MODIFIED Fc REGION OF ANTIBODY

Abstract

Polypeptides with improved stability as compared to that of a parent polypeptide were successfully obtained by modifying at least one amino acid in a loop region of the antibody Fc region. Furthermore, by combining multiple amino acid modifications in the loop region, polypeptides with maintained or enhanced FcγR-binding activity as well as improved thermal stability, polypeptides with decreased FcγR-binding activity as well as improved thermal stability, and polypeptides with not only improved thermal stability and adjusted FcγR-binding activity but also decreased aggregate content, as compared to those of a parent polypeptide, were successfully obtained.

1 .- 62 . (canceled) 
     
     
         63 . A polypeptide comprising a modified antibody Fc region, wherein the amino acid sequence of the modified antibody Fc region differs from the sequence of a naturally occurring IgG Fc region at one or more positions including at least one position in a loop region of the IgG Fc region, and wherein the thermal stability of the polypeptide is greater than the thermal stability of a control protein identical to the polypeptide except at the at least one position in the loop region. 
     
     
         64 . The polypeptide of  claim 63 , wherein the thermal stability is determined by measuring the melting temperature (Tm) of the polypeptide. 
     
     
         65 . The polypeptide of  claim 63 , wherein the polypeptide binds to a human FcγR receptor with a binding activity that is the same as or greater than the binding activity of the control protein for the human FcγR receptor. 
     
     
         66 . The polypeptide of  claim 63 , wherein the polypeptide binds to a human FcγR receptor with a binding activity that is lower than the binding activity of the control protein for the human FcγR receptor. 
     
     
         67 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 235, 236, 237, 238, 239, 247, 250, 265, 266, 267, 268, 269, 270, 271, 295, 296, 298, 300, 307, 309, 315, 324, 325, 326, 327, 329, 330, 333, 335, 337, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering). 
     
     
         68 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 235, 239, 266, 267, 268, 269, 270, 295, 296, 298, 300, 324, 325, 326, and 330 (all positions by EU numbering). 
     
     
         69 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 266, 268, 269, 270, 295, 300, 324, 326, and 330 (all positions by EU numbering). 
     
     
         70 . The polypeptide of  claim 69 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Ile at position 234,   Ile at position 266,   Gln at position 268,   Asp at position 269,   Glu at position 270,   Met or Leu at position 295,   Glu at position 300,   His at position 324,   Ser or Ala at position 326, and   His or Tyr at position 330.   
     
     
         71 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 235, 239, 267, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         72 . The polypeptide of  claim 71 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Lys or Arg at position 234,   Lys or Arg at position 235,   Lys at position 239,   Pro at position 267,   Met or Lys at position 268,   Phe at position 270,   Met at position 295,   Gly at position 296,   Gly at position 298, and   Gly, His, or Met at position 325.   
     
     
         73 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 295, 326, and 330 (all positions by EU numbering). 
     
     
         74 . The polypeptide of  claim 73 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Met or Leu at position 295,   Ser or Ala at position 326, and   His or Tyr at position 330.   
     
     
         75 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 235, 239, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         76 . The polypeptide of  claim 75 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Lys at position 234,   Lys or Arg at position 235,   Lys or Ser at position 239,   Lys or His at position 268,   Phe or Asp at position 270,   Met at position 295,   Gly at position 296,   Gly at position 298, and   His or Gly at position 325.   
     
     
         77 . The polypeptide of  claim 75 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Lys at position 234,   Lys or Arg at position 235,   Lys at position 239,   Lys at position 268,   Phe at position 270,   Met at position 295,   Gly at position 296,   Gly at position 298, and   His or Gly at position 325.   
     
     
         78 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 235, 239, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         79 . The polypeptide of  claim 78 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Lys at position 234,   Lys or Arg at position 235,   Lys at position 239,   Met at position 295,   Gly at position 296,   Gly at position 298, and   His or Gly at position 325.   
     
     
         80 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 235, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         81 . The polypeptide of  claim 80 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Lys at position 234,   Lys or Arg at position 235,   Lys at position 268,   Phe at position 270,   Met at position 295,   Gly at position 296,   Gly at position 298, and   His or Gly at position 325.   
     
     
         82 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 234, 235, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         83 . The polypeptide of  claim 82 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Lys at position 234,   Lys or Arg at position 235,   Met at position 295,   Gly at position 296,   Gly at position 298, and   His or Gly at position 325.   
     
     
         84 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 247, 250, 307, 309, 315, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering). 
     
     
         85 . The polypeptide of  claim 84 , wherein one or more of the following positions (EU numbering) in the modified antibody Fc region is occupied by the indicated amino acid:
 Val at position 247,   Phe, Ile, Met, Val, Trp, or Tyr at position 250,   Ala, Gln, or Pro at position 307,   Ala, Arg, or Pro at position 309,   Ala at position 315,   His at position 360,   Asp at position 385,   Pro at position 386,   Glu at position 387,   Ser at position 389,   His, Trp, Tyr, or Phe at position 428, and   Lys at position 433.   
     
     
         86 . The polypeptide of  claim 63 , wherein the at least one position is selected from positions 298 and 309 (all positions by EU numbering). 
     
     
         87 . The polypeptide of  claim 86 , wherein one or both of the following positions (EU numbering) in the modified antibody Fc region are occupied by the indicated amino acid:
 Gly at position 298, and   Asp at position 309.   
     
     
         88 . A method of producing the polypeptide of  claim 63 , comprising:
 identifying a first nucleotide sequence encoding a first polypeptide comprising a first antibody Fc region comprising one or more loop regions, wherein the amino acid sequences of the one or more loop regions of the first antibody Fc region are identical to the amino acid sequences of the corresponding loop regions of a naturally occurring IgG Fc region;   generating a DNA comprising a second nucleotide sequence encoding a second polypeptide that differs from the first polypeptide at one or more positions, including one or more positions in at least one loop region;   expressing the DNA, thereby producing the second polypeptide; and   collecting the second polypeptide.   
     
     
         89 . The method of  claim 88 , further comprising measuring the melting temperature of the second polypeptide; determining that the melting temperature of the second polypeptide is higher than the melting temperature of the first polypeptide; and determining, based on the relative melting temperatures of the first and second polypeptides, that the second polypeptide is thermally more stable than the first polypeptide. 
     
     
         90 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 235, 236, 237, 238, 239, 247, 250, 265, 266, 267, 268, 269, 270, 271, 295, 296, 298, 300, 307, 309, 315, 324, 325, 326, 327, 329, 330, 333, 335, 337, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering). 
     
     
         91 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 235, 239, 266, 267, 268, 269, 270, 295, 296, 298, 300, 324, 325, 326, and 330 (all positions by EU numbering). 
     
     
         92 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 266, 268, 269, 270, 295, 300, 324, 326, and 330 (all positions by EU numbering). 
     
     
         93 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 235, 239, 267, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         94 . The method of  claim 88 , wherein the one or more positions are selected from positions 295, 326, and 330 (all positions by EU numbering). 
     
     
         95 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 235, 239, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         96 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 235, 239, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         97 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 235, 268, 270, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         98 . The method of  claim 88 , wherein the one or more positions are selected from positions 234, 235, 295, 296, 298, and 325 (all positions by EU numbering). 
     
     
         99 . The method of  claim 88 , wherein the one or more positions are selected from positions 247, 250, 307, 309, 315, 360, 385, 386, 387, 389, 428, and 433 (all positions by EU numbering). 
     
     
         100 . The method of  claim 88 , wherein the one or more positions are selected from positions 298 and 309 (by EU numbering). 
     
     
         101 . A nucleic acid that encodes a polypeptide comprising a modified antibody Fc region, wherein the amino acid sequence of the modified antibody Fc region differs from the sequence of a naturally occurring IgG Fc region at one or more positions including at least one position in a loop region of the IgG Fc region, and wherein the thermal stability of the polypeptide is greater than the thermal stability of a control protein identical to the polypeptide except at the at least one position in the loop region. 
     
     
         102 . A vector comprising the nucleic acid of  claim 101 . 
     
     
         103 . A host cell comprising the vector of  claim 102 . 
     
     
         104 . A pharmaceutical composition comprising the polypeptide of  claim 63 . 
     
     
         105 . A method for treating an immune-inflammatory disease in a subject in need of such treatment, the method comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 104 . 
     
     
         106 . The method of  claim 105 , wherein the immune-inflammatory disease is selected from rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune bullous dermatosis, autoimmune adrenocortical inflammation, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megalocytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis, autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, Paget's disease, osteoporosis, multiple myeloma, uveitis, acute and chronic spondylitis, gouty arthritis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), psoriasis, Crohn's disease, Basedow's disease, juvenile diabetes, Addison's disease, myasthenia gravis, lenticular uveitis, systemic lupus erythematosus, allergic rhinitis, allergic dermatitis, ulcerative colitis, hypersensitivity, asthma, myodegeneration, cachexia, systemic scleroderma, localized scleroderma, Sjogren's syndrome, Behcet's disease, Reiter's syndrome, type I and type II diabetes, bone resorption disease, graft versus host reaction, ischemia reperfusion injury, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, malgias due to staining, aplastic anemia, hemolytic anemia, sudden thrombocytopenia, Goodpasture's syndrome, Guillain-Barre syndrome, Hashimoto's disease, pemphigus, IgA nephropathy, pollinosis, antiphospholipid antibody syndrome, polymyositis, Wegener's sarcoma, arteritis nodosa, mixed connective tissue disease, and fibromyalgia. 
     
     
         107 . A method for treating cancer in a subject in need of such treatment, the method comprising administering to the subject an effective amount of the pharmaceutical composition of  claim 104 . 
     
     
         108 . The method of  claim 107 , wherein the cancer is selected from pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatoma, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, cholangioma, bladder cancer, ureteral cancer, thyroid cancer, adrenal carcinoma, renal cancer, other glandular tissue cancers, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, or other solid organ tumors.