Multispecific antigen binding proteins and methods of use thereof

Abstract

Disclosed herein are multispecific, such as bispecific, antigen binding proteins comprising a first antigen binding domain comprising a heavy chain variable domain and a light chain variable domain, and a second antigen binding domain comprising a single-domain antibody. Pharmaceutical compositions comprising the multispecific antigen binding proteins, kits and methods of use thereof are further provided.

What is claimed is: 
     
         1 . A multispecific antigen binding protein (MABP) comprising:
 (a) a first antigen binding portion comprising a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H  and V L  together form an antigen-binding site that specifically binds a first epitope, and   (b) a second antigen binding portion comprising a single-domain antibody (sdAb) that specifically binds a second epitope, and   (c) an Fc region,   wherein the first antigen binding portion and the second antigen binding portion are fused to each other, and wherein the first antigen binding portion is connected to the Fc region via the second antigen binding portion.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The MABP of  claim 1 , wherein the MABP is bispecific. 
     
     
         5 . The MABP of  claim 1 , wherein:
 (i) the first antigen binding portion is a full-length antibody consisting of two heavy chains and two light chains; or   (ii) the first antigen binding portion is an antibody fragment comprising a heavy chain comprising the V H  and a light chain comprising the V L .   
     
     
         6 - 11 . (canceled) 
     
     
         12 . The MABP of  claim 1 , wherein the second antigen binding portion is a Fab-like domain comprising:
 (i) a first polypeptide chain comprising a sdAb fused to a C H 1 domain; or   (ii) a first polypeptide chain comprising a sdAb fused to a C H 1 domain, and a second polypeptide chain comprising a second single-domain antibody fused to a C L  domain.   
     
     
         13 - 17 . (canceled) 
     
     
         18 . The MABP of  claim 1 , wherein the first antigen binding portion and the second antigen binding portion are fused to each other via a peptide bond or a peptide linker. 
     
     
         19 . The MABP of  claim 18 , wherein the peptide linker is no more than about 30 amino acids long. 
     
     
         20 . The MABP of  claim 19 , wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 1, or 13. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The MABP of  claim 1 , wherein:
 (i) the first epitope is from an immune checkpoint molecule;   (ii) the second epitope is from an immune checkpoint molecule; or   (iii) the first epitope and the second epitope are from an immune checkpoint molecule.   
     
     
         24 . The MABP of  claim 23 , wherein the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, TIM-3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR, and CD40. 
     
     
         25 . The MABP of  claim 24 , wherein the first antigen binding portion is an anti-PD-1 antibody or antigen binding fragment thereof or an anti-PD-L1 antibody or antigen binding fragment thereof. 
     
     
         26 - 30 . (canceled) 
     
     
         31 . The MABP of claim  30 , wherein the second antigen binding portion comprises an anti-CTLA-4 sdAb. 
     
     
         32 . The MABP of  claim 1 , wherein the first epitope is from a tumor antigen or a pro-inflammatory molecule. 
     
     
         33 . The MABP of  claim 32 , wherein:
 (i) the tumor antigen is selected from the group consisting of HER2, BRAF, EGFR, VEGFR2, CD20, RANKL, CD38, and CD52; or   (ii) the pro-inflammatory molecule is selected from the group consisting of IL-1β, TNF-α, IL-5, IL-6, IL-6R, and eotaxin-1.   
     
     
         34 - 36 . (canceled) 
     
     
         37 . The MABP of  claim 1 , wherein:
 (i) the first antigen binding portion is an anti-Ang2 antibody or antigen binding fragment thereof; and/or   (ii) the second antigen binding portion is an anti-VEGF sdAb.   
     
     
         38 - 51 . (canceled) 
     
     
         52 . A pharmaceutical composition comprising the MABP of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         53 . A method of treating a disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of  claim 52 . 
     
     
         54 - 57 . (canceled) 
     
     
         58 . The MABP of  claim 1 , wherein:
 (i) the first antigen binding portion comprises a Fab,
 wherein the N-terminus of the sdAb is fused to the C-terminus of the Fab, and the C-terminus of the sdAb is fused to the N-terminus of the Fc region; 
   (ii) (a) the first antigen binding portion is a Fab;
 (b) the second antigen binding portion is a Fab-like domain comprising a sdAb,
 wherein the N-termini of the Fab-like domain is fused to the C-termini of the Fab, and the C-termini of the Fab-like domain is fused to the N-terminus of the Fc region; or 
 
   (iii) (a) the first antigen binding portion is a scFv;
 (b) the second antigen binding portion is a Fab-like domain comprising a sdAb,
 wherein the N-termini of the Fab-like domain is fused to the C-terminus of the scFv, and the C-termini of the Fab-like domain is fused to an N-terminus of the Fc region. 
 
   
     
     
         59 . The MABP of  claim 1 , comprising two identical first polypeptides and two identical second polypeptides, wherein:
 (i) (a) the two identical first polypeptides each comprise, from the N-terminus to the C-terminus: V H -C H 1-an optional peptide linker-sdAb-C H 2-C H 3; and
 (b) the two identical second polypeptides each comprise a V L -C L ; 
   (ii) (a) the two identical first polypeptides each comprise, from the N-terminus to the C-terminus: V H -C H 1-an optional peptide linker-sdAb-C H 1-C H 2-C H 3; and
 (b) the two identical second polypeptides each comprise, from the N-terminus to the C-terminus: V L -C L -an optional peptide linker-sdAb-C L ; or, 
   (iii) (a) the two identical first polypeptides each comprise, from the N-terminus to the C-terminus: scFv-an optional peptide linker-sdAb-C H 1-C H 2-C H 3; and
 (b) the two identical second polypeptides each comprise, from the N-terminus to the C-terminus: sdAb-C L . 
   
     
     
         60 . The MABP of  claim 1 , comprising four polypeptide chains with structures from the N-terminus to the C-terminus as follows:
 (i) (1) V L -C L ; (2) V H -C H 1-V H H-C H 2-C H 3; (3) V H -C H 1-V H H-C H 2-C H 3; and (4) V L -C L , wherein V H  and V L  of polypeptide chains (1) and (2) forms an antigen binding site that specifically binds a first copy of the first epitope, V H  and V L  of polypeptide chains (3) and (4) forms an antigen binding site that specifically binds a second copy of the first epitope, and each V H H specifically binds a copy of the second epitope;   (ii) (1) V L -C L -V H H-C L ; (2) V H -C H 1-V H H-C H 1-C H 2-C H 3; (3) V H -C H 1-V H H-C H 1-C H 2-C H 3; and (4) V L -C L -V H H-C L , wherein V H  and V L  of polypeptide chains (1) and (2) forms an antigen binding site that specifically binds a first copy of the first epitope, V H  and V L  of polypeptide chains (3) and (4) forms an antigen binding site that specifically binds a second copy of the first epitope, and each V H H specifically binds a copy of the second epitope; or   (iii) (1) V H H-C L ; (2) V L -V H -V H H-C H 1-C H 2-C H 3; (3) V L -V H -V H H-C H 1-C H 2-C H 3; and (4) V H H-C L , wherein V H  and V L  of polypeptide chains (2) and (3) each forms an scFv that specifically binds a copy of the first epitope, and each V H H specifically binds a copy of the second epitope.   
     
     
         61 . The method of  claim 53 , wherein the disease is cancer, an inflammatory disease, or an autoimmune disease.