Fabs-in-tandem immunoglobulin and uses thereof

Abstract

The present invention provides multivalent and multispecific binding proteins that are capable of binding two or more antigens, or two or more epitopes. The present invention also provides methods of making and using such multivalent and multispecific binding proteins, including methods of using such binding proteins for prevention or treatment of various diseases, or for detecting specific antigens in vitro or in vivo.

What is claimed is: 
     
         1 . A binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein the first, second, and third polypeptide chains comprise, respectively, the following amino acid sequences:   
       
         
           
                 
                 
                 
               
                     
                 
                   first polypeptide 
                   second polypeptide 
                   third polypeptide 
                 
                   chain sequence 
                   chain sequence 
                   chain sequence 
                 
                     
                 
                   SEQ ID NO: 15, 
                   SEQ ID NO: 21, and 
                   SEQ ID NO: 23; 
                 
                   SEQ ID NO: 41, 
                   SEQ ID NO: 44, and 
                   SEQ ID NO: 46; 
                 
                   SEQ ID NO: 87, 
                   SEQ ID NO: 89, and 
                   SEQ ID NO: 91; or 
                 
                   SEQ ID NO: 92, 
                   SEQ ID NO: 95, and 
                   SEQ ID NO: 97; 
                 
                     
                 
             
                
                
                
                
               
               
                
                
                
                
                
               
            
           
         
         wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions; and 
         wherein VL A  is a light chain variable domain of a first parental antibody that binds epitope or antigen A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds epitope or antigen B, CH1 is a first constant domain of an antibody heavy chain, VH A  is a heavy chain variable domain of said first parental antibody that binds epitope or antigen A, and VL B  is a light chain variable domain of said second parental antibody that binds epitope or antigen B, and wherein said bispecific binding protein binds both epitope or antigen A and epitope or antigen B. 
       
     
     
         2 . A binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, either (i) VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , or (ii) VH B -CH1-VL A -CL-Fc, wherein CH1 is fused directly to VL A , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A —CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein the binding protein is capable of binding two or more epitopes or antigens, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a first constant domain of a heavy chain, A is a first epitope or antigen, and B is a second epitope or antigen, and wherein A and B are different epitopes of the same antigen or are different antigens;   wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions, and wherein said binding protein binds both epitope or antigen A and epitope or antigen B; and   wherein the binding protein is capable of binding pairs of epitope or antigen targets selected from the group consisting of: IL-1a and IL-1β; IL-12 and IL-18; TNFα and IL-23; TNFα and IL-13; TNF and IL-18; TNF and IL-12; TNF and IL-1beta; TNF and MIF; TNF and IL-6; TNF and IL-6 Receptor; TNF and IL-17; IL-17 and IL-20; IL-17 and IL-23; TNF and IL-15; TNF and VEGF; VEGFR and EGFR; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS; and TNFα and PGE4; IL-13 and PED2; TNF and PEG2; CD138 and CD20; CD138 and CD40; CD19 and CD20; CD19 and CD22; CD20 and CD3; CD3 and CD33; CD16 and CD33; CD3 and CD133; CD38 and CD138; CD38 and CD20; CD20 and CD22; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CSPGs and RGM A; IGF1 and IGF2; IGF1/2 and ErbB2; IGF-1R and EGFR; EGFR and CD13; IGF-1R and ErbB3; EGFR-2 and IGFR; VEGFR-2 and c-Met; VEGF-A and Angiopoietin-2 (Ang-2); IL-12 and TWEAK; IL-13 and IL-1beta; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; CTLA-4 and BTNO2; PD-1 and CTLA-4; PD-L1 and CTLA-4; LAG-3 and CTLA-4; ICOS and CTLA-4; PD-1 and TIM-3; CD137 and CD20; CD137 and EGFR; CD137 and Her-2; CD137 and PD-1; CD137 and PD-L1; CD137 and CD27; CD137 and GITR; CD137 and HVEM; CD137 and OX40; CD27 and HVEM; CD137 and LAG-3; CD137 and TIM-3; CD137 and VISTA; c-Met and PD-L1; Lag-3 and PD-L1; TIM-3 and PD-L1; EGFR and PD-L1; ICOS and PD-L1; Her2 and PD-L1; PD-L1 and PD-L2; EGFR and PD-1; PD-1 and PD-L1; OX40 and PD-L1; VEGF and PD-L1; LAG-3 and TIM-3; OX40 and PD-1; BTLA and PD-1; VISTA and PD-1; TIM3 and PD-1; LAG-3 and PD-1; LAG-3 and Her-2; LAG-3 and EGFR; LAG-3 and BTLA; LAG-3 and VISTA; OX40 and GITR; OX40 and CD27; OX40 and HVEM; GITR and HYEM; EGFR and DLL-4; VEGF and DLL-4; PDGFR and VEGF; EpCAM and CD3; Her2 and CD3; CD19 and CD3; CD19 and CD20; CEA and EGFR; EpCAM and EGFR; PSMA and PSCA; EphA2 and Her2, EpCAM and CD3; EGFR and Her3; CD16a and CD30; CD30 and PSMA; EGFR and CD3; CEA and CD3; TROP-2 and HSG; TROP-2 and CD3; VEGF and EGFR; HGF and VEGF; VEGF and VEGF (a different epitope); VEGF and HGF; EGFR and cMet; PDGF and VEGF; ICOS and PD-1, Lag-3 and PD-1, CD47 and CD20, RGM A and RGM B; Te38 and TNFα; TNFα and Blys; TNFα and CD-22; TNFα and CTLA-4 domain; TNFα and GP130; TNFα and IL-12p40; and TNFα and RANK ligand.   
     
     
         3 . The binding protein according to  claim 2  capable of binding cMet and EGFR. 
     
     
         4 . A pharmaceutical composition comprising the binding protein according to any one of  claims 1 - 3 , and one or more pharmaceutically acceptable carriers. 
     
     
         5 . The pharmaceutical composition of  claim 4 , wherein the composition further comprises at least one additional therapeutic agent selected from the group consisting of: a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulation molecule blocker; an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof, methotrexate, cyclosporin, rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist. 
     
     
         6 . The binding protein of any one of  claims 1 - 3 , wherein the binding protein is conjugated to an agent selected from the group consisting of: an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. 
     
     
         7 . The binding protein conjugate of  claim 6 , wherein the imaging agent is selected from the group consisting of: a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. 
     
     
         8 . A method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a binding protein, said binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, either (i) VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , or (ii) VH B -CH1-VL A -CL-Fc, wherein CH1 is fused directly to VL A , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A —CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein the binding protein is capable of binding two or more epitopes or antigens related to said disease or disorder, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a first constant domain of a heavy chain, A is a first epitope or antigen, and B is a second epitope or antigen, and wherein A and B are different epitopes of the same antigen or are different antigens; and   wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions, and wherein said binding protein binds both epitope or antigen A and epitope or antigen B.   
     
     
         9 . The method of  claim 8 , wherein the disease or disorder is an inflammatory disease, autoimmune disease, neurodegenerative disease, cancer, sepsis, a metabolic disorder, or spinal cord injury. 
     
     
         10 . The method of  claim 8 , wherein the disease or disorder is selected from the group consisting of: asthma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Alzheimer's disease, and Parkinson's disease. 
     
     
         11 . The method of  claim 8 , wherein the disease or disorder is an autoimmune or inflammatory disease selected from the group consisting of: Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoriatic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, ankylosing spondylitis, spondylosing arthropathy, systemic lupus erythematosus, uveitis, sepsis, neurodegenerative diseases, neuronal regeneration, spinal cord injury, primary and metastatic cancers, a respiratory disorder asthma, allergic and nonallergic asthma, asthma due to infection, asthma due to infection with respiratory syncytial virus (RSV), chronic obstructive pulmonary disease (COPD), a condition involving airway inflammation, eosinophilia, fibrosis and excess mucus production, cystic fibrosis, pulmonary fibrosis, an atopic disorder, atopic dermatitis, urticaria, eczema, allergic rhinitis, allergic enterogastritis, an inflammatory and/or autoimmune condition of the skin, an inflammatory and/or autoimmune condition of gastrointestinal organs, inflammatory bowel diseases (IBD), ulcerative colitis, an inflammatory and/or autoimmune condition of the liver, liver cirrhosis, liver fibrosis, liver fibrosis caused by hepatitis B and/or C virus, scleroderma, tumors or cancers, hepatocellular carcinoma, glioblastoma, lymphoma, Hodgkin's lymphoma, a viral infection, a bacterial infection, a parasitic infection, HTLV-1 infection, suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination. 
     
     
         12 . A method of treating a disease selected from the group consisting of rheumatoid arthritis, psoriasis, psoriatic arthritis, osteoporosis, stroke, liver disease, and oral cancer in a subject in need thereof, the method comprising administering a binding protein to the subject, wherein the binding protein is capable of binding IL-17 and IL-20, said binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, either (i) VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , or (ii) VH B -CH1-VL A -CL-Fc, wherein CH1 is fused directly to VL A , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A —CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a first constant domain of a heavy chain,   wherein A is IL-17 and B is IL-20, or A is IL-20 and B is IL-17; and   wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions.   
     
     
         13 . A method of treating a B cell cancer in a subject in need thereof, the method comprising administering to the subject a binding protein, wherein the binding protein is capable of binding CD3 and CD20, said binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, either (i) VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , or (ii) VH B -CH1-VL A -CL-Fc, wherein CH1 is fused directly to VL A , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A —CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a first constant domain of a heavy chain,   wherein A is CD3 and B is CD20, or A is CD20 and B is CD3; and   wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions.   
     
     
         14 . The method of  claim 13 , wherein the B cell cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), precursor B cell lymphoblastic leukemia/lymphoma, mature B cell neoplasms, B cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicle center lymphoma, marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma. 
     
     
         15 . A method of treating autoimmune or inflammatory disease in a subject in need thereof, the method comprising administering to the subject a binding protein, wherein the binding protein is capable of binding TNF and IL-17, said binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, either (i) VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , or (ii) VH B -CH1-VL A -CL-Fc, wherein CH1 is fused directly to VL A , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A —CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a first constant domain of a heavy chain,   wherein A is TNF and B is IL-17, or A is IL-17 and B is TNF; and   
       wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions. 
     
     
         16 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a binding protein, wherein the binding protein is capable of binding CTLA-4 and PD-1, said binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, either (i) VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , or (ii) VH B -CH1-VL A -CL-Fc, wherein CH1 is fused directly to VL A , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a first constant domain of a heavy chain,   wherein A is CTLA-4 and B is PD-1, or A is PD-1 and B is CTLA-4; and   
       wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions. 
     
     
         17 . The method of  claim 16 , wherein the cancer is melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer, esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, or other neoplastic malignancies. 
     
     
         18 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a binding protein, wherein the binding protein is capable of binding cMet and EGFR, said binding protein comprising three polypeptide chains, wherein:
 the first polypeptide chain comprises, from amino terminus to carboxyl terminus, either (i) VL A -CL-VH B -CH1-Fc, wherein CL is fused directly to VH B , or (ii) VH B -CH1-VL A -CL-Fc, wherein CH1 is fused directly to VL A , and wherein there are no linkers inserted between the variable domains and constant domains;   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A —CH1, wherein there is no linker inserted between VH A  and CH1; and   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, wherein there is no linker inserted between VL B  and CL;   wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a first constant domain of a heavy chain,   wherein A is cMet and B is EGFR, or A is EGFR and B is cMet; and   
       wherein, on expression in a mammalian host cell, two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains associate to provide a monomeric, tetravalent, and bispecific binding protein comprising six polypeptide chains, having four functional Fab binding regions.