disease is cancer. 
     
     
         67 . 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  or  29  in the presence of a T cell. 
     
     
         68 . (canceled)