Multispecific polypeptide constructs having constrained cd3 binding and related methods and uses

Abstract

The invention relates generally to multispecific polypeptides having constrained CD3 binding. In some embodiments, components of the multispecific polypeptides are connected by a non-cleavable linker. Also provided are methods of making and using these multispecific polypeptides in a variety of therapeutic, diagnostic and prophylactic indications.

What is claimed: 
     
         1 . A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein:
 the CD3 binding region is an anti-CD3 antibody or antigen binding fragment that is an Fv antibody fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL);   the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide and the VH and VL of the anti-CD3 antibody or antigen binding fragment are linked to opposite polypeptides of the heterodimeric Fc;   the first and second components are coupled by a non-cleavable linker, wherein the Fc region is positioned N-terminal to the CD3-binding region; and   the first component comprises a first antigen binding domain and the second component comprises a second antigen binding domain, wherein each of the antigen binding domains bind a tumor associated antigen (TAA).   
     
     
         2 . (canceled) 
     
     
         3 . The multispecific polypeptide construct of  claim 1 , wherein the first antigen binding domain is positioned amino-terminally relative to the Fc region of the multispecific polypeptide construct and the second antigen binding domain is positioned carboxy-terminally relative to the CD3 binding region of the multispecific polypeptide construct. 
     
     
         4 . The multispecific polypeptide construct of  claim 1 , wherein the multispecific polypeptide construct comprises in order, from N-terminus to C-terminus:
 the first antigen binding domain that binds to a tumor-associated antigen (TAA);   the immunoglobulin Fc region;   the non-cleavable linker;   the CD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε); and   the second antigen binding domain that binds a tumor-associated antigen (TAA).   
     
     
         5 . A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein:
 the CD3 binding region is an anti-CD3 antibody or antigen binding fragment that is a disulfide-stabilized Fv antibody fragment (dsFv) comprising a variable heavy chain (VH) and a variable light chain (VL);   the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide and the VH and VL of the anti-CD3 antibody or antigen binding fragment are linked to opposite polypeptides of the heterodimeric Fc;   the first and second components are coupled by a non-cleavable linker, wherein the Fc region is positioned N-terminal to the CD3-binding region; and   one or both of the first and second components comprises an antigen binding domain that binds a tumor associated antigen (TAA).   
     
     
         6 . A multispecific polypeptide construct, the multispecific polypeptide construct comprising a first component comprising an immunoglobulin Fc region and a second component comprising a CD3-binding region, wherein:
 the CD3 binding region is an anti-CD3 antibody or antigen binding fragment that is a an Fv antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL);   the Fc is a heterodimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide and the VH and VL of the anti-CD3 antibody or antigen binding fragment are linked to opposite polypeptides of the heterodimeric Fc;   the first and second components are coupled by a non-cleavable linker, wherein the Fc region is positioned N-terminal to the CD3-binding region; and   one or both of the first and second components comprises an antigen binding domain that binds a tumor associated antigen (TAA), wherein the antigen-binding domain is a single chain antibody fragment.   
     
     
         7 . The multispecific polypeptide construct of  claim 6 , wherein the single chain antibody fragment is a single domain antibody or is a single chain variable fragment (scFv). 
     
     
         8 . (canceled) 
     
     
         9 . The multispecific polypeptide construct of  claim 5 , wherein the multispecific polypeptide construct is selected from:
 (A) a multispecific polypeptide construct that comprises in order, from N-terminus to C-terminus:   the first antigen binding domain that binds to a tumor-associated antigen (TAA);   the immunoglobulin Fc region;   the non-cleavable linker;   the CD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε); and   the second antigen binding domain that binds a tumor-associated antigen (TAA); or   (B) a multispecific polypeptide construct that comprises in order, from N-terminus to C-terminus:   the immunoglobulin Fc region;   the non-cleavable linker;   the CD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε); and   an antigen binding domain that binds a tumor-associated antigen (TAA); or   (C) a multispecific polypeptide construct that comprises in order, from N-terminus to C-terminus:   the antigen binding domain that binds to a tumor-associated antigen (TAA);   the immunoglobulin Fc region;   the non-cleavable linker; and   the CD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε).   
     
     
         10 . The multispecific polypeptide construct of  claim 6 , wherein the multispecific polypeptide construct is selected from:
 (A) a multispecific polypeptide construct that comprises in order, from N-terminus to C-terminus:   the first antigen binding domain that binds to a tumor-associated antigen (TAA);   the immunoglobulin Fc region;   the non-cleavable linker;   the CD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε); and   the second antigen binding domain that binds a tumor-associated antigen (TAA); or   (B) a multispecific polypeptide construct that comprises in order, from N-terminus to C-terminus:   the immunoglobulin Fc region;   the non-cleavable linker;   the CD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε); and   an antigen binding domain that binds a tumor-associated antigen (TAA); or   (C) a multispecific polypeptide construct that comprises in order, from N-terminus to C-terminus:   the antigen binding domain that binds to a tumor-associated antigen (TAA);   the immunoglobulin Fc region;   the non-cleavable linker; and   the CD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε).   
     
     
         11 - 14 . (canceled) 
     
     
         15 . The multispecific polypeptide construct of  claim 6 , wherein the heterodimeric Fc region comprises at least one modification selected from among the following: a steric modification(s), a knob-into-hole modification(s), a charge mutation(s) to increase electrostatic complementarity of the polypeptides, a modification(s) to alter the isoelectric point (pI variant), or combinations thereof. 
     
     
         16 - 35 . (canceled) 
     
     
         36 . The multispecific polypeptide construct of  claim 6 , wherein the linker is a polypeptide linker. 
     
     
         37 . (canceled) 
     
     
         38 . The multispecific polypeptide construct of  claim 36 , wherein the linker is a polypeptide of from or from about 2 to 24 amino acids. 
     
     
         39 - 43 . (canceled) 
     
     
         44 . The multispecific polypeptide construct of  claim 6 , wherein the non-cleavable linker comprises:
 (GGS)n, wherein n is 1 to 10;   (GGGGS)n (SEQ ID NO: 173), wherein n is 1 to 10; or   (GGGGGS)n (SEQ ID NO:172), wherein n is 1 to 4.   
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . The multispecific polypeptide construct of  claim 6 , wherein the non-cleavable linker is or comprises an amino acid sequence selected from among 
       
         
           
                 
                 
               
                     
                   GGS; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 149) 
                 
                     
                     GGGGS ; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 135) 
                 
                     
                     GGGGGS ; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 10) 
                 
                     
                     (GGS) 2   ; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 11) 
                 
                     
                     GGSGGSGGS ; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 12) 
                 
                     
                     GGSGGSGGSGGS ; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 13) 
                 
                     
                     GGSGGSGGSGGSGGS ; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 119) 
                 
                     
                   GGGGGSGGGGGSGGGGGS; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 147) 
                 
                     
                     GGSGGGGSGGGGSGGGGS ; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 170) 
                 
                     
                     GGGGSGGGGSGGGGS . 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         48 - 56 . (canceled) 
     
     
         57 . The multispecific polypeptide construct of  claim 6 , wherein the multispecific polypeptide construct comprises at least (i) a first polypeptide comprising the first Fc polypeptide of the heterodimeric Fc region, the linker and the VH domain of the anti-CD3 antibody or antigen binding fragment thereof; and (ii) a second polypeptide comprising the second Fc polypeptide of the heterodimeric Fc region, the linker and the VL domain of the anti-CD3 antibody or antigen binding fragment thereof, wherein one or both of the first and second polypeptide comprise at least one antigen-binding domain that binds to a tumor associated antigen (TAA). 
     
     
         58 . The multispecific polypeptide construct of  claim 6 , wherein the VH of the anti-CD3 antibody or antigen-binding fragment is on the same polypeptide as the at least one antigen-binding domain that binds to a tumor associated antigen (TAA). 
     
     
         59 . The multispecific polypeptide construct of  claim 58 , wherein the polypeptide comprising the VL of the anti-CD3 antibody or antigen-binding fragment does not contain the at least one antigen-binding domain that binds to a tumor associated antigen (TAA). 
     
     
         60 . (canceled) 
     
     
         61 . (canceled) 
     
     
         62 . The multispecific polypeptide construct of  claim 6 , wherein only one of the first and second polypeptide comprises the at least one antigen-binding domain that binds a TAA. 
     
     
         63 . The multispecific polypeptide construct of  claim 6 , wherein:
 the at least one antigen binding domain is positioned amino-terminally relative to the Fc region and/or is positioned carboxy-terminally relative to the CD3 binding region of one of the first or second polypeptide of the multispecific polypeptide construct; or   the at least one antigen binding domain comprises a first and a second antigen binding domain and the first antigen binding domain is positioned amino-terminally relative to the Fc region of the multispecific polypeptide construct and the second antigen binding domain is positioned carboxy-terminally relative to the CD3 binding region of the multispecific polypeptide construct.   
     
     
         64 . (canceled) 
     
     
         65 . The multispecific polypeptide construct of  claim 1 , wherein the antigen binding domain, or independently each of the antigen binding domains:
 comprises an extracellular domain or binding fragment thereof of the native cognate binding partner of the TAA, or a variant thereof that exhibits binding activity to the TAA; or   is an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.   
     
     
         66 . (canceled) 
     
     
         67 . The multispecific polypeptide construct of  claim 65 , wherein the antibody or antigen-binding fragment thereof is a Fv, a scFv, a Fab, or a single domain antibody (sdAb). 
     
     
         68 - 77 . (canceled) 
     
     
         78 . The multispecific polypeptide construct of  claim 6 , wherein the antigen binding domain, or independently each of the antigen binding domains, binds to a tumor antigen selected from among 1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors, Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM, Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R, Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin, MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, Notch Receptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2, Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3. 
     
     
         79 . The multispecific polypeptide construct of any of  claim 6 , wherein the multispecific polypeptide construct comprises:
 at least a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain and second antigen binding domain bind to the same TAA;   at least a first antigen binding domain and a second antigen binding domain wherein the first antigen binding domain and the second antigen binding domain binds different epitopes of the same TAA;   at least a first antigen binding domain and a second antigen binding domain wherein the first antigen binding domain and the second antigen binding domain bind the same epitope of the same TAA; or   at least a first antigen binding domain and a second antigen binding domain wherein the first antigen binding domain and the second antigen binding domain bind a different TAA.   
     
     
         80 - 86 . (canceled) 
     
     
         87 . The multispecific polypeptide construct of  claim 6 , wherein the Fv antibody fragment comprises a disulfide stabilized anti-CD3 binding Fv fragment (dsFv). 
     
     
         88 - 91 . (canceled) 
     
     
         92 . The multispecific polypeptide construct of  claim 6 , wherein the multispecific polypeptide construct is conjugated to an agent selected from among the following: a therapeutic agent, an antineoplastic agent, a toxin or fragment thereof, a detectable moiety, and a diagnostic agent. 
     
     
         93 . (canceled) 
     
     
         94 . (canceled) 
     
     
         95 . A polynucleotide(s) encoding the mutispecific polypeptide constructs of  claim 6 . 
     
     
         96 - 101 . (canceled) 
     
     
         102 . A vector, comprising the polynucleotide of  claim 95 . 
     
     
         103 . (canceled) 
     
     
         104 . (canceled) 
     
     
         105 . A cell, comprising the polynucleotide or polynucleotides of  claim 95 . 
     
     
         106 - 108 . (canceled) 
     
     
         109 . A method of producing a multispecific polypeptide construct, the method comprising introducing into a cell the polynucleotide or polynucleotides of  claim 95  and culturing the cell under conditions to produce the multispecific polypeptide construct. 
     
     
         110 - 113 . (canceled) 
     
     
         114 . A pharmaceutical composition comprising the multispecific polypeptide construct of  claim 6  and a pharmaceutically acceptable carrier. 
     
     
         115 . (canceled) 
     
     
         116 . A method of stimulating or inducing an immune response, the method comprising contacting a target cell and a T cell with the multispecific polypeptide construct of  claim 6 , said target cell expressing a tumor associated antigen recognized by the multispecific polypeptide construct. 
     
     
         117 - 125 . (canceled) 
     
     
         126 . A method of treating a disease or condition in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of the multispecific polypeptide construct of  claim 6 . 
     
     
         127 . The method of  claim 126 , wherein the disease or condition is a tumor or a cancer. 
     
     
         128 . (canceled) 
     
     
         129 . The multispecific polypeptide construct of  claim 1 , wherein the Fv antibody fragment comprises a disulfide stabilized anti-CD3 binding Fv fragment (dsFv). 
     
     
         130 . The multispecific polypeptide construct of  claim 5 , wherein the antigen binding domain, or independently each of the antigen binding domains:
 comprises an extracellular domain or binding fragment thereof of the native cognate binding partner of the TAA, or a variant thereof that exhibits binding activity to the TAA; or   is an antibody or antigen-binding fragment thereof selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.   
     
     
         131 . The multispecific polypeptide construct of  claim 1 , wherein the non-cleavable linker is or comprises an amino acid sequence selected from among GGS; GGGGS (SEQ ID NO: 149); GGGGGS (SEQ ID NO: 135); (GGS)2 (SEQ ID NO: 10); GGSGGSGGS (SEQ ID NO: 11); GGSGGSGGSGGS (SEQ ID NO: 12); GGSGGSGGSGGSGGS (SEQ ID NO: 13); GGGGGSGGGGGSGGGGGS (SEQ ID NO: 119); GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147); and GGGGSGGGGSGGGGS (SEQ ID NO:170). 
     
     
         132 . The multispecific polypeptide construct of  claim 5 , wherein the non-cleavable linker is or comprises an amino acid sequence selected from among GGS; GGGGS (SEQ ID NO: 149); GGGGGS (SEQ ID NO: 135); (GGS)2 (SEQ ID NO: 10); GGSGGSGGS (SEQ ID NO: 11); GGSGGSGGSGGS (SEQ ID NO: 12); GGSGGSGGSGGSGGS (SEQ ID NO: 13); GGGGGSGGGGGSGGGGGS (SEQ ID NO: 119); GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147); and GGGGSGGGGSGGGGS (SEQ ID NO:170). 
     
     
         133 . A polynucleotide(s) encoding the mutispecific polypeptide constructs of  claim 1 . 
     
     
         134 . A cell, comprising the polynucleotide or polynucleotides of  claim 133 . 
     
     
         135 . A method of producing a multispecific polypeptide construct, the method comprising introducing into a cell the polynucleotide or polynucleotides of  claim 133  and culturing the cell under conditions to produce the multispecific polypeptide construct. 
     
     
         136 . A pharmaceutical composition comprising the multispecific polypeptide construct of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         137 . A method of stimulating or inducing an immune response, the method comprising contacting a target cell and a T cell with the multispecific polypeptide construct of  claim 1 , said target cell expressing a tumor associated antigen recognized by the multispecific polypeptide construct. 
     
     
         138 . A method of treating a disease or condition in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of the multispecific polypeptide construct-of  claim 1 . 
     
     
         139 . A polynucleotide(s) encoding the mutispecific polypeptide constructs of  claim 5 . 
     
     
         140 . A cell, comprising the polynucleotide or polynucleotides of  claim 139 . 
     
     
         141 . A method of producing a multispecific polypeptide construct, the method comprising introducing into a cell the polynucleotide or polynucleotides of  claim 139  and culturing the cell under conditions to produce the multispecific polypeptide construct. 
     
     
         142 . A pharmaceutical composition comprising the multispecific polypeptide construct of  claim 5  and a pharmaceutically acceptable carrier. 
     
     
         143 . A method of stimulating or inducing an immune response, the method comprising contacting a target cell and a T cell with the multispecific polypeptide construct of  claim 5 , said target cell expressing a tumor associated antigen recognized by the multispecific polypeptide construct. 
     
     
         144 . A method of treating a disease or condition in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of the multispecific polypeptide construct-of  claim 5 .