Heterodimeric fc-fused proteins

Abstract

The present invention provides Fc-fused protein constructs, which as monovalent dimers have a higher serum half-life compared to a native/natural molecule, and are, therefore, advantageous for achieving higher titers of the proteins during production, higher stability during storage, and improved efficacy when used as a therapeutic. Also provided are Fc-fused protein constructs having mutations in the Fc region that reduce effector functions, which have increased activity to inhibit tumor growth and are, therefore, advantageous when used as a cancer therapy.

1 . A heterodimeric Fc-fused protein comprising:
 (a) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit protein; and   (b) a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of the multisubunit protein,   wherein the first and second antibody Fc domain polypeptides each comprise different mutations promoting heterodimerization,   wherein the first antibody Fc domain polypeptide comprises mutation K360E, numbered according to the EU numbering system,   wherein the second antibody Fc domain polypeptide comprises mutation Q347R, numbered according to the EU numbering system,   wherein the first and second antibody Fc domain polypeptides comprise mutations that reduce an effector function of an Fc selected from the group consisting of:   (i) L234A and L235A;   (ii) L234A, L235A, and P329A;   (iii) L234A, L235A, and P329G; and   (iv) L234A, L235E, G237A, A330S, and P331S,   wherein the first subunit and second, different subunit of the multisubunit protein are bound to each other,   wherein the first and second antibody Fc domain polypeptides are optionally:   (i) IgG1, IgG2, IgG3, or IgG4 Fc domain polypeptides; and/or   (ii) human antibody Fc domain polypeptides, and   optionally wherein the first and second antibody Fc domain polypeptides are further mutated to introduce an inter-chain disulfide bond, optionally wherein:   (i) the first antibody Fc domain polypeptide comprises mutation Y349C, and the second antibody Fc domain polypeptide comprises mutation S354C; or   (ii) the first antibody Fc domain polypeptide comprises mutation S354C, and the second antibody Fc domain polypeptide comprises mutation Y349C, and   optionally wherein the heterodimeric Fc-fused protein is fucosylated.   
     
     
         2 . The heterodimeric Fc-fused protein according to  claim 1 ,
 wherein the effector function comprises the ability of an Fc to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).   
     
     
         3 .- 34 . (canceled) 
     
     
         35 . The heterodimeric Fc-fused protein according to  claim 1 ,
 wherein the first subunit of the multisubunit protein is fused by a first linker to the first antibody Fc domain polypeptide and the second, different subunit of the multisubunit protein is optionally fused to the second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, wherein the first linker comprises a first amino acid sequence, wherein the first amino acid sequence comprises amino acid sequence;   (a) PKSSDKTHTCPPCPAPEX 1 X 2 GX 3  (SEQ ID NO:237), wherein X 1  represents L or A, X 2  represents L, E, or A, and X 3  represents A or G,   wherein when X 1  represents L and/or X 2  represents L; or   (b) RVESKYGPPCPPCPAPEFXGG (SEQ ID NO:1), wherein X represents L or E.   
     
     
         36 . The heterodimeric Fc-fused protein according to  claim 35 , wherein:
 (a) the first amino acid sequence comprises or consists of amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO:239), and the second amino acid sequence optionally comprises or consists of amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO:10),   (b) the first amino acid sequence comprises or consists of amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO:238), and the second amino acid sequence optionally comprises or consists of amino acid sequence:   
       
         
           
                 
                 
               
                     
                   (i) 
                 
                     
                   (SEQ ID NO: 8) 
                 
                     
                   GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG 
                 
                     
                     
                 
                     
                   (ii) 
                 
                     
                   (SEQ ID NO: 15) 
                 
                     
                   GGGGSEPKSSDKTHTCPPCPAPELLGG; 
                 
                     
                     
                 
                     
                   (iii) 
                 
                     
                   (SEQ ID NO: 16) 
                 
                     
                   GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG; 
                 
                     
                     
                 
                     
                   (iv) 
                 
                     
                   (SEQ ID NO: 65) 
                 
                     
                   GGGGSEPKSSDKTHTCPPCPAPEAAGG; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (v) 
                 
                     
                   (SEQ ID NO: 66) 
                 
                     
                   GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
          or 
         (c) the first amino acid sequence comprises or consists of amino acid sequence PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO:240), and the second amino acid sequence optionally comprises or consists of amino acid sequence: 
       
       
         
           
                 
                 
               
                     
                   (i) 
                 
                     
                   (SEQ ID NO: 12) 
                 
                     
                   GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA; 
                 
                     
                   iii) 
                 
                     
                   (SEQ ID NO: 67) 
                 
                     
                   GGGGSEPKSSDKTHTCPPCPAPEAEGA; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (iii) 
                 
                     
                   (SEQ ID NO: 68) 
                 
                     
                   GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         37 .- 60 . (canceled) 
     
     
         61 . The heterodimeric Fc-fused protein according to  claim 35 , wherein the first amino acid sequence comprises or consists of amino acid sequence:
 (a) RVESKYGPPCPPCPAPEFLGG (SEQ ID NO:2), and the second amino acid sequence optionally comprises or consists of amino acid sequence:   
       
         
           
                 
                 
               
                     
                   (i) 
                 
                     
                   (SEQ ID NO: 3) 
                 
                     
                   GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG, 
                 
                     
                   iii) 
                 
                     
                   (SEQ ID NO: 13)  
                 
                     
                   GGGGSRVESKYGPPCPPCPAPEFLGG; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (iii) 
                 
                     
                   (SEQ ID NO: 14) 
                 
                     
                   GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
          or 
         (b) RVESKYGPPCPPCPAPEFEGG (SEQ ID NO:4), and the second amino acid sequence optionally comprises or consists of amino acid sequence: 
       
       
         
           
                 
                 
               
                     
                   (i) 
                 
                     
                   (SEQ ID NO: 5) 
                 
                     
                   GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG; 
                 
                     
                   iii) 
                 
                     
                   (SEQ ID NO: 63) 
                 
                     
                   GGGGSRVESKYGPPCPPCPAPEFEGG; 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (iii) 
                 
                     
                   (SEQ ID NO: 64) 
                 
                     
                   GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         62 .- 89 . (canceled) 
     
     
         90 . The heterodimeric Fc-fused protein according to  claim 1 , wherein the first antibody Fc domain polypeptide further comprises mutation K409W, and the second antibody Fc domain polypeptide further comprises one or more mutation(s) selected from the group consisting of D399V, F405T, and any combination thereof, numbered according to the EU numbering system. 
     
     
         91 . The heterodimeric Fc-fused protein according to  claim 90 , wherein the first antibody Fc domain polypeptide comprises mutations K360E and K409W, and the second antibody Fc domain polypeptide comprises mutations Q347R, D399V, and F405T. 
     
     
         92 .- 104 . (canceled) 
     
     
         105 . The heterodimeric Fc-fused protein according to  claim 1 , wherein the multisubunit protein is a multisubunit cytokine, wherein the multisubunit cytokine is optionally IL-12 wherein:
 (a) the first subunit of the multisubunit cytokine is a p40 subunit of human IL-12, and the second, different subunit of the multisubunit cytokine is a p35 subunit of human IL-12, or   (b) the first subunit of the multisubunit cytokine is a p35 subunit of human IL-12, and the second, different subunit of the multisubunit cytokine is a p40 subunit of human IL-12.   
     
     
         106 .- 110 . (canceled) 
     
     
         111 . The heterodimeric Fc-fused protein of  claim 1 , further comprising an antibody variable domain,
 wherein the antibody variable domain optionally comprises an antibody heavy chain variable domain, optionally wherein the antibody heavy chain variable domain is:   (a) fused to the N-terminus of the first antibody Fc domain polypeptide or the second antibody Fc domain polypeptide, optionally wherein the antibody heavy chain variable domain:
 (i) binds an antibody light chain variable domain to form an Fab; or 
 (ii) is further fused to an antibody light chain variable domain to form an scFv, 
   wherein the first subunit and second, different subunit of the multisubunit protein are optionally fused to the C-terminus of the first antibody Fc domain polypeptide and the C-terminus of the second antibody Fc domain polypeptide, respectively, or   (b) fused to the C-terminus of the first antibody Fc domain polypeptide or the second antibody Fc domain polypeptide, optionally wherein the antibody heavy chain variable domain:
 (i) is further fused to an antibody light chain variable domain to form an scFv, 
   wherein the first subunit and second, different subunit of the multisubunit protein are optionally fused to the N-terminus of the first antibody Fc domain polypeptide and the N-terminus of the second antibody Fc domain polypeptide, respectively.   
     
     
         112 .- 129 . (canceled) 
     
     
         130 . The heterodimeric Fc-fused protein according to  claim 1 , further comprising:
 (a) a proteoglycan-binding domain, optionally wherein the proteoglycan-binding domain binds one or more proteoglycan(s) specifically expressed in a tumor,   wherein the proteoglycan-binding domain optionally binds one or more proteoglycan(s) selected from syndecan, serglycin, CSPG4, betaglycan, glypican, perlecan, versican, brevican, and small leucine-rich proteoglycans (SLRPs),   wherein the SLRPs are optionally selected from decorin, biglycan, asporin, fibrodulin, and lumican;   (b) a collagen-binding domain, optionally wherein the collagen-binding domain binds one or more collagen(s) specifically expressed in a tumor; and/or   (c) a hyaluronic acid-binding domain,   optionally wherein the proteoglycan-binding domain, collagen-binding domain, or hyaluronic acid-binding domain is fused to the C-terminus of the first antibody Fc domain polypeptide or the second antibody Fc domain polypeptide.   
     
     
         131 .- 146 . (canceled) 
     
     
         147 . A pharmaceutical composition comprising the heterodimeric Fc-fused protein according to  claim 1 , and a pharmaceutically acceptable carrier, optionally wherein the composition is suitable for intravenous, subcutaneous, intraperitoneal, or intratumoral administration. 
     
     
         148 . (canceled) 
     
     
         149 . A method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to  claim 147 . 
     
     
         150 .- 151 . (canceled) 
     
     
         152 . The method according to  claim 149 , further comprising administering to the patient a checkpoint blocker selected from the group consisting of: nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, ipilimumab, tremelimumab, lirilumab, and monalizumab. 
     
     
         153 .- 156 . (canceled) 
     
     
         157 . The method according to  claim 152 , wherein the checkpoint blocker is nivolumab. 
     
     
         158 .- 162 . (canceled) 
     
     
         163 . The method of according to  claim 149 , wherein the cancer is selected from the group consisting of breast cancer, esophageal cancer, Ewing's sarcoma, follicular lymphoma, gastric cancer, head and neck cancer, melanoma, renal cell carcinoma, non-small cell lung cancer, ovarian cancer, prostate cancer, small cell lung cancer, urothelial cancer, head and neck squamous cell carcinoma (HNSCC), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), and endometrial cancer. 
     
     
         164 .- 244 . (canceled) 
     
     
         245 . A nucleic acid encoding a polypeptide comprising an antibody Fc domain polypeptide and a subunit of a multisubunit protein,
 wherein the antibody Fc domain polypeptide comprises mutation K360E, numbered according to the EU numbering system,   wherein the antibody Fc domain polypeptide comprises mutations that reduce an effector function of an Fc selected from the group consisting of:   (i) L234A and L235A;   (ii) L234A, L235A, and P329A;   (iii) L234A, L235A, and P329G; and   (iv) L234A, L235E, G237A, A330S, and P331S,   wherein the antibody Fc domain polypeptide is optionally:   (i) an IgG1, IgG2, IgG3, or IgG4 Fc domain polypeptide; and/or   (ii) a human antibody Fc domain polypeptide,   optionally wherein the antibody Fc domain polypeptide is further mutated to introduce an inter-chain disulfide bond, and   optionally wherein the antibody Fc domain polypeptide comprises mutation Y349C, or mutation S354C.   
     
     
         246 . A nucleic acid encoding a polypeptide comprising an antibody Fc domain polypeptide and a subunit of the multisubunit protein,
 wherein the antibody Fc domain polypeptide comprises mutation Q347R, numbered according to the EU numbering system,   wherein the antibody Fc domain polypeptide comprises mutations that reduce an effector function of an Fc selected from the group consisting of:   (i) L234A and L235A;   (ii) L234A, L235A, and P329A;   (iii) L234A, L235A, and P329G; and   (iv) L234A, L235E, G237A, A330S, and P331S,   wherein the antibody Fc domain polypeptide is optionally:   (i) an IgG1, IgG2, IgG3, or IgG4 Fc domain polypeptide; and/or   (ii) a human antibody Fc domain polypeptide,   optionally wherein the antibody Fc domain polypeptide is further mutated to introduce an inter-chain disulfide bond, and   optionally wherein the antibody Fc domain polypeptide comprises mutation Y349C, or mutation S354C.   
     
     
         247 . An expression vector comprising:
 (a) a nucleic acid encoding a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit protein; and/or   (b) a nucleic acid encoding a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of the multisubunit protein,   wherein the first and second antibody Fc domain polypeptides each comprise different mutations promoting heterodimerization,   wherein the first antibody Fc domain polypeptide comprises mutation K360E, numbered according to the EU numbering system,   wherein the second antibody Fc domain polypeptide comprises mutation Q347R, numbered according to the EU numbering system,   wherein the first and second antibody Fc domain polypeptides each comprise mutations that reduce an effector function of an Fc selected from the group consisting of:   (i) L234A and L235A;   (ii) L234A, L235A, and P329A;   (iii) L234A, L235A, and P329G; and   (iv) L234A, L235E, G237A, A330S, and P331S,   wherein the first subunit and second, different subunit of the multisubunit protein are bound to each other,   wherein the first and second antibody Fc domain polypeptides are optionally:   (i) IgG1, IgG2, IgG3, or IgG4 Fc domain polypeptides; and/or   (ii) human antibody Fc domain polypeptides, and   optionally wherein the first and second antibody Fc domain polypeptides are further mutated to introduce an inter-chain disulfide bond, optionally wherein:   (i) the first antibody Fc domain polypeptide comprises mutation Y349C, and the second antibody Fc domain polypeptide comprises mutation S354C; or   (ii) the first antibody Fc domain polypeptide comprises mutation S354C, and the second antibody Fc domain polypeptide comprises mutation Y349C.   
     
     
         248 . A host cell comprising the expression vector according to  claim 247 . 
     
     
         249 . A method of producing a heterodimeric Fc fused protein, the method comprising culturing the host cell according to  claim 248  under conditions suitable for expression of the heterodimeric Fc fused protein. 
     
     
         250 . The method according to  claim 149 , wherein the cancer is selected from the group consisting of breast cancer, melanoma, and non-small cell lung cancer.