Cancer treatment using antibodies that bind cell surface grp78

Abstract

This application provides, inter alia, antibodies or antigen-binding fragments thereof, targeting cell surface GRP78 expressed on tumor cells, tumor endothelial cells, and tumor initiating cancer cells. These anti-GRP78 antibodies, or antigen-binding fragments thereof, have a high affinity for GRP78 and are less immunogenic compared to their unmodified parent antibodies in a given species, e.g., a human, and function to inhibit GRP78. Importantly, these isolated novel antibodies and antigen-binding fragments thereof, attenuate PI3K signaling and promote apoptosis in tumor cells, while leaving normal cells unaffected. The antibodies and antigen-binding fragments are useful for UPR-targeted cancer therapeutic treatments.

1 . An isolated antibody or antigen-binding fragment thereof that binds to human cell surface glucose regulated protein 78 (GRP78)(SEQ ID NO: 1), wherein said antibody or antigen-binding fragment binds to an epitope depicted in SEQ ID NO: 31. 
     
     
         2 . An isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein said antibody or antigen-binding fragment comprises the CDR regions V H CDR 1 , V H CDR 2 , V H CDR 3 , having the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, or a combination of two or three thereof and/or wherein said antibody or antigen-binding fragment comprises CDR regions V L CDR 1 , V L CDR 2 , and V L CDR 3  having the amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, or a combination of two or three thereof. 
     
     
         3 - 6 . (canceled) 
     
     
         7 . An isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of the sequences set forth in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 21 and a light chain variable region selected from the group consisting of the sequences set forth in SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 29. 
     
     
         8 - 13 . (canceled) 
     
     
         14 . An isolated antibody or antigen-binding fragment thereof which competes for binding to the epitope depicted in SEQ ID NO: 31 or SEQ ID NO: 32, with an antibody which comprises the heavy chain variable region sequence set forth in SEQ ID NO: 9 and the light chain variable region sequence set forth in SEQ ID NO: 11. 
     
     
         15 . (canceled) 
     
     
         16 . An isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein the heavy chain variable region includes
 (a) an FR1 selected from the group consisting of amino acids 1-30 of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21 or an FR1 selected from the group consisting of amino acids 1-23 of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29;   (b) an FR2 selected from the group consisting of amino acids 36-49 of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21 or an FR2 selected from the group consisting of amino acids 35-49 of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29;   (c) an FR3 selected from the group consisting of amino acids 67-98 of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21 or an FR3 selected from the group consisting of amino acids 55-86 of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29; and   (d) an FR4 selected from the group consisting of amino acids 109-119 of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21 or an FR4 selected from the group consisting of amino acids 96-105 of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29.   
     
     
         17 . (canceled) 
     
     
         18 . An isolated antibody or antigen-binding fragment thereof according to  claim 1 ,
 wherein said antibody or antigen-binding fragment thereof is less immunogenic in a human subject than the monoclonal antibody which comprises the heavy chain variable region sequence set forth in SEQ ID NO: 9 and the light chain variable region sequence set forth in SEQ ID NO: 11;   wherein said antibody or antigen-binding fragment thereof binds said cell surface GRP78 with a similar or greater binding affinity than the monoclonal antibody which comprises the heavy chain variable region sequence set forth in SEQ ID NO: 9 and the light chain variable region sequence set forth in SEQ ID NO: 11; and/or   wherein said antibody or antigen-binding fragment thereof binds to said cell surface GRP78 with a dissociation constant (K D ) of at least about 1×10- 3  M, at least about 1×10- 4  M, at least about 1×10- 5  M, at least about 1×10- 6  M, at least about 1×10- 7  M, at least about 1×10- 8  M, at least about 1×10- 9  M, at least about 1×10- 10  M, at least about 1×10- 11 M, or at least about 1×10- 12  M.   
     
     
         19 . (canceled) 
     
     
         20 . An isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein said antibody or antigen-binding fragment thereof is a polyclonal antibody, a monoclonal antibody or antigen-binding fragment thereof, a recombinant antibody, a diabody, a chimerized or chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, a single chain antibody, an Fv, an Fd, an Fab, an Fab′, or an F(ab′) 2 , or synthetic or semi-synthetic antibodies. 
     
     
         21 . (canceled) 
     
     
         22 . A method of detecting cancer or confirming the diagnosis of cancer in a subject, comprising: contacting a sample from the subject with the isolated human monoclonal antibody or antigen-binding fragment thereof according to  claim 1 ; and detecting binding of the isolated human monoclonal antibody or antigen-binding fragment to the sample, wherein an increase in binding of the isolated human monoclonal antibody or antigen-binding fragment to the sample as compared to binding of the isolated human monoclonal antibody or antigen-binding fragment to a control sample detects cancer in the subject or confirms the diagnosis of cancer in the subject. 
     
     
         23 . The method of  claim 22 , wherein the cancer is prostate cancer, uterine cancer, breast cancer, myeloid leukemia, lymphatic leukemia, small cell lung cancer, colon cancer, pancreatic cancer, glioma, or head-neck cancer. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 22 , wherein the sample is a blood, urine, biopsy, serum, sputum, plasma, or a cerebral spinal fluid sample. 
     
     
         26 . A method for inhibiting PI3K/AKT signaling in a cell of a human subject or reducing the growth rate of a tumor in a human subject, comprising administering to said cell or tumor an effective amount of an antibody or antigen fragment thereof according to  claim 1  to a cell or tumor that expresses cell surface GRP78. 
     
     
         27 . (canceled) 
     
     
         28 . A method of treating a human subject suffering from cancer, comprising (a) identifying in the subject a tumor having a plurality of cancer cells that express cell surface GRP78; and (b) administering to said subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to  claim 1 , thereby treating the cancer that expresses cell surface GRP78. 
     
     
         29 . A method according to  claim 28 , wherein said cancer is a cancer with high P13K activity selected from the group consisting of prostate cancer, uterine cancer, breast cancer, ovarian cancer, myeloid leukemia, lymphatic leukemia, small cell lung cancer, colon cancer, pancreatic cancer, glioma, and head-neck cancer. 
     
     
         30 . A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to  claim 1  in pharmaceutically acceptable carriers or excipients. 
     
     
         31 . (canceled) 
     
     
         32 . An isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein said antibody or antigen-binding fragment is labeled, wherein said label is a fluorescent label, a radioactive label, or a label having a distinctive nuclear magnetic resonance signature. 
     
     
         33 . An isolated immunoconjugate comprising an antibody or antigen-binding fragment according to  claim 1  linked to an effector molecule. 
     
     
         34 . An immunoconjugate of  claim 33 , wherein the effector molecule is an immunotoxin, cytokine, chemokine, therapeutic agent, or a chemotherapeutic agent. 
     
     
         35 . (canceled) 
     
     
         36 . An isolated or recombinant polynucleotide encoding an antibody or antigen-binding fragment thereof of  claim 1 . 
     
     
         37 . The isolated or recombinant polynucleotide of  claim 36 , wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises the nucleic acid sequence set forth in SEQ ID NO: 22 or a degenerate variant thereof, and the light chain variable region of the antibody or antigen-binding fragment comprises the nucleic acid sequence set forth in SEQ ID NO: 24 or a degenerate variant thereof. 
     
     
         38 . The isolated or recombinant polynucleotide of  claim 36 , operably linked to a heterologous promoter. 
     
     
         39 . An expression vector comprising the isolated or recombinant polynucleotide of  claim 38 . 
     
     
         40 . An isolated host cell transformed with the polynucleotide of  claim 38 .