Affinity matured anti-ccr4 humanized monoclonal antibodies and methods of use

Abstract

The present invention provides affinity matured humanized monoclonal antibodies, bi-specific antibodies, antibody conjugates, and fusion proteins that bind to the chemokine receptor CCR4. This antibody is derived from mAb 1567 and recognizes the same epitope. Binding of the antibodies disclosed herein to CCR4 inhibits ligand-mediated activities and is used to treat symptoms of cancer. Moreover, the antibody is used in combination with vaccines to suppress the activity of regulatory T cells.

1 . A recombinant antibody having:
 a. a heavy chain with three CDRs comprising the amino acid sequences GYTFASQW (SEQ ID NO:22), INPGNVNT (SEQ ID NO:27), and STWYRPLDY (SEQ ID NO:30) respectively and a light chain with three CDRs comprising the amino acid sequences QSILYSSNQKNY (SEQ ID NO:26), WASTRE (SEQ ID NO:28), and HQYISSYT (SEQ ID NO:35) respectively;   b. a heavy chain with three CDRs comprising the amino acid sequences GYTFASSW (SEQ ID NO:23), INPGNVNT (SEQ ID NO:27), and STWYRPNDY (SEQ ID NO:31) respectively and a light chain with three CDRs comprising the amino acid sequences QSILYSSNQKNY (SEQ ID NO:26), WASTRE (SEQ ID NO:28), and HQYKSSYT (SEQ ID NO:36) respectively;   c. a heavy chain with three CDRs comprising the amino acid sequences GYTFASSW (SEQ ID NO:23), INPGNVNT (SEQ ID NO:27), and TTRYRPLDY (SEQ ID NO: 32) respectively and a light chain with three CDRs comprising the amino acid sequences QSILYSSNQKNY (SEQ ID NO:26), WASTRE (SEQ ID NO:28), and HQYRSSYT (SEQ ID NO:37) respectively;   d. a heavy chain with three CDRs comprising the amino acid sequences GYTFASQY (SEQ ID NO:24), INPGNVNT (SEQ ID NO:27), and LTYYRPPDY (SEQ ID NO:33) respectively and a light chain with three CDRs comprising the amino acid sequences QSILYSSNQKNY (SEQ ID NO:26), WASTRE (SEQ ID NO:28), and HQYYSSYT (SEQ ID NO:38) respectively; or   e. a heavy chain with three CDRs comprising the amino acid sequences GYTFASAW (SEQ ID NO:25), INPGNVNT (SEQ ID NO:27), and STYYRPLDY (SEQ ID NO: 29) respectively and a light chain with three CDRs comprising the amino acid sequences QSILYSSNQKNY (SEQ ID NO:26), WASTRE (SEQ ID NO:28), and HQYMSSYT (SEQ ID NO:39) respectively;   wherein said antibody hinds human CCR4.   
     
     
         2 . The antibody of  claim 1 , wherein said antibody is monovalent or bivalent. 
     
     
         3 . The antibody of  claim 1 , wherein said antibody is a single chain antibody. 
     
     
         4 . A single chain antibody comprising:
 a. a VH nucleotide sequence comprising SEQ ID NO: 1 and a VL nucleotide sequence comprising SEQ ID NO: 3;   b. a VH nucleotide sequence comprising SEQ ID NO: 5 and a VL nucleotide sequence comprising SEQ ID NO:7;   c. a VH nucleotide sequence comprising SEQ ID NO: 9 and a VL nucleotide sequence comprising SEQ ID NO: 11;   d. a VH nucleotide sequence comprising SEQ ID NO: 13 and a VL nucleotide sequence comprising SEQ ID NO: 15;   e. a VH nucleotide sequence comprising SEQ ID NO: 17 and a VL nucleotide sequence comprising SEQ ID NO: 19;   
     
     
         5 . A single chain antibody comprising
 a. a VH amino acid sequence comprising SEQ ID NO: 2 and a VL amino acid sequence comprising SEQ ID NO: 4;   b. a VH amino acid sequence comprising SEQ ID NO: 6 and a VL amino acid sequence comprising SEQ ID NO: 8;   c. a VH amino acid sequence comprising SEQ ID NO: 10 and a VL amino acid sequence comprising SEQ ID NO: 12;   d. a VH amino acid sequence comprising SEQ ID NO: 14 and a VL amino acid sequence comprising SEQ ID NO: 16; or   e. a VH amino acid sequence comprising SEQ ID NO: 18 and a VL amino acid sequence comprising SEQ ID NO: 20.   
     
     
         6 . The antibody of  claim 1 , wherein said antibody has a binding affinity of about 1.5 nM −1  or less. 
     
     
         7 . The antibody according to any one of  claims 1 - 6  linked to a therapeutic agent. 
     
     
         8 . The antibody of  claim 7 , wherein said therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. 
     
     
         9 . The antibody of  claim 8 , wherein said cytokine is IL-2 or TGF-beta. 
     
     
         10 . The antibody of  claim 1 , wherein said antibody is a bi-specific antibody that also immunospecifically binds to a second antigen. 
     
     
         11 . The antibody of  claim 10 , wherein the second antigen is CA-IX or PD-L1. 
     
     
         12 . A cell producing the antibody of any one of  claims 1 - 11 . 
     
     
         13 . A method of selectively killing a tumor cell comprising contacting said cell with the antibody of any one of  claims 1 - 11 . 
     
     
         14 . The method of  claim 13 , wherein said selective killing occurs by antibody-dependent cellular toxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP). 
     
     
         15 . The method of  claim 14 , wherein said tumor cell expresses CCR4. 
     
     
         16 . The method of  claim 15 , wherein said tumor cell is a T-cell. 
     
     
         17 . A method of decreasing suppressor T-cell activity comprising contacting said cell with the antibody of any one of  claims 1 - 11 . 
     
     
         18 . A method of augmenting an immune response to an antigen comprising contacting a T cell with the antibody of any one of  claims 1 - 11 . 
     
     
         19 . The method of  claim 18 , wherein said antigen is a viral antigen, a bacterial antigen or a tumor associated antigen. 
     
     
         20 . The method of  claim 19 , wherein said viral antigen is HIV. 
     
     
         21 . The method of  claim 18 , wherein said antibody is administered prior to or after exposure to the antigen. 
     
     
         22 . The method of  claim 18 , wherein said administration of said antibody causes an increase in antigen specific T cell activity. 
     
     
         23 . The method of  claim 18 , wherein said administration of said antibody causes an increase in T cell proliferation. 
     
     
         24 . A method of increasing T cell proliferation comprising contacting a T cell with the antibody of any one of  claims 1 - 11 . 
     
     
         25 . A method of reversing regulatory T cell-mediated suppression of effector T cell proliferation comprising contacting a T cell with the antibody of any one of  claims 1 - 11 . 
     
     
         26 . A method of increasing cytokine production or secretion comprising contacting a T cell with the antibody of any one of  claims 1 - 11 . 
     
     
         27 . The method of any of  claims 18  and  22 - 26 , wherein said T-cell is an effector T-cell. 
     
     
         28 . The method of  claim 26 , wherein the cytokine is IFN-gamma. 
     
     
         29 . The method of  claim 26 , wherein secretion of IFN-gamma is increased and wherein secretion of IL-10, IL-4, or TGF-beta is unchanged or decreased. 
     
     
         30 . A method of increasing vaccine efficiency comprising administering to a subject an antibody according to any of  claims 1 - 11  and a vaccine. 
     
     
         31 . The method of  claim 30  wherein said antibody and said vaccine are administered sequentially or concurrently. 
     
     
         32 . The method of  claim 30 , wherein said vaccine is a tumor vaccine, a bacterial vaccine, or a viral vaccine. 
     
     
         33 . A method of treating or alleviating a symptom of cancer, comprising administering to a subject in need thereof a composition comprising an antibody according to any one of  claims 1 - 11 . 
     
     
         34 . The method of  claim 33 , wherein said cancer secretes CCL22. 
     
     
         35 . The method of  claim 33 , wherein said cancer is a solid cancer or a hematologic cancer. 
     
     
         36 . The method of  claim 35 , wherein said hematologic cancer is cutaneous T-cell Lymphoma (CTCL), mycosis fungoides (MF), primary cutaneous anaplastic large cell Lymphoma (cutaneous ALCL), Sezary syndrome, or adult T cell Leukemia/Lymphoma (ATLL). 
     
     
         37 . The method of treating or alleviating a symptom of a cancer, comprising administering to a subject in need thereof a composition comprising a bi-specific antibody according to  claim 10  or  11 , wherein the bi-specific antibody also binds to CA IX or PD-L1. 
     
     
         38 . The method of  claim 37 , wherein the cancer is a solid cancer or a cancer that overexpresses CA IX or PD-L1. 
     
     
         39 . The method of  claim 35  or  37 , were said solid cancer is renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney or stomach cancer. 
     
     
         40 . A method of treating or alleviating a symptom of an autoimmune disease comprising administering to a subject in need thereof a composition comprising an antibody according to any one of  claims 1 - 9 , wherein said antibody is linked to a regulatory T-cell expansion agent. 
     
     
         41 . The method of  claim 40 , wherein said regulatory T-cell expansion agent is a cytokine. 
     
     
         42 . The method of  claim 41 , wherein said cytokine is TGF-beta. 
     
     
         43 . A method of decreasing regulatory T-cell activity comprising contacting a regulatory T-cell with an antibody according to any one of  claims 1 - 9 , wherein said antibody is linked to toxin. 
     
     
         44 . A fusion protein comprising the monoclonal antibody of  claim 1 , or a functional fragment thereof, operably linked to a cytokine. 
     
     
         45 . The fusion protein of  claim 44 , wherein the cytokine is IL-2 or TGF-beta. 
     
     
         46 . The method of increasing T cell proliferation comprising contacting a T cell with the fusion protein of  claim 44 . 
     
     
         47 . The method of  claim 46 , wherein the T cell is a regulatory T cell. 
     
     
         48 . A nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19. 
     
     
         49 . A nucleic acid sequence encoding the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. 
     
     
         50 . A polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. 
     
     
         51 . A vector comprising the nucleic acid  claim 48  or  49 . 
     
     
         52 . A cell comprising the vector of  claim 51 .