Bispecific, tetravalent antigen binding proteins

Abstract

The present invention relates to bispecific, tetravalent antigen binding proteins, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

1 - 12 . (canceled) 
     
     
         13 . One or more nucleic acid molecules encoding a bispecific, tetravalent antigen binding protein, wherein the antigen binding protein comprises:
 a) a first heavy chain of a first antibody, wherein said first antibody specifically binds to a first antigen, and wherein said first heavy chain is fused through its C-terminus to the N-terminus of a moiety comprising VH-CH1 domains of said first antibody;   b) two first light chains of said first antibody of a);   c) a second heavy chain of a second antibody, wherein said second antibody specifically binds to a second antigen, wherein said second heavy chain is fused through its C-terminus to the N-terminus of a moiety comprising VH-CL domains of said second antibody, and wherein the CH1 domain of said second heavy chain is replaced by the CL domain of said second antibody; and   d) two second light chains of said second antibody of c),   
       wherein the CL domain of each of said second light chains is replaced by the CH1 domain of said second antibody. 
     
     
         14 . The one or more nucleic acid molecules of  claim 13 , wherein
 i) the CH3 domain of one of the first or second heavy chains is altered,   so that an amino acid residue in the CH3 domain of said first or second heavy chain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain   and   ii) the CH3 domain of the other heavy chain is altered,   so that an amino acid residue in the CH3 domain of the other heavy chain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within which a protuberance as set forth in i) above is positionable.   
     
     
         15 . The one or more nucleic acid molecules of  claim 14 , wherein said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W) and said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V). 
     
     
         16 . The one or more nucleic acid molecules of  claim 15 , wherein
 both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.   
     
     
         17 . The one or more nucleic acid molecules of  claim 13 , wherein the fusion of the first heavy chain and the fusion of the second heavy chain are by linkage with a peptide linker. 
     
     
         18 . The one or more nucleic acid molecules of  claim 17 , wherein the peptide linker comprises (G x S) n G m , wherein x=3; n=3, 4, 5, or 6; and m=0, 1, 2 or 3, or wherein x=4; n=2, 3, 4, or 5; and m=0, 1, 2 or 3. 
     
     
         19 . The one or more nucleic acid molecules of  claim 17 , wherein the peptide linker comprises a sequence of any one of SEQ ID NOs: 5-10. 
     
     
         20 . The one or more nucleic acid molecules of  claim 13 , wherein said first antibody and said second antibody are selected from the group consisting of:
 a) a human IgG1 subclass antibody;   b) a human IgG1 subclass antibody with the mutations L234A and L235A;   c) a human IgG2 subclass antibody;   d) a human IgG3 subclass antibody;   e) a human IgG4 subclass antibody;   f) a human IgG4 subclass antibody with the mutation S228P;   g) an antibody that has reduced FcR binding compared to an IgG1 antibody;   h) an antibody that has reduced FcγRIIIa binding, comprising mutations at P238, D265, D270, N297, P329, L234, L235, G236, G237, 1253, 5254, K288, T307, Q311, N434 and/or H435; and   i) an antibody with the mutations S228P, L234A, L235A, L235E and/or PVA236;   
       numbering according to EU index of Kabat. 
     
     
         21 . The one or more nucleic acid molecules of  claim 13 , wherein said first antigen is Ang-2 and said second antigen is VEGF-A. 
     
     
         22 . One or more nucleic acid molecules encoding a bispecific, tetravalent antigen binding protein that recognizes Ang-2 and VEGF-A, wherein the antigen binding protein comprises:
 a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1;   b) two first light chains, each comprising the amino acid sequence of SEQ ID NO: 2;   c) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 3; and   d) two second light chains, each comprising the amino acid sequence of SEQ ID NO: 4.   
     
     
         23 . One or more vectors comprising the one or more nucleic acid molecules of  claim 13 . 
     
     
         24 . One or more vectors comprising the one or more nucleic acid molecules of  claim 22 . 
     
     
         25 . A host cell comprising the one or more nucleic acids of  claim 22 . 
     
     
         26 . The host cell of  claim 25 , wherein the host cell comprises one or more vectors comprising the one or more nucleic acid molecules. 
     
     
         27 . The host cell of  claim 25 , wherein the host cell is prokaryotic. 
     
     
         28 . The host cell of  claim 25 , wherein the host cell is eukaryotic. 
     
     
         29 . A method for the preparation of a bispecific, tetravalent antigen binding protein, comprising:
 a) culturing the host cell of  claim 25  under conditions that allow synthesis of said antigen binding protein molecule; and   b) recovering said antigen binding protein molecule from said culture.   
     
     
         30 . A bispecific, tetravalent antigen binding protein prepared by the method of  claim 29 . 
     
     
         31 . A bispecific, tetravalent antigen binding protein, comprising:
 a) a first heavy chain of a first antibody, wherein said first antibody specifically binds to a first antigen, and wherein said first heavy chain is fused through its C-terminus to the N-terminus of a moiety comprising VH-CH1 domains of said first antibody;   b) two light chains of said first antibody of a);   c) a second heavy chain of a second antibody, wherein said second antibody specifically binds to a second antigen, wherein said second heavy chain is fused through its C-terminus to the N-terminus of a moiety comprising VH-CL domains of said second antibody, and wherein the CH1 domain of said second heavy chain is replaced by the CL domain of said second antibody; and   d) two second light chains of said second antibody of c),   wherein the CL domain of each of said second light chains is replaced by the CH1 domain of said second antibody;   wherein the first antibody of a) and the second antibody of c) are selected from the group consisting of:   i) a human IgG1 subclass antibody with the mutations L234A and L235A;   ii) a human IgG4 subclass antibody with the mutation S228P;   iii) an antibody that has reduced FcR binding compared to an IgG1 antibody;   iv) an antibody that has reduced FcγRIIIa binding, comprising mutations at P238, D265, D270, N297, P329, L234, L235, G236, G237, 1253, S254, K288, T307, Q311, N434 and/or H435; and   v) an antibody with the mutations S228P, L234A, L235A, L235E and/or PVA236;   numbering according to EU index of Kabat.   
     
     
         32 . A method for the treatment of a patient suffering from cancer, comprising administering to the patient a therapeutically effective amount of a bispecific, tetravalent antigen binding protein comprising:
 a) a first heavy chain of a first antibody, wherein said first antibody specifically binds to a first antigen, and wherein said first heavy chain is fused through its C-terminus to the N-terminus of a moiety comprising VH-CH1 domains of said first antibody;   b) two light chains of said first antibody of a);   c) a second heavy chain of a second antibody, wherein said second antibody specifically binds to a second antigen, wherein said second heavy chain is fused through its C-terminus to the N-terminus of a moiety comprising VH-CL domains of said second antibody, and wherein the CH1 domain of said second heavy chain is replaced by the CL domain of said second antibody; and   d) two second light chains of said second antibody of c),   wherein the CL domain of each of said second light chains is replaced by the CH1 domain of said second antibody.   
     
     
         33 . The method of  claim 32 , wherein the bispecific, tetravalent antigen binding protein is in a composition comprising the antigen binding protein and at least one pharmaceutically acceptable excipient. 
     
     
         34 . The method of  claim 33 , wherein the composition further comprises an adjuvant. 
     
     
         35 . The method of  claim 32 , wherein the bispecific, tetravalent antigen binding protein is administered parenterally. 
     
     
         36 . The method of  claim 32 , wherein said first antigen is Ang-2 and said second antigen is VEGF-A, and wherein the cancer is selected from the group consisting of lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwanoma, ependymona, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewings sarcoma.