Antibodies comprising modified heavy constant regions

Abstract

Provided herein are heavy chain constant regions (referred to as “modified heavy chain constant regions”), or functionally equivalent fragments thereof, that enhance biological properties of antibodies relative to the same antibodies in unmodified form. An exemplary modified heavy chain constant region includes an IgG2 hinge and three constant domains (i.e., CH1, CH2, and CH3 domains), wherein one or more of the constant region domains are of a non-IgG2 isotype (e.g., IgG1, IgG3 or IgG4). The heavy chain constant region may comprise wildtype human IgG domain sequences, or variants of these sequences. Also provided herein are methods for enhancing certain biological properties of antibodies that comprise a non-IgG2 hinge, such as internalization, agonism and antagonism, wherein the method comprises replacing the non-IgG2 hinge of the antibody with an IgG2 hinge.

We claim: 
     
         1 . An antibody comprising a modified heavy chain constant region, wherein the modified heavy chain constant region comprises a CH1 domain, a hinge, a CH2 domain, and a CH3 domain in order from N- to C-terminus, wherein:
 (a) the hinge (i) is a wildtype human IgG2 hinge and comprises the amino acid substitution C219S, or (ii) comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of a wildtype human IgG2 hinge and comprises the amino acid substitution C219S;   (b) the CH1 domain (i) is a wildtype human IgG2 CH1 domain, or (ii) comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of a wildtype human IgG2 CH1 domain; and   (c) the CH2 and CH3 domains (i) are wildtype human IgG1 CH2 and CH3 domains, or (ii) comprise an amino acid sequence that is at least 95% identical to the amino acid sequence of a wildtype human IgG1 CH2 and CH3 domains.   
     
     
         2 . The antibody of  claim 1 , wherein the hinge comprises the amino acid sequence of any one of SEQ ID NO: 21, 129 or 144. 
     
     
         3 . The antibody of  claim 1 , wherein the IgG2 CH1 domain comprises the amino acid sequence 
       
         
           
                 
               
                   (SEQ ID NO: 7) 
                 
                   ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 
                 
                     
                 
                   HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV. 
                 
             
                
                
                
                
               
            
           
         
       
     
     
         4 . The antibody of  claim 1 , wherein the CH2 domain comprises one or more modifications which reduces or eliminates effector functions. 
     
     
         5 . The antibody of  claim 1 , wherein the CH2 domain comprises amino acid substitutions A330S and P331S. 
     
     
         6 . The antibody of  claim 13 , wherein the CH2 domain comprises the amino acid sequence 
       
         
           
                 
               
                   (SEQ ID NO: 4) 
                 
                   PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 
                 
                     
                 
                   KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 
                 
                     
                 
                   KAK. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         7 . The antibody of  claim 1 , wherein the CH3 domain comprises the amino acid sequence 
       
         
           
                 
               
                   (SEQ ID NO: 5) 
                 
                   GQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENN 
                 
                     
                 
                   YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS 
                 
                     
                 
                   LSLSPGK. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         8 . The antibody of  claim 1 , wherein the CH3 domain comprises amino acid substitutions E356D and M358L. 
     
     
         9 . The antibody of  claim 1 , wherein the antibody comprises a modified heavy chain constant region selected from the group of SEQ ID NOs: 35, 37, 81, 84, 109, and 110. 
     
     
         10 . The antibody of  claim 1 , which (i) binds specifically to a costimulatory receptor; (ii) binds specifically to a cell surface molecule and triggers antibody mediated internalization of the cell surface molecule; (iii) binds specifically to an inhibitory receptor; (iv) binds specifically to a cell surface molecule and triggers intracellular signaling; (v) binds specifically to a cell surface molecule and triggers formation of high molecular weight antibody-cell surface molecule complexes; or (vi) binds specifically to a cell surface molecule and triggers clustering or oligomerization of the cell surface molecule. 
     
     
         11 . The antibody of  claim 10 , wherein the costimulatory receptor is GITR, OX40, 4-1BB, CD28, ICOS, CD40L, CD27 or any other TNFR superfamily member; the cell surface molecule is CD73; the inhibitory receptor is CTLA-4, PD-1, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4; or the intracellular signaling mediates agonist activity, antagonist activity, internalization of the cell surface molecule, or ADCC. 
     
     
         12 . The antibody of  claim 10 , wherein the antibody exhibits enhanced or altered agonist activity relative to an antibody having the same variable regions and light chain, but comprising an IgG1 heavy chain constant region; the antibody possesses enhanced or altered internalization properties relative to an antibody having the same variable regions and light chain, but comprising an IgG1 heavy chain constant region; the antibody exhibits more potent or altered antagonist activity or introduces a new activity relative to the same antibody having an IgG1 heavy chain constant region; the antibody triggers more potent intracellular signaling relative to an antibody having the same variable regions and light chain, but comprising an IgG1 heavy chain constant region; the antibody triggers formation of higher molecular weight complexes relative to an antibody having the same variable regions and light chain, but comprising an IgG1 heavy chain constant region; or the antibody triggers more clustering or oligomerization of the cell surface molecule relative to an antibody having the same variable regions and light chain, but comprising an IgG1 heavy chain constant region. 
     
     
         13 . A bispecific molecule comprising the antibody of  claim 1 , linked to a molecule having a second binding specificity. 
     
     
         14 . An immunoconjugate comprising the antibody of  claim 1 , linked to a second agent. 
     
     
         15 . A composition comprising the antibody of  claim 1 , and a carrier. 
     
     
         16 . The composition of  claim 15 , further comprising one or more additional therapeutic agents. 
     
     
         17 . A method of treating a subject, comprising administering the antibody of  claim 1 , wherein the subject is treated.