Multispecific antigen binding proteins

Abstract

Multimeric multispecific proteins formed from dimerization between CH1 and CK domains and that bind two target antigens are provided. The proteins have advantages in production and in the treatment of disease, notably cancer or infectious disease.

1 - 52 . (canceled) 
     
     
         53 . An isolated hetero-multimeric protein, which does not bind to CD16, and which comprises a first antigen-binding domain (ABD) that monovalently binds to a first antigen of interest and further comprises a second ABD which monovalently binds to a second antigen of interest, comprising:
 (a) a first polypeptide chain comprising a first variable domain (V) specific to the first antigen fused to a CH1 or CK constant region, a second variable domain specific to the second antigen and comprised within the second ABD, and an Fc domain or portion thereof interposed between the first and second variable domains; and   (b) a second polypeptide chain comprising a first variable domain (V) specific to the first antigen fused to a CH1 or CK constant region selected to be complementary to the CH1 or CK constant region of the first polypeptide chain such that the first and second polypeptides form a CH1-CK heterodimer in which the first variable domain of the first polypeptide chain and the first variable domain of the second polypeptide form a first ABD that binds the first antigen of interest, and   wherein the first polypeptide chain comprises a third variable domain fused to the second variable domain, wherein the first and second polypeptide form a CH1-CK heterodimer, the first variable domain of the first polypeptide chain and the first variable domain of the second polypeptide chain form an antigen binding domain specific for the first antigen of interest, and the second and third variable domains of the first polypeptide chain form an scFv specific for the second antigen of interest, and still further wherein   wherein the hetero-multimeric polypeptide is a dimer with a dimeric Fc domain, which dimeric Fc domain does not bind to CD16, and wherein said hetero-multimeric polypeptide has the domain arrangement:   V1-(CH1 or CK)-Fc domain-V2-V3   V1-(CH1 or CK)-Fc domain   wherein each V1, V2 and V3 is a heavy or light chain variable region; wherein the Fc domains comprise a CH2 and a CH3 domain capable of CH3-CH3 dimerization; and further wherein one of the V1 of the first polypeptide chain and the V1 of the second polypeptide chain is a light chain variable domain and the other is a heavy chain variable domain.   
     
     
         54 . An isolated hetero-multimeric protein, which does not bind to CD16, which comprises a first antigen-binding domain (ABD) that monovalently binds to a first antigen of interest and further comprises a second ABD which monovalently binds to a second antigen of interest, said hetero-multimeric protein comprising:
 (a) a first polypeptide chain comprising a first variable domain (V) specific to the first antigen fused to a CH1 or CK constant region, a second variable domain specific to the second antigen and comprised within the second ABD, and an Fc domain or portion thereof interposed between the first and second variable domains which does not bind to CD16; and   (b) a second polypeptide chain comprising a first variable domain (V) specific to the first antigen fused to a CH1 or CK constant region selected to be complementary to the CH1 or CK constant region of the first polypeptide chain such that the first and second polypeptides form a CH1-CK heterodimer in which the first variable domain of the first polypeptide chain and the first variable domain of the second polypeptide form a first ABD that binds the first antigen of interest, wherein the hetero-multimeric polypeptide is a trimeric polypeptide, comprising:   (a) a first polypeptide chain comprising a first variable domain (V) fused to a first CH1 or CK constant region, a second variable domain fused to a second CH1 or CK constant region, and an Fc domain or portion thereof interposed between the first and second variable domains;   (b) a second polypeptide chain comprising a variable domain fused at its C-terminus to a CH1 or CK constant region selected to be complementary to the first CH1 or CK constant region of the first polypeptide chain such that the first and second polypeptides form a CH1-CK heterodimer; and   (c) a third polypeptide chain comprising a variable domain fused at its C-terminus to a CH1 or CK constant region, wherein the variable domain and the constant region are selected to be complementary to the second variable domain and second CH1 or CK constant region of the first polypeptide chain such that the first polypeptide chain and the third polypeptide chain form a CH1-CK heterodimer bound by disulfide bond(s) formed between the CH1 or CK constant region of the third polypeptide and the second CH1 or CK constant region of the first polypeptide, but not between the CH1 or CK constant region of the third polypeptide and the first CH1 or CK constant region of the first polypeptide such that the first, second and third polypeptides form a CH1-CK heterotrimer, and   wherein the first variable domain of the first polypeptide chain and the variable domain of the second polypeptide chain form an antigen binding domain specific for the first antigen of interest, and the second variable domain of the first polypeptide chain and the variable domain of the third polypeptide chain form an antigen binding domain specific for the second antigen of interest;   and further wherein the trimeric hetero-multimeric polypeptide is a trimer with comprises a dimeric Fc domain which does not bind CD16, which trimeric hetero-multimeric polypeptide has having the domain arrangement:   
       
         
           
           
               
               
           
         
         wherein each V-V pairing occurs between a light chain variable domain and a heavy chain variable domain, and wherein each constant region pairing occurs between a CH1 and a CK. 
       
     
     
         55 . The isolated protein of  claim 53 , wherein one of the first and second antigens of interest is an activating receptor expressed by an effector cell and-the other antigen of interest is a cancer antigen. 
     
     
         56 . The isolated protein of  claim 54 , wherein one of the first and second antigens of interest is an activating receptor expressed by an effector cell and-the other antigen of interest is a cancer antigen. 
     
     
         57 . A pharmaceutical composition comprising an isolated protein according to  claim 53 , and a pharmaceutically acceptable carrier. 
     
     
         58 . A pharmaceutical composition comprising an isolated protein according to  claim 54 , and a pharmaceutically acceptable carrier. 
     
     
         59 . A method of treating cancer in a subject comprising administering to the subject a composition comprising an isolated hetero-multimeric protein according to  claim 53 . 
     
     
         60 . A method of treating cancer in a subject comprising administering to the subject a composition comprising an isolated hetero-multimeric protein according to  claim 54 . 
     
     
         61 . A method of making a heterodimeric protein, comprising:
 (a) providing a first nucleic acid encoding a first polypeptide chain according to  claim 53 ;   (b) providing a second nucleic acid encoding a second polypeptide chain according to  claim 53 ;   (c) expressing said first and second nucleic acids in a host cell to produce a protein comprising said first and second polypeptide chains, respectively; and   (d) loading the protein produced onto an affinity purification support, optionally a Protein-A support, and recovering a heterodimeric protein.   
     
     
         62 . A method of making a heterotrimeric protein, comprising:
 (a) providing a first nucleic acid encoding a first polypeptide chain according to  claim 54 ;   (b) providing a second nucleic acid encoding a second polypeptide chain according to  claim 54 ;   (c) providing a third nucleic acid comprising a third polypeptide chain according to  claim 54 ;   (d) expressing said first, second and third nucleic acids in a host cell to produce a protein comprising said first, second and third polypeptide chains, respectively; and   (e) loading the protein produced onto an affinity purification support, optionally a Protein-A support, and recovering a heterotrimeric protein.   
     
     
         63 . An expression vector or vectors which comprise nucleic acids which in combination provide for the expression of a hetero-multimeric protein according to  claim 53 , said expression vector or vectors comprising:
 (a) a first nucleic acid encoding a first polypeptide chain according to  claim 53 ;   (b) a second nucleic acid encoding a second polypeptide chain according to  claim 53 ; and   (c) which first and second nucleic acids when expressed in combination in a host cell produce a hetero-multimeric protein according to  claim 53 .   
     
     
         64 . An expression vector or vectors which comprise nucleic acids which in combination provide for the expression of a hetero-multimeric protein according to  claim 54 , said expression vector or vectors comprising:
 (a) a first nucleic acid encoding a first polypeptide chain according to  claim 54 ;   (b) a second nucleic acid encoding a second polypeptide chain according to  claim 54 ;   (c) a third nucleic acid comprising a third polypeptide chain according to  claim 54 ; and   (d) which first, second and third nucleic acids when expressed in combination in a host cell produce a hetero-multimeric protein according to  claim 54 .   
     
     
         65 . An isolated or recombinant host cell which comprises an expression vector or vectors according to  claim 63 . 
     
     
         66 . An isolated or recombinant host cell which comprises an expression vector or vectors according to  claim 64 .