Structural Variants of Antibodies for Improved Therapeutic Characteristics

Abstract

Substituted humanized, chimeric or human anti-CD20 antibodies or antigen binding fragments and bispecific antibodies or fusion proteins comprising the substituted antibodies or antigen binding fragments are disclosed. They are useful for treatment of B-cell disorders, such as B-cell malignancies and autoimmune diseases, as well as GVHD, organ transplant rejection, and hemolytic anemia and cryoglobulinemia. Substitution of an aspartate residue at Kabat position 101 of CDR3 V H (CDRH3), produces improved therapeutic properties, including decreased dissociation rates and improved CDC activity, apoptosis, B-cell depletion and therapeutic efficacy at very low dosages. Veltuzumab, a humanized antibody that incorporates such sequence variation, exhibits improved therapeutic efficacy compared to similar antibodies of different CDRH3 sequence, allowing therapeutic effect at dosages as low as 200 mg or less, preferably 100 mg or less, preferably 80 mg or less, preferably 50 mg or less, most preferably 30 mg or less of naked antibody administered i.v. or s.c.

What is claimed is: 
     
         1 . A method of treating a disease in a subject comprising:
 a) obtaining a bispecific antibody or antibody fusion protein comprising (i) a first antibody or fragment thereof which is a substituted chimeric, humanized or human anti-CD20 antibody or antigen binding fragment thereof made by a method comprising making one amino acid substitution in the third complementarity determining region (CDR) sequence of the heavy chain of a chimeric, humanized or human anti-CD20 antibody or antigen binding fragment thereof to make a substituted antibody or antigen binding fragment thereof, wherein the antibody is substituted at Kabat position 101 of CDR3 and the substituted antibody or antigen binding fragment thereof has at least one improved characteristic selected from the group consisting of a slower off-rate, slower antigen dissociation rate, higher CDC activity, higher ADCC activity, higher apoptotic activity, greater ability to induce cell death in vitro in the absence of cross-linking and greater ability to kill or inhibit the growth of CD20-positive cells in vivo when administered to a subject with CD20-positive cells and (ii) a second antibody or fragment thereof:   b) administering the bispecific antibody or antibody fusion protein to a subject; and   c) treating the disease in the subject, wherein the disease is selected from the group consisting of B-cell mediated immune disease, autoimmune disease, B-cell lymphoma and leukemia, graft-versus-host disease, organ transplant rejection, immune hemolytic anemia, allosensitization, and cryoglobulinemia.   
     
     
         2 . The method of  claim 1 , wherein the disease is immune thrombocytopenic purpura, systemic lupus erythematosus, Sjögren's syndrome, Evans syndrome, arthritis, arteritis, pemphigus vulgaris, renal graft rejection, cardiac graft rejection, rheumatoid arthritis, Burkitt lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, small lymphocytic lymphoma, diffuse B-cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, Type I diabetes mellitus, GVHD, multiple sclerosis and multiple myeloma. 
     
     
         3 . The method of  claim 1 , further comprising administering at least one therapeutic agent to the subject before, concurrently with or after the administration of the bispecific antibody or antibody fusion protein. 
     
     
         4 . The method of  claim 1 , wherein the bispecific antibody or antibody fusion protein is conjugated to at least one therapeutic agent. 
     
     
         5 . The method of  claim 1 , wherein the bispecific antibody or antibody fusion protein is administered parenterally to the subject at a dosage of 200 mg or less, wherein the administration is effective to treat the B-cell mediated immune disease, autoimmune disease, other B-cell related immune diseases, B-cell lymphomas or leukemias. 
     
     
         6 . The method of  claim 5 , wherein the bispecific antibody or antibody fusion protein is administered to the subject two or more times at an interval of one to three weeks. 
     
     
         7 . The method of  claim 5 , wherein the administration is intravenous or subcutaneous. 
     
     
         8 . The method of  claim 5 , wherein the administration is subcutaneous and wherein subcutaneous administration is more effective than intravenous administration at killing or inhibiting the growth of CD20-positive cells in vivo when administered to a subject with CD20-positive cells. 
     
     
         9 . The method of  claim 5 , wherein administration of the bispecific antibody or antibody fusion protein is effective to deplete peripheral B-cell levels in the subject. 
     
     
         10 . The method of  claim 9 , wherein the administration is effective to deplete peripheral B-cells in the subject with a single dose of 80 mg/m 2 i.v. or 80 mg s.c. 
     
     
         11 . The method of  claim 9 , wherein the administration is effective to deplete peripheral B-cells in the subject at a dosage less than 80 mg/m 2  i.v. or less than 80 mg s.c. when administered at least once, preferably when administered two to four times to the subject. 
     
     
         12 . The method of  claim 1 , further comprising repeating the administration as needed to prevent or treat relapse of the subject. 
     
     
         13 . The method of  claim 1 , wherein the first antibody is veltuzumab and administration is to a subject who is refractory to rituximab. 
     
     
         14 . The method of  claim 4 , wherein the therapeutic agent is selected from the group consisting of a radionuclide, boron, an immunomodulator, a cytokine, a hormone, a hormone antagonist, an enzyme, an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxic drug, a toxin, an angiogenesis inhibitor, an oligonucleotide, an interference RNA, a second antibody or fragment thereof and a combination thereof. 
     
     
         15 . The method of  claim 3 , wherein the therapeutic agent is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, interferon-alpha, interferon-beta interferon-gamma, TNF-alpha and the stem cell growth factor designated “S1 factor”. 
     
     
         16 . The method of  claim 1 , wherein the second antibody or fragment thereof binds to an antigen selected from the group consisting of carbonic anhydrase IX, B7, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-d, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM6, B7, ED-B fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, Ia, insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, NCA-66, NCA-95, NCA-90, Ia, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAIL receptors (R1 and R2), VEGFR, EGFR, P1GF, complement factors C3, C3a, C3b, C5a, C5, and oncogene products, including bcl-2, Kras and cMET. 
     
     
         17 . The method of  claim 1 , wherein the second antibody or fragment thereof is selected from the group consisting of LL1, LL2, RFB4, hA20, 1F5, L243, MN-3, MN-15, L19, G250, and L243 or a fragment of one of these. 
     
     
         18 . The method of  claim 1 , wherein the bispecific antibody or antibody fusion protein is administered to the subject at least twice a week. 
     
     
         19 . The method of  claim 1 , wherein the first antibody is veltuzumab and the second antibody or fragment thereof binds to CD20. 
     
     
         20 . The method of  claim 1 , wherein the first antibody or fragment thereof is veltuzumab or a fragment thereof and the second antibody or fragment thereof is epratuzumab or a fragment thereof. 
     
     
         21 . The method of  claim 1 , wherein the bispecific antibody or antibody fusion protein is administered parenterally to the subject at a dosage of 1 to 10 mg/kg. 
     
     
         22 . The method of  claim 5 , wherein the bispecific antibody or antibody fusion protein is administered parenterally to the subject at a dosage of 100 mg or less. 
     
     
         23 . The method of  claim 5 , wherein the bispecific antibody or antibody fusion protein is administered parenterally to the subject at a dosage of 80 mg or less. 
     
     
         24 . The method of  claim 5 , wherein the bispecific antibody or antibody fusion protein is administered parenterally to the subject at a dosage of 50 mg or less. 
     
     
         25 . The method of  claim 5 , wherein the bispecific antibody or antibody fusion protein is administered parenterally to the subject at a dosage of 30 mg or less.