T cell activating bispecific 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, and which comprises at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected from the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34;   (ii) a second antigen binding moiety capable of specific binding to Folate Receptor 1 (FolR1).   
     
     
         2 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the first antigen binding moiety comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 36 and a variable light chain comprising an amino acid sequence of SEQ ID NO: 31. 
     
     
         3 . The T cell activating bispecific antigen binding molecule of  claim 1  or  2 , additionally comprising
 (i) a third antigen binding moiety capable of specific binding to FolR1. 
 
     
     
         4 . The T cell activating bispecific antigen binding molecule of  claim 3 , wherein the second and third antigen binding moiety capable of specific binding to FolR1 comprise identical heavy chain complementarity determining region (CDR) and light chain CDR sequences. 
     
     
         5 . The T cell activating bispecific antigen binding molecule of  claim 4 , wherein the third antigen binding moiety is identical to the second antigen binding moiety. 
     
     
         6 . The T cell activating bispecific antigen binding molecule of  claim 3 , wherein at least one of the second and third antigen binding moiety is a Fab molecule. 
     
     
         7 . The T cell activating bispecific antigen binding molecule of  claim 1 , additionally comprising
 (i) 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 7 , wherein the first antigen binding moiety and the second antigen binding moiety are each connected at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. 
     
     
         9 . The T cell activating bispecific antigen binding molecule of  claim 7  or  8 , wherein a third antigen binding moiety is connected at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety via a peptide linker. 
     
     
         10 .- 21 . (canceled) 
     
     
         22 . The T cell activating bispecific antigen binding molecule of  claim 1 , which binds to a human FolR1, a cynomolgus monkey FolR1 and a murine FolR1. 
     
     
         23 . The T cell activating bispecific antigen binding molecule of  claim 1 , which binds to a human FolR1 and a cynomolgus monkey FolR1 and not a murine FolR1. 
     
     
         24 . 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. 
     
     
         25 . 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. 
     
     
         26 . The T cell activating bispecific antigen binding molecule of  claim 7 , wherein the first and the second antigen binding moiety and the Fc domain are part of an immunoglobulin molecule. 
     
     
         27 . The T cell activating bispecific antigen binding molecule of  claim 26 , wherein the Fc domain is an IgG class immunoglobulin. 
     
     
         28 . The T cell activating bispecific antigen binding molecule of  claim 27 , wherein the Fc domain is a human IgG 1  or IgG 4  Fc domain. 
     
     
         29 . The T cell activating bispecific antigen binding molecule of  claim 7  or  28 , wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. 
     
     
         30 . The T cell activating bispecific antigen binding molecule of  claim 29 , 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. 
     
     
         31 . The T cell activating bispecific antigen binding molecule of  claim 7 , wherein the Fc domain comprises at least one amino acid substitution that reduces binding to an Fc receptor and/or effector function, as compared to a native IgG 1  Fc domain. 
     
     
         32 . The T cell activating bispecific antigen binding molecule of  claim 31 , 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 numbering). 
     
     
         33 . (canceled) 
     
     
         34 . The T cell activating bispecific antigen binding molecule of  claim 31 , wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). 
     
     
         35 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the T cell activating bispecific antigen binding molecule induces at least one of
 (a) proliferation of a human CD3 positive T cell in vitro;   (b) human peripheral blood mononuclear cell mediated killing of a FolR1-expressing human tumor cell in vitro;   (c) T cell-mediated killing of a FolR1-expressing human tumor cell in vitro; and   (d) upregulation of cell surface expression of at least one of CD25 and CD69 on the T cell as measured by flow cytometry.   
     
     
         36 .- 37 . (canceled) 
     
     
         38 . The T cell activating bispecific antigen binding molecule of  claim 35 , wherein the FolR1-expressing human tumor cell is a Hela, Skov-3, or a cell. 
     
     
         39 . The T cell activating bispecific antigen binding molecule of  claim 35  or  38 , wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing human tumor cell in vitro with an EC50 of between about 36 pM and about 39573 pM after 24 hours. 
     
     
         40 . The T cell activating bispecific antigen binding molecule of  claim 39 , wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 36 pM after 24 hours. 
     
     
         41 . The T cell activating bispecific antigen binding molecule of  claim 39 , wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 178.4 pM after 24 hours. 
     
     
         42 . The T cell activating bispecific antigen binding molecule of  claim 39 , wherein the T cell activating bispecific antigen binding molecule induces T cell mediated killing of the FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pM or greater after 48 hours. 
     
     
         43 . (canceled) 
     
     
         44 . The T cell activating bispecific antigen binding molecule of  claim 35 , wherein the T cell is a CD4 positive T cell or a CD8 positive T cell. 
     
     
         45 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the T cell activating bispecific antigen binding molecule binds human FolR1 with an apparent K D  of about 5.36 pM to about 4 nM. 
     
     
         46 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the T cell activating bispecific antigen binding molecule binds human and cynomolgus FolR1 with an apparent K D  of about 4 nM. 
     
     
         47 . (canceled) 
     
     
         48 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the T cell activating bispecific antigen binding molecule binds human FolR1 with a monovalent binding K D  of at least about 1000 nM. 
     
     
         49 . (canceled) 
     
     
         50 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the T cell activating bispecific antigen binding molecule binds to a conformational epitope of a human FolR1 expressed on a human tumor cell. 
     
     
         51 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the T cell activating bispecific antigen binding molecule does not bind to human Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3). 
     
     
         52 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the antigen binding moiety binds to a FolR1 polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ ID NO:227). 
     
     
         53 . The T cell activating bispecific antigen binding molecule of  claim 1 , wherein the FolR1 antigen binding moiety binds to a FolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moiety does not bind to a FolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228 and 229. 
     
     
         54 .- 103 . (canceled) 
     
     
         104 . An isolated polynucleotide encoding the T cell activating bispecific antigen binding molecule of  claim 1  or a fragment thereof. 
     
     
         105 .- 109 . (canceled) 
     
     
         110 . A polypeptide encoded by the polynucleotide of  claim 104 . 
     
     
         111 . A vector comprising the polynucleotide of  claim 104 . 
     
     
         112 . A host cell comprising the polynucleotide of  claim 104  or the vector of  claim 111 . 
     
     
         113 . 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 112  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. 
     
     
         114 . A T cell activating bispecific antigen binding molecule produced by the method of  claim 113 . 
     
     
         115 . A pharmaceutical composition comprising the T cell activating bispecific antigen binding molecule of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         116 .- 119 . (canceled) 
     
     
         120 . 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. 
     
     
         121 . The method of  claim 120 , wherein said disease is cancer. 
     
     
         122 . A method for inducing lysis of a FolR1 +  target cell, comprising contacting a target cell with the T cell activating bispecific antigen binding molecule of  claim 1  in the presence of a CD3 +  T cell. 
     
     
         123 . (canceled)