Antigen-binding molecule containing modified antibody variable region

Abstract

The present inventors have successfully prepared an antigen-binding molecule comprising an antibody variable region that has binding activity against a molecule expressed on the surface of a T cell and a molecule expressed on the surface of any other immunocyte, but does not bind to these molecules at the same time. The present invention allows the preparation of an antigen-binding molecule capable of circumventing adverse reactions that may be caused by the cross-linking of T cells to other immunocytes, and provides an antigen-binding molecule suitable as a drug.

1 . An antigen-binding molecule comprising:
 an antibody variable region that is capable of binding to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen at the same time; and   a variable region binding to a third antigen different from the first antigen and the second antigen.   
     
     
         2 . An antigen-binding molecule comprising an antibody variable region with an amino acid altered in a heavy chain variable domain such that the variable region is capable of binding to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen at the same time. 
     
     
         3 . The antigen-binding molecule according to  claim 1  or  2 , wherein the variable region that does not bind to the first antigen and the second antigen at the same time is a variable region that does not bind to the first antigen and the second antigen each expressed on a different cell, at the same time. 
     
     
         4 . The antigen-binding molecule according to any of  claims 1  to  3 , further comprising an antibody Fc region. 
     
     
         5 . The antigen-binding molecule according to  claim 4 , wherein the Fc region is an Fc region having reduced binding activity against FcγR as compared with the Fc region of a naturally occurring human IgG1 antibody. 
     
     
         6 . The antigen-binding molecule according to any of  claims 1  to  5 , wherein the antigen-binding molecule is a multispecific antibody. 
     
     
         7 . The antigen-binding molecule according to any of  claims 1  to  6 , wherein the antibody variable region capable of binding to the first antigen and the second antigen is a variable region having alteration of at least one amino acid. 
     
     
         8 . The antigen-binding molecule according to  claim 7 , wherein the alteration is substitution or insertion of at least one amino acid. 
     
     
         9 . The antigen-binding molecule according to  claim 7  or  8 , wherein the alteration is substitution of a portion of the amino acid sequence of a variable region binding to the first antigen by an amino acid sequence binding to the second antigen, or insertion of an amino acid sequence binding to the second antigen to the amino acid sequence of a variable region binding to the first antigen. 
     
     
         10 . The antigen-binding molecule according to  claim 8  or  9 , wherein the number of amino acids to be inserted is 1 to 25. 
     
     
         11 . The antigen-binding molecule according to any of  claims 7  to  10 , wherein the amino acid to be altered is an amino acid in a CDR1, CDR2, CDR3, or FR3 region of the antibody variable region. 
     
     
         12 . The antigen-binding molecule according to any of  claims 7  to  11 , wherein the amino acid to be altered is an amino acid in a loop. 
     
     
         13 . The antigen-binding molecule according to any of  claims 7  to  11 , wherein the amino acid to be altered is at least one amino acid selected from Kabat numbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in an antibody H chain variable domain, and Kabat numbering positions 24 to 34, 50 to 56, and 89 to 97 in an L chain variable domain. 
     
     
         14 . The antigen-binding molecule according to any of  claims 1  to  13 , wherein any one of the first antigen and the second antigen is a molecule specifically expressed on the surface of a T cell, and the other antigen is a molecule expressed on the surface of a T cell or any other immunocyte. 
     
     
         15 . The antigen-binding molecule according to  claim 14 , wherein any one of the first antigen and the second antigen is CD3, and the other antigen is FcγR, TLR, lectin, IgA, an immune checkpoint molecule, a TNF superfamily molecule, a TNFR superfamily molecule, or an NK receptor molecule. 
     
     
         16 . The antigen-binding molecule according to  claim 14  or  15 , wherein the third antigen is a molecule specifically expressed in a cancer tissue. 
     
     
         17 . A pharmaceutical composition comprising an antigen-binding molecule according to any of  claims 1  to  16  and a pharmaceutically acceptable carrier. 
     
     
         18 . A method for producing an antigen-binding molecule according to any of  claims 1  to  16 , the method comprising steps (i) to (iv):
 (i) preparing a library of antigen-binding molecules with at least one amino acid altered in their antibody variable regions each binding to the first antigen or the second antigen, wherein the altered variable regions differ in at least one amino acid from each other; 
 (ii) selecting, from the prepared library, an antigen-binding molecule comprising a variable region that has binding activity against the first antigen and the second antigen, but does not bind to the first antigen and the second antigen at the same time; 
 (iii) culturing a host cell comprising a nucleic acid encoding the variable region of the antigen-binding molecule selected in the step (ii), and/or a nucleic acid encoding a variable region of an antigen-binding molecule binding to the third antigen, to express an antigen-binding molecule comprising the antibody variable region that is capable of binding to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen at the same time, and/or the variable region binding to the third antigen; and 
 (iv) recovering the antigen-binding molecule from the host cell cultures. 
 
     
     
         19 . The method according to  claim 18 , wherein the variable region that does not bind to the first antigen and the second antigen at the same time, contained in the antigen-binding molecule selected in the step (ii) is a variable region that does not bind to the first antigen and the second antigen each expressed on a different cell, at the same time. 
     
     
         20 . The method according to  claim 18  or  19 , wherein the host cell cultured in the step (iii) further comprises a nucleic acid encoding an antibody Fc region. 
     
     
         21 . The method according to  claim 20 , wherein the Fc region is an Fc region having reduced binding activity against FcγR as compared with the Fc region of a naturally occurring human IgG1 antibody. 
     
     
         22 . The method according to any of  claims 18  to  21 , wherein the antigen-binding molecule to be produced is a multispecific antibody. 
     
     
         23 . The method according to any of  claims 18  to  22 , wherein the at least one amino acid altered in the variable regions in the step (i) is a substituted or inserted amino acid. 
     
     
         24 . The method according to  claim 23 , wherein the number of inserted amino acids is 1 to 25. 
     
     
         25 . The method according to any of  claims 18  to  24 , wherein the alteration is alteration of an amino acid in a CDR1, CDR2, CDR3, or FR3 region of the antibody variable region. 
     
     
         26 . The method according to any of  claims 18  to  25 , wherein the alteration is alteration of an amino acid in a loop. 
     
     
         27 . The method according to any of  claims 18  to  25 , wherein the alteration is alteration of at least one amino acid selected from Kabat numbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in an antibody H chain variable domain, and Kabat numbering positions 24 to 34, 50 to 56, and 89 to 97 in an L chain variable domain. 
     
     
         28 . The method according to any of  claims 18  to  27 , wherein any one of the first antigen and the second antigen is a molecule specifically expressed on the surface of a T cell, and the other antigen is a molecule expressed on the surface of a T cell or any other immunocyte. 
     
     
         29 . The method according to  claim 28 , wherein any one of the first antigen and the second antigen is CD3, and the other antigen is FcγR, TLR, IgA, lectin, an immune checkpoint molecule, a TNF superfamily molecule, a TNFR superfamily molecule, or an NK receptor molecule. 
     
     
         30 . The method according to  claim 28  or  29 , wherein the third antigen is a molecule specifically expressed in a cancer tissue.