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 method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising 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 ,   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1,   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL,   wherein 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, which antigen or antigens are related to said disease;   wherein VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B;   wherein the binding protein binds to both A and B; and   wherein the binding protein comprises two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains under non-reducing conditions; and   wherein 90% or more of the binding protein in the composition is a single, monomeric, tetravalent, and bispecific antibody as determined by size exclusion chromatography.   
     
     
         2 . The method according to  claim 1 , wherein said disease is selected from the group consisting of: an inflammatory disease, an autoimmune disease, a neurodegenerative disease, a cancer, sepsis, an infection, a metabolic disorder, and a spinal cord injury. 
     
     
         3 . The method according to  claim 2 , wherein the inflammatory disease, autoimmune disease, or neurodegenerative disease is selected from the group consisting of: asthma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Alzheimer's disease, and Parkinson's disease. 
     
     
         4 . The method according to  claim 2 , wherein the binding protein is capable of binding TNF and IL-17, wherein A is TNF or IL-17 and B is TNF or IL-17 and wherein A and B are different, and wherein the autoimmune disease or inflammatory disease is selected from the group consisting of: Crohn's disease, psoriasis, arthritis, multiple sclerosis, ankylosing spondylitis, spondylosing arthropathy, systemic lupus erythematosus, uveitis, neurodegenerative diseases, neuronal regeneration, spinal cord injury, 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, suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination. 
     
     
         5 . The method according to  claim 4 , wherein said psoriasis is plaque psoriasis. 
     
     
         6 . The method according to  claim 4 , wherein said arthritis is selected from the group consisting of: rheumatoid arthritis, psoriatic arthritis, osteoarthritis, and juvenile idiopathic arthritis. 
     
     
         7 . The method according to  claim 2 , wherein the binding protein is capable of binding TNF and IL-17, wherein A is TNF or IL-17 and B is TNF or IL-17 and wherein A and B are different, and wherein the cancer is selected from the group consisting of: a primary cancer, a metastatic cancer, hepatocellular carcinoma, glioblastoma, lymphoma, and Hodgkin's lymphoma. 
     
     
         8 . The method according to  claim 2 , wherein the binding protein is capable of binding TNF and IL-17, wherein A is TNF or IL-17 and B is TNF or IL-17 and wherein A and B are different, and wherein the infection is selected from the group consisting of: a viral infection, a bacterial infection, a parasitic infection, and HTLV-1 infection. 
     
     
         9 . The method according to any one of  claims 4 - 8 , wherein:
 the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:87,   the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:88, and   the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91.   
     
     
         10 . A method of treating or preventing 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 to the subject an effective amount of a composition comprising a binding protein, wherein the binding protein is capable of binding IL-17 and IL-20, and wherein the binding protein comprises 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 ,   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1,   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL,   wherein A is IL-17 or IL-20 and B is IL-17 or IL-20, and wherein A and B are different;   wherein VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B; and   wherein the binding protein comprises two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains under non-reducing conditions; and   wherein 90% or more of the binding protein in the composition is a single, monomeric, tetravalent, and bispecific antibody as determined by size exclusion chromatography.   
     
     
         11 . The method according to  claim 10  wherein:
 the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:15, 
 the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:21, and 
 the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:23. 
 
     
     
         12 . A method of treating or preventing a B cell cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a binding protein, wherein the binding protein is capable of binding CD3 and CD20, and wherein the binding protein comprises 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 ,   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1,   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL,   wherein A is CD3 or CD20 and B is CD3 or CD20, and wherein A and B are different;   wherein VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B; and   wherein the binding protein comprises two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains under non-reducing conditions; and   wherein 90% or more of the binding protein in the composition is a single, monomeric, tetravalent, and bispecific antibody as determined by size exclusion chromatography.   
     
     
         13 . The method according to  claim 12 , 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. 
     
     
         14 . The method according to  claim 12  or  claim 13  wherein:
 the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:41, 
 the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:44, and 
 the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:46. 
 
     
     
         15 . A method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a binding protein, wherein the binding protein is capable of binding CTLA-4 and PD-1, and wherein the binding protein comprises 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 ,   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1,   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL,   wherein A is CTLA-4 or PD-1 and B is CTLA-4 or PD-1, and wherein A and B are different;   wherein VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B; and   wherein the binding protein comprises two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains under non-reducing conditions; and   wherein 90% or more of the binding protein in the composition is a single, monomeric, tetravalent, and bispecific antibody as determined by size exclusion chromatography.   
     
     
         16 . The method according  claim 15  wherein the cancer is selected from the group consisting of: 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, and neoplastic malignancies. 
     
     
         17 . The method according to  claim 15  or  16 , wherein:
 the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:92, 
 the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:95, and 
 the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:97. 
 
     
     
         18 . A method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a binding protein, wherein the binding protein is capable of binding cMet and EGFR, and wherein the binding protein comprises 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 ,   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1,   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL,   wherein A is cMet or EGFR and B is cMet or EGFR, and wherein A and B are different;   wherein VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B; and   wherein the binding protein comprises two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains under non-reducing conditions; and   wherein 90% or more of the binding protein in the composition is a single, monomeric, tetravalent, and bispecific antibody as determined by size exclusion chromatography.   
     
     
         19 . An isolated nucleic acid comprising the coding sequence for a first polypeptide chain 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 ,   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1,   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL,   wherein 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 VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B;   wherein the binding protein binds to both A and B; and   wherein the binding protein comprises two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains under non-reducing conditions.   
     
     
         20 . The isolated nucleic acid according to  claim 19  further comprising a coding sequence for said second polypeptide chain of said binding protein and a coding sequence for said third polypeptide chain. 
     
     
         21 . An expression vector comprising the isolated nucleic acid according to  claim 19 . 
     
     
         22 . The expression vector according to  claim 21 , wherein said expression vector is selected from the group consisting of: pcDNA; pTT; pTT3; pEFBOS; pBV; pJV; pcDNA3.1 TOPO, pEF6 TOPO and pBJ. 
     
     
         23 . An isolated host cell comprising an expression vector according to  claim 21 . 
     
     
         24 . An isolated host cell transfected with nucleic acid encoding three polypeptide chains of a binding protein,
 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 ,   wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1,   wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL,   wherein 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 VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B;   wherein two of said first polypeptide chain, two of said second polypeptide chain, and two of said third polypeptide chain are capable of associating to provide a bispecific, tetravalent 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 said transfected host cell produces said binding protein and 90% or more of the binding protein produced by said transfected host cell is a single, monomeric, tetravalent and bispecific antibody as determined by size exclusion chromatography.   
     
     
         25 . The isolated host cell according to  claim 24 , wherein said host cell is a prokaryotic host cell. 
     
     
         26 . The isolated host cell according to  claim 25 , wherein said prokaryotic host cell is  Escherichia coli.    
     
     
         27 . The isolated host cell according to  claim 24 , wherein said host cell is a eukaryotic host cell. 
     
     
         28 . The isolated host cell according to  claim 27 , wherein said eukaryotic host cell is a mammalian host cell. 
     
     
         29 . The isolated host cell according to  claim 28 , wherein said mammalian host cell is selected from the group consisting of: a 293 cell, a COS cell, an NSO cell, and a CHO cell. 
     
     
         30 . A method of producing a binding protein comprising culturing an isolated host cell according to  claim 24  in culture medium under conditions sufficient to produce the 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 , 
 wherein the second polypeptide chain comprises, from amino terminus to carboxyl terminus, VH A -CH1, 
 wherein the third polypeptide chain comprises, from amino terminus to carboxyl terminus, VL B -CL, 
 wherein 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 VL A  is a light chain variable domain of a first parental antibody that binds A, CL is an antibody light chain constant domain, VH B  is a heavy chain variable domain of a second parental antibody that binds 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 A, and VL B  is a light chain variable domain of said second parental antibody that binds B; 
 wherein the binding protein binds to both A and B; 
 wherein the binding protein comprises two of said first polypeptide chains, two of said second polypeptide chains, and two of said third polypeptide chains under non-reducing conditions; and 
 wherein 90% or more of the binding protein produced by said host cell is a single, monomeric, tetravalent, and bispecific antibody as determined by size exclusion chromatography.