Antibody variants having modifications in the constant region

Abstract

The present invention relates to positions in the constant region of antibodies, in particular the CH3 region of IgG4, which affect the strength of CH3-CH3 interactions. Mutations that either stabilize or destabilize this interaction are disclosed.

1 - 91 . (canceled) 
     
     
         92 . A method of treating a disorder, comprising administering to a patient in need thereof a therapeutically effective amount of a monovalent antibody comprising a single heavy chain and a single light chain, wherein the antibody is a human IgG4 isotype comprising a modified heavy chain constant region,
 wherein the heavy chain constant region has been modified relative to the sequence set forth in SEQ ID NO: 4 by one or more of the following amino acid substitutions: Glu (E) in position 225 has been replaced by Ala (A) or Val (V); Ser (S) in position 232 has been replaced by Arg (R); Leu (L) in position 236 has been replaced by Glu (E), Gly (G), Ser (S), or Thr (T); Asp (D) in position 267 has been replaced by Ala (A) or Ser (S); Phe (F) in position 273 has been replaced by Asp (D), Thr (T), Arg (R), Gln (Q), or Tyr (Y); or Tyr (Y) in position 275 has been replaced by Glu (E), Gln (Q), or Lys (K); and   wherein the heavy chain constant region further comprises a hinge region modified such that all amino acid residues in the hinge region which are capable of forming a disulfide bond with an identical constant region in the presence of polyclonal human IgG have been deleted or substituted with other amino acid residues, including modification of all cysteine residues in the hinge region.   
     
     
         93 . The method of  claim 92 , wherein the monovalent antibody is a human antibody. 
     
     
         94 . The method of  claim 92 , wherein the monovalent antibody binds to a target selected from erythropoietin, beta-amyloid, thrombopoietin, interferon-alpha (2a and 2b), interferon-beta (1b), interferon-gamma, TNFR I (CD120a), TNFR II (CD120b), IL-1 R type 1 (CD121a), IL-1 R type 2 (CD121b), IL-2, IL-2R (CD25), IL-2R-beta (CD123), IL-3, IL-4, IL-3R (CD123), IL-4R (CD124), IL-5R (CD125), IL-6R-alpha (CD126), IL-6R-beta (CD130), IL-8, IL-10, IL-11, IL-15, IL-15BP, IL-15R, IL-20, IL-21, TCR variable chain, RANK, RANK-L, CTLA4, CXCR4R, CCR5R, TGF-beta1, TGF-beta2, TGF-beta3, G-CSF, GM-CSF, MIF-R (CD74), M-CSF-R (CD115), GM-CSF-R (CD116), soluble FcRI, soluble FcRII, soluble FcRIII, FcRn, Factor VII, Factor VIII, Factor IX, VEGF, VEGFxxxb, alpha-4 integrin, CD11a, CD18, CD20, CD38, CD79, CD81, FcalphaRI, FcepsilonRI, acetylcholine receptor, fas, fasL, TRAIL, hepatitis C virus, envelope E2 of hepatitis C virus, tissue factor, a complex of tissue factor and Factor VII, EGFr, CD4, CD28, VLA-1, VLA-2, VLA-3, VLA-4, LFA-1, MAC-1, I-selectin, PSGL-1, ICAM-1, P-selectin, periostin, CD33 (Siglec 3), Siglec 8, TNF, CCL1, CCL2, CCL3, CCL4, CCLS, CCL11, CCL13, CCL17, CCL18, CCL20, CCL22, CCL26, CCL27, CX3CL1, LIGHT, EGF, TGFalpha, HGF, PDGF, NGF, C1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL, factor B, a matrix metallo protease (MMP), CD32b, CD200, CD200R, a killer immunoglobulin-like receptor (KIR), NKG2D, a leukocyte-associated immunoglobulin-like receptor (LAIR), ly49, PD-L2, CD26, BST-2, melanoma inhibitor of apoptosis protein (ML-IAP), cathepsin D, CD40, CD40R, CD86, a B cell receptor, c-Met, PD-1, a T cell receptor, erb-B1, erb-B2, erb-B3, erb-B4, ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-A6, ephrin-A7, ephrin-A8, ephrin-B1, ephrin-B2, ephrin-B3, ephrin-B4, ephrin-B5, ephrin-B6, TLR-3, TLR-9, angiopoietin-1, angiopoietin-2, CD30, CD95, an adrenocorticosteroid, insulin, GIP, GLP-1, a thyroid hormone, growth hormone, ACTH, oestrogen, testosterone, anti-diuretic hormone, heparin, EPO, an opiate, morphine, vitamin C, LH, and FSH. 
     
     
         95 . The method of  claim 92 , wherein the monovalent antibody is conjugated to a therapeutic moiety, an immunosuppressant or a radioisotope. 
     
     
         96 . The method of  claim 92 , wherein the monovalent antibody comprises all the listed amino acid substitutions relative to the sequence set forth in SEQ ID NO: 4. 
     
     
         97 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified such that all cysteine residues in the hinge region have been substituted with amino acid residues that have an uncharged polar side chain or a nonpolar side chain. 
     
     
         98 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 by the following amino acid substitutions: Phe (F) in position 273 has been replaced by Asp (D) or Thr (T), and Tyr (Y) in position 275 has been replaced by Glu (E). 
     
     
         99 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 such that Phe (F) in position 273 has been replaced by Asp (D). 
     
     
         100 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 such that Phe (F) in position 273 has been replaced by Thr (T). 
     
     
         101 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 such that Tyr (Y) in position 275 has been replaced by Glu (E). 
     
     
         102 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 by the following amino acid substitutions: Asp (D) in position 267 has been replaced by Ser (S), and Tyr (Y) in position 275 has been replaced by Glu (E), Gln (Q), or Lys (K). 
     
     
         103 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 such that Asp (D) in position 267 has been replaced by Ser (S). 
     
     
         104 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 such that Tyr (Y) in position 275 has been replaced by Glu (E). 
     
     
         105 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 such that Tyr (Y) in position 275 has been replaced by Gln (Q). 
     
     
         106 . The method of  claim 92 , wherein the heavy chain constant region of the monovalent antibody has been modified relative to the sequence set forth in SEQ ID NO: 4 such that Tyr (Y) in position 275 has been replaced by Lys (K). 
     
     
         107 . The method of  claim 92 , wherein the monovalent antibody is administered with one or more pharmaceutically acceptable excipients, diluents, and/or carriers. 
     
     
         108 . The method of  claim 92 , wherein the monovalent antibody is administered alone or in combination with one or more additional therapeutic agents. 
     
     
         109 . The method of  claim 108 , wherein the one or more additional therapeutic agents are selected from one or more antibodies, one or more cytotoxic agents, one or more radiotoxic agents, one or more anti-angiogenic agents, one or more cytokines, one or more growth inhibitory agents, one or more anti-inflammatory agents, one or more disease modifying anti-rheumatic drugs (DMARDs), and one or more immunosuppressants, or any combination thereof. 
     
     
         110 . The method of  claim 108 , wherein the monovalent antibody and the one or more additional therapeutic agents are administered simultaneously. 
     
     
         111 . The method of  claim 110 , wherein the monovalent antibody and the one or more additional therapeutic agents are in the same formulation. 
     
     
         112 . The method of  claim 110 , wherein the monovalent antibody and the one or more additional therapeutic agents are in separate formulations. 
     
     
         113 . The method of  claim 108 , wherein the monovalent antibody and the one or more additional therapeutic agents are administered sequentially. 
     
     
         114 . The method of  claim 113 , wherein the monovalent antibody is administered before administration of the one or more additional therapeutic agents, after administration of the one or more additional therapeutic agents, or both. 
     
     
         115 . The method of  claim 92 , wherein the disorder is selected from a neoplastic disorder or malignancy, an inflammatory or immune disorder, an angiogenic disorder, an allergic disorder, a neurological or neurogenerative disorder, a gastrointestinal disorder, a hepatic disorder, a hematological disorder, a skin disorder, a pulmonary disorder, an endocrine disorder, a vascular disorder, an infectious disease or disorder, a kidney disorder, an ophthalmologic disorder, a cardiac disorder, a circulatory disorder, a metabolic disorder, a bone disorder, a muscle disorder, a connective tissue disorder, a gynecological-obstetrical disorder, a male reproductive disorder, a transplantation-derived disorder, and a viral infection. 
     
     
         116 . The method of  claim 115 , wherein the disorder is a neoplastic disorder or malignancy selected from a leukemia, a lymphoid neoplasm, an ovarian cancer, an endometrial cancer, a lung cancer, a brain cancer, a spinal cord cancer, a breast cancer, an oral cancer, a colorectal cancer, a pancreatic cancer, a head and neck cancer, a kidney cancer, a thymoma, a lung cancer, a skin cancer, a larynx cancer, a liver cancer, a gastric cancer, an esophageal cancer, a prostate cancer, a bladder cancer, a sarcoma, a cancer of the testis, and a parotid tumor, or any metastases thereof. 
     
     
         117 . The method of  claim 115 , wherein the disorder is selected from B-cell chronic lymphocytic leukemia, acute myeloid leukemia, B-cell lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, non-cutaneous T-cell lymphoma, non-small cell lung cancer, glioblastoma, glioma, oral squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, immune mediated cytopenia, HIV infection, AIDS, cystic fibrosis, osteomyelitis, glomerulonephritis, fibrosis, acute respiratory distress syndrome, chorioretinitis, palmoplanar pustulosis, alcoholic hepatitis, acute pancreatitis, transplant rejection, graft-versus-host disease, Alzheimer's disease, Parkinson's disease, myocardial vascular disease, cerebral vascular disease, retinopathy, macular degeneration, cardiovascular disease, post-thrombotic vein wall fibrosis, ischemia reperfusion injury, atherosclerosis, stroke, cerebral aneurysm, asthma, allergic rhinitis, hay fever, atopic dermatitis, eczema, hives, urticaria, allergic conjunctivitis, a seasonal allergy, a nasal allergy, a contact allergy, an ocular allergy, a food or drug allergy, a latex allergy, an insect allergy, an IgA nephropathy, corneal wound healing, a degenerative genetic eye disease, dental caries, periodontitis, rheumatoid arthritis, gout, multiple sclerosis, inflammatory bowel disease, type-1 diabetes, systemic lupus erythematosus, psoriasis, atopic dermatitis, chronic obstructive pulmonary disease, sepsis, hepatitis C virus infection, Crohn's disease, Guillain-Barre syndrome, ulcerative colitis, hemolytic anemia, paroxysmal nocturnal hemoglobinuria, a heart attack, a burn injury, myasthenia gravis, and anti-phospholipid syndrome.