Novel modulators and methods of use

Abstract

Novel modulators, including antibodies and derivatives thereof, and methods of such modulators to treat hyperproliferative disorders are provided.

1 . An isolated EFNA modulator. 
     
     
         2 . The isolated EFNA modulator of  claim 1 , wherein the EFNA modulator comprises an EFNA antagonist. 
     
     
         3 . The isolated EFNA modulator of  claim 1 , wherein the EFNA modulator comprises an antibody or immunoreactive fragment thereof. 
     
     
         4 . The isolated EFNA modulator of  claim 3  wherein the antibody or immunoreactive fragment thereof comprises a monoclonal antibody. 
     
     
         5 . The isolated EFNA modulator of  claim 4  wherein the monoclonal antibody is selected from the group consisting of chimeric antibodies, CDR-grafted antibodies, humanized antibodies and human antibodies. 
     
     
         6 . The isolated EFNA modulator of  claim 4  wherein said monoclonal antibody comprises a neutralizing antibody. 
     
     
         7 . The isolated EFNA modulator of  claim 4  wherein said monoclonal antibody comprises an internalizing antibody. 
     
     
         8 . The isolated EFNA modulator of  claim 4  wherein said monoclonal antibody comprises a depleting antibody. 
     
     
         9 . The isolated EFNA modulator of  claim 4  wherein said monoclonal antibody comprises an antibody that associates with EFNA4. 
     
     
         10 . The isolated EFNA modulator of  claim 9  wherein said monoclonal antibody comprises a light chain variable region having three complementarity determining regions and a heavy chain variable region having three complementarity determining regions wherein the heavy and light chain complementarity determining regions comprise complementarity determining regions set forth in  FIG. 7A . 
     
     
         11 . The isolated EFNA modulator of  claim 9  wherein said monoclonal antibody comprises a light chain variable region and a heavy chain variable region wherein said light chain variable region comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from the group consisting of amino acid sequences as set forth in SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 163 and wherein said heavy chain variable region comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from the group consisting of amino acid sequences as set forth in SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157 and SEQ ID NO: 161. 
     
     
         12 . The isolated EFNA modulator of  claim 9 ,  10  or  11  further comprising a cytotoxic agent. 
     
     
         13 . A nucleic acid encoding an amino acid heavy chain variable region or an amino acid light chain variable region of  claim 11 . 
     
     
         14 . A vector comprising the nucleic acid of  claim 13 . 
     
     
         15 . A host cell comprising the vector of  claim 14 . 
     
     
         16 . The isolated EFNA modulator of  claim 1  comprising an amino acid sequence as set forth in SEQ ID NO: 2 or a fragment thereof. 
     
     
         17 . The isolated EFNA modulator of  claim 16  wherein the EFNA modulator further comprises at least a portion of an immunoglobulin constant region. 
     
     
         18 . The isolated EFNA modulator of  claim 1  wherein said modulator reduces the frequency of tumor initiating cells upon administration to a subject in need thereof. 
     
     
         19 . The isolated EFNA modulator of  claim 18  wherein the reduction in frequency is determined using flow cytometric analysis of tumor cell surface markers known to enrich for tumor initiating cells. 
     
     
         20 . The isolated EFNA modulator of  claim 18  wherein the reduction in frequency is determined using immunohistochemical detection of tumor cell surface markers known to enrich for tumor initiating cells. 
     
     
         21 . The isolated EFNA modulator of  claim 18  wherein said tumor initiating cells comprise tumor perpetuating cells. 
     
     
         22 . The isolated EFNA modulator of  claim 1  further comprising a cytotoxic agent. 
     
     
         23 . The isolated EFNA modulator of  claim 1  wherein said EFNA modulator comprises a pan-EFNA modulator. 
     
     
         24 . A pharmaceutical composition comprising the isolated EFNA modulator of  claim 1 . 
     
     
         25 . An isolated EFNA4 modulator. 
     
     
         26 . The isolated EFNA4 modulator of  claim 25  wherein said EFNA4 modulator comprises a pan-EFNA4 modulator. 
     
     
         27 . A pharmaceutical composition comprising the isolated EFNA4 modulator of  claim 25 . 
     
     
         28 . A method of treating an EFNA associated disorder comprising administering a therapeutically effective amount of an EFNA modulator to a subject in need thereof. 
     
     
         29 . The method of  claim 28  wherein said EFNA modulator comprises an EFNA antagonist. 
     
     
         30 . The method of  claim 28  wherein said EFNA modulator comprises an antibody or immunoreactive fragment thereof. 
     
     
         31 . The method of  claim 30  wherein the antibody or immunoreactive fragment thereof comprises a monoclonal antibody. 
     
     
         32 . The method of  claim 31  wherein the monoclonal antibody is selected from the group consisting of chimeric antibodies, CDR-grafted antibodies, humanized antibodies and human antibodies. 
     
     
         33 . The method of  claim 32  wherein said monoclonal antibody comprises a light chain variable region having three complementarity determining regions and a heavy chain variable region having three complementarity determining regions wherein the heavy and light chain complementarity determining regions comprise complementarity determining regions set forth in  FIG. 7A . 
     
     
         34 . The method of  claim 31  wherein said monoclonal antibody associates with EFNA4. 
     
     
         35 . The method of  claim 31  wherein said monoclonal antibody comprises a neutralizing antibody. 
     
     
         36 . The method of  claim 31  wherein said monoclonal antibody comprises an internalizing antibody. 
     
     
         37 . The method of  claim 36  wherein said internalizing antibody comprises a cytotoxic agent. 
     
     
         38 . The method of  claim 28  wherein said EFNA associated disorder comprises a hyperproliferative disorder. 
     
     
         39 . The method of  claim 38  wherein said hyperproliferative disorder comprises a neoplastic disorder. 
     
     
         40 . The method of  claim 39  wherein said neoplastic disorder comprises a solid tumor. 
     
     
         41 . The method of  claim 40  wherein neoplastic disorder comprises adrenal cancer, bladder cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, prostate cancer or breast cancer. 
     
     
         42 . The method of  claim 39  wherein said neoplastic disorder comprises a hematologic malignancy. 
     
     
         43 . The method of  claim 42  wherein said hematologic malignancy comprises leukemia or lymphoma. 
     
     
         44 . The method of  claim 39  wherein the subject suffering said neoplastic disorder exhibits tumors comprising tumor initiating cells. 
     
     
         45 . The method of  claim 44  further comprising the step of reducing the frequency of tumor initiating cells in said subject. 
     
     
         46 . The method of  claim 45  wherein the reduction in frequency is determined using flow cytometric analysis of tumor cell surface markers known to enrich for tumor initiating cells or immunohistochemical detection of tumor cell surface markers known to enrich for tumor initiating cells. 
     
     
         47 . The method of  claim 45  wherein the reduction in frequency is determined using in vitro or in vivo limiting dilution analysis. 
     
     
         48 . The method of  claim 47  wherein the reduction in frequency is determined using in vivo limiting dilution analysis comprising transplant of live human tumor cells into immunocompromised mice. 
     
     
         49 . The method of  claim 48  wherein the reduction of frequency determined using in vivo limiting dilution analysis comprises quantification of tumor initiating cell frequency using Poisson distribution statistics. 
     
     
         50 . The method of  claim 47  wherein the reduction of frequency is determined using in vitro limiting dilution analysis comprising limiting dilution deposition of live human tumor cells into in vitro colony supporting conditions. 
     
     
         51 . The method of  claim 50  wherein the reduction of frequency determined using in vitro limiting dilution analysis comprises quantification of tumor initiating cell frequency using Poisson distribution statistics. 
     
     
         52 . The method of  claim 28  further comprising the step of administering an anti-cancer agent. 
     
     
         53 . The method of  claim 28  wherein said EFNA modulator comprises an amino acid sequence as set forth in SEQ ID NO: 2 or a fragment thereof. 
     
     
         54 . The method of  claim 28  wherein said EFNA modulator comprises a pan-EFNA modulator. 
     
     
         55 . A method of reducing the frequency of tumor initiating cells in a subject in need thereof comprising the step of administering an EFNA modulator to said subject. 
     
     
         56 . The method of  claim 55  wherein the tumor initiating cells comprise tumor perpetuating cells. 
     
     
         57 . The method of  claim 56  wherein said tumor perpetuating cells are CD44 +  or CD133 +  cells. 
     
     
         58 . The method of  claim 55  wherein said EFNA modulator comprises an antibody. 
     
     
         59 . The method of  claim 58  wherein said antibody comprises a monoclonal antibody. 
     
     
         60 . The method of  claim 59  wherein said EFNA modulator comprises an anti-EFNA4 antibody. 
     
     
         61 . The method of  claim 55  wherein the subject is suffering from a neoplastic disorder selected from the group consisting of adrenal cancer, bladder cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, prostate cancer and breast cancer. 
     
     
         62 . The method of  claim 55  wherein the subject is suffering from a hematologic malignancy. 
     
     
         63 . The method of  claim 55  wherein the frequency of tumor initiating cells is reduced by at least 10%. 
     
     
         64 . The method of  claim 55  wherein the reduction in frequency is determined using flow cytometric analysis of tumor cell surface markers known to enrich for tumor initiating cells or immunohistochemical detection of tumor cell surface markers known to enrich for tumor initiating cells. 
     
     
         65 . The method of  claim 55  wherein the reduction in frequency is determined using in vitro or in vivo limiting dilution analysis. 
     
     
         66 . A method of treating a subject suffering from a hematologic malignancy comprising the step of administering an EFNA modulator to said subject. 
     
     
         67 . The method of  claim 66  wherein said EFNA modulator is an EFNA4 modulator. 
     
     
         68 . A method of sensitizing a tumor in a subject for treatment with an anti-cancer agent comprising the step of administering an EFNA modulator to said subject. 
     
     
         69 . The method of  claim 68  wherein said EFNA modulator comprises an antibody. 
     
     
         70 . The method of  claim 68  wherein said tumor is a solid tumor. 
     
     
         71 . The method of  claim 68  wherein said anti-cancer agent comprises a chemotherapeutic agent. 
     
     
         72 . The method of  claim 68  wherein said anti-cancer agent comprises an immunotherapeutic agent. 
     
     
         73 . A method of diagnosing a hyperproliferative disorder in a subject in need thereof comprising the steps of:
 a. obtaining a tissue sample from said subject;   b. contacting the tissue sample with at least one EFNA modulator; and   c. detecting or quantifying the EFNA modulator associated with the sample.   
     
     
         74 . The method of  claim 73  wherein the EFNA modulator comprises a monoclonal antibody. 
     
     
         75 . The method of  claim 74  wherein the antibody is operably associated with a reporter. 
     
     
         76 . An article of manufacture useful for diagnosing or treating an EFNA associated disorder comprising a receptacle comprising an EFNA modulator and instructional materials for using said EFNA modulator to treat or diagnose the EFNA associated disorder. 
     
     
         77 . The article of manufacture of  claim 76  wherein said EFNA modulator is a monoclonal antibody. 
     
     
         78 . The article of manufacture of  claim 76  wherein the receptacle comprises a readable plate. 
     
     
         79 . A method of treating a subject suffering from neoplastic disorder comprising the step of administering a therapeutically effective amount of at least one internalizing EFNA modulator. 
     
     
         80 . The method of  claim 79  wherein said EFNA modulator comprises an antibody. 
     
     
         81 . The method of  claim 80  wherein said antibody comprises a monoclonal antibody. 
     
     
         82 . The method of  claim 81  wherein the monoclonal antibody further comprises a cytotoxic agent. 
     
     
         83 . The method of  claim 81  wherein the monoclonal antibody associates with EFNA4. 
     
     
         84 . A method of treating a subject suffering from neoplastic disorder comprising the step of administering a therapeutically effective amount of at least one neutralizing EFNA modulator. 
     
     
         85 . The method of  claim 84  wherein said EFNA modulator comprises an antibody. 
     
     
         86 . The method of  claim 85  wherein said antibody comprises a monoclonal antibody. 
     
     
         87 . The method of  claim 86  wherein said monoclonal antibody comprises an anti-EFNA4 antibody. 
     
     
         88 . The method of  claim 87  wherein said EFNA4 antibody comprises a pan-EFNA antibody. 
     
     
         89 . The method of  claim 84  wherein the neoplastic disorder is selected from the group consisting of adrenal cancer, bladder cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, prostate cancer and breast cancer. 
     
     
         90 . A method of identifying, isolating, sectioning or enriching a population of tumor initiating cells comprising the step of contacting said tumor initiating cells with an EFNA modulator. 
     
     
         91 . The method of  claim 90  wherein said EFNA modulator comprises an antibody. 
     
     
         92 . A composition comprising a humanized antibody variable region substantially similar to a humanized variable region found on an antibody selected from the group consisting of hSC4.5, hSC4.15, hSC4.22 and hSC4.47 and a pharmaceutically acceptable carrier. 
     
     
         93 . An anti-EFNA4 antibody comprising a light chain variable region and a heavy chain variable region wherein said light chain variable region comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from the group consisting of amino acid sequences as set forth in SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 163 and wherein said heavy chain variable region comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from the group consisting of amino acid sequences as set forth in SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157 and SEQ ID NO: 161. 
     
     
         94 . A method inhibiting or preventing metastasis in a subject in need thereof comprising the step of administering a pharmaceutically effective amount of an EFNA modulator. 
     
     
         95 . The method of  claim 94  wherein the subject undergoes a debulking procedure before or after administration of the EFNA modulator. 
     
     
         96 . The method of  claim 94  wherein said debulking procedure comprises the administration of at least one anti-cancer agent. 
     
     
         97 . A method of performing maintenance therapy on a subject in need thereof comprising the step of administering a pharmaceutically effective amount of an EFNA modulator. 
     
     
         98 . The method of  claim 97  wherein said subject was treated for a neoplastic disorder prior to the administration of the EFNA modulator. 
     
     
         99 . A method of depleting tumor cells in a subject suffering from a hyperproliferative disorder comprising the step of administering an EFNA modulator. 
     
     
         100 . The method of  claim 99  wherein said tumor cells comprise tumor initiating cells. 
     
     
         101 . A method of diagnosing, detecting or monitoring an EFNA associated disorder in vivo in a subject in need thereof comprising the step of administering an EFNA modulator.