Fluorescent chlorotoxin conjugate and method for intra-operative visualization of cancer

Abstract

A chlorotoxin conjugate detectable by fluorescence imaging that allows for intra-operative visualization of cancerous tissues, compositions that include the chlorotoxin conjugate, and methods for using the chlorotoxin conjugate.

1 - 20 . (canceled) 
     
     
         21 . A chlorotoxin conjugate comprising a fluorescent moiety coupled to a chlorotoxin or a variant or fragment thereof, wherein the conjugate has the binding activity of chlorotoxin and is capable of passing across the blood-brain barrier. 
     
     
         22 . The chlorotoxin conjugate of  claim 21 , wherein the fluorescent moiety is a red or near-infrared emitting fluorescent moiety covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         23 . The chlorotoxin conjugate of  claim 21 , wherein the red or near-infrared emitting fluorescent moiety is a cyanine moiety. 
     
     
         24 . The chlorotoxin conjugate of  claim 23 , wherein the red or near-infrared emitting fluorescent moiety is Cy5.5. 
     
     
         25 . The chlorotoxin conjugate of  claim 21 , wherein the conjugate comprises from one to three red or near-infrared emitting fluorescent moieties covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         26 . The chlorotoxin conjugate of  claim 21 , wherein the chlorotoxin, variant, or fragment is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, and recombinant chlorotoxin. 
     
     
         27 . The chlorotoxin conjugate of  claim 26 , wherein the chlorotoxin or variant comprises 36 amino acids. 
     
     
         28 . The chlorotoxin conjugate of  claim 21 , wherein the chlorotoxin conjugate is detectable by fluorescence imaging. 
     
     
         29 . A chlorotoxin conjugate comprising a red or near-infrared emitting fluorescent moiety covalently coupled to a chlorotoxin. 
     
     
         30 . The chlorotoxin conjugate of  claim 29 , wherein the red or near-infrared emitting fluorescent moiety is a cyanine moiety. 
     
     
         31 . The chlorotoxin conjugate of  claim 30 , wherein the cyanine moiety is Cy5.5. 
     
     
         32 . The chlorotoxin conjugate of  claim 29  consisting of from one to three red or near-infrared emitting fluorescent moieties covalently coupled to a chlorotoxin. 
     
     
         33 . The chlorotoxin conjugate of  claim 29 , wherein the chlorotoxin is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, and recombinant chlorotoxin. 
     
     
         34 . The chlorotoxin conjugate of  claim 33 , wherein the chlorotoxin comprises 36 amino acids. 
     
     
         35 . A composition comprising a pharmaceutically acceptable carrier and a chlorotoxin conjugate, wherein the conjugate comprises a fluorescent moiety coupled to a chlorotoxin or a variant or fragment thereof, wherein the conjugate has the binding activity of chlorotoxin and is capable of passing across the blood-brain barrier. 
     
     
         36 . The composition of  claim 35 , wherein the fluorescent moiety is a red or near-infrared emitting fluorescent moiety covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         37 . The composition of  claim 36 , wherein the red or near-infrared emitting fluorescent moiety is a cyanine moiety. 
     
     
         38 . The composition of  claim 37 , wherein the cyanine moiety is Cy5.5. 
     
     
         39 . The composition of  claim 35 , wherein the conjugate comprises from one to three red or near-infrared emitting fluorescent moieties covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         40 . The composition of  claim 35 , wherein the chlorotoxin, variant, or fragment is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, and recombinant chlorotoxin. 
     
     
         41 . The composition of  claim 40 , wherein the chlorotoxin or variant comprises 36 amino acids. 
     
     
         42 . The composition of  claim 35 , wherein the chlorotoxin conjugate is detectable by fluorescence imaging. 
     
     
         43 . A composition comprising a pharmaceutically acceptable carrier and a chlorotoxin conjugate, wherein the chlorotoxin conjugate comprises a red or near-infrared emitting fluorescent moiety covalently coupled to a chlorotoxin, variant, or fragment thereof. 
     
     
         44 . The composition of  claim 43 , wherein the red or near-infrared emitting fluorescent moiety is a cyanine moiety. 
     
     
         45 . The composition of  claim 44 , wherein the cyanine moiety is Cy5.5. 
     
     
         46 . The composition of  claim 43 , wherein the conjugate comprises from one to three red or near-infrared emitting fluorescent moieties covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         47 . The composition of  claim 43 , wherein the chlorotoxin, variant, or fragment is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, and recombinant chlorotoxin. 
     
     
         48 . The composition of  claim 47 , wherein the chlorotoxin or variant comprises 36 amino acids. 
     
     
         49 . The composition of  claim 43 , wherein the chlorotoxin conjugate is detectable by fluorescence imaging. 
     
     
         50 . A method for detecting cancer, the method comprising:
 (a) contacting a tissue of interest with a chlorotoxin conjugate, wherein the chlorotoxin conjugate comprises a fluorescent moiety coupled to a chlorotoxin or a variant or fragment thereof, wherein the chlorotoxin conjugate has the binding activity of chlorotoxin and is capable of passing across the blood brain barrier, and wherein the chlorotoxin conjugate binds a chlorotoxin binding site on a cancer cell; and   (b) detecting the level of binding of the chlorotoxin conjugate in the tissue, wherein an elevated level of chlorotoxin conjugate, relative to normal tissue, is indicative that the tissue is cancerous.   
     
     
         51 . The method of  claim 50 , wherein the fluorescent moiety is a red or near-infrared emitting fluorescent moiety covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         52 . The method of  claim 50 , wherein the red or near-infrared emitting fluorescent moiety is a cyanine moiety. 
     
     
         53 . The method of  claim 52 , wherein the cyanine moiety is Cy5.5. 
     
     
         54 . The method of  claim 50 , wherein the chlorotoxin conjugate comprises from one to three red or near-infrared emitting fluorescent moieties covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         55 . The method of  claim 50 , wherein the chlorotoxin, variant, or fragment is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, and recombinant chlorotoxin. 
     
     
         56 . The method of  claim 55 , wherein the chlorotoxin or variant comprises 36 amino acids. 
     
     
         57 . The method of  claim 50 , wherein the chlorotoxin conjugate is detected by fluorescence imaging. 
     
     
         58 . The method of  claim 50 , wherein the method further comprises differentiating foci of cancers that express chlorotoxin binding sites from non-neoplastic tissue. 
     
     
         59 . The method of  claim 50 , wherein the cancer that expresses chlorotoxin binding sites is selected from the group consisting of gliomas, astrocytomas medulloblastomas, choroids plexus carcinomas, ependymomas, brain tumors, neuroblastomas, head and neck cancers, lung cancers, breast cancers, intestinal cancers, pancreatic cancers, liver cancers, kidney cancers, sarcomas, osteosarcomas, rhabdomyosarcomas, Ewing's sarcoma, carcinomas, melanomas, ovarian cancers, cervical cancers, lymphomas, thyroid cancers, anal cancers, colo-rectal cancers, endometrial cancers, germ cell tumors, laryngeal cancers, multiple myelomas, prostate cancers, retinoblastomas, gastric cancers, testicular cancers, and Wilm's tumor. 
     
     
         60 . The method of  claim 50 , wherein the cancer that expresses chlorotoxin binding sites is selected from the group consisting of gliomas, medulloblastomas, sarcomas, prostate cancer, and intestinal cancer. 
     
     
         61 . The method of  claim 50 , wherein the chlorotoxin conjugate is combined with a pharmaceutically acceptable carrier prior to contacting a tissue of interest. 
     
     
         62 . A method for determining the location of a cancer cell that expresses a chlorotoxin binding site in a patient intra-operatively, the method comprising:
 (a) administering a pharmaceutical composition to a patient, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an amount of a chlorotoxin conjugate sufficient to image a cancer cell that expresses a chlorotoxin binding site in vivo, wherein the chlorotoxin conjugate comprises a fluorescent moiety coupled to a chlorotoxin or a variant or fragment thereof, wherein the chlorotoxin conjugate has the binding activity of chlorotoxin and is capable of passing across the blood brain barrier; and   (b) detecting the level of binding of the chlorotoxin conjugate to determine the location of a cancer cell that expresses a chlorotoxin binding site, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of a cancer cell that expresses a chlorotoxin binding site.   
     
     
         63 . The method of  claim 62 , wherein the method further comprises:
 (c) surgically removing from the patient at least some cells that express a chlorotoxin binding site.   
     
     
         64 . The method of  claim 62 , wherein the level of binding of the chlorotoxin conjugate is measured by fluorescence imaging. 
     
     
         65 . The method of  claim 62 , wherein the fluorescent moiety is a red or near-infrared emitting fluorescent moiety covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         66 . The method of  claim 65 , wherein the red or near-infrared emitting fluorescent moiety is a cyanine moiety. 
     
     
         67 . The method of  claim 66 , wherein the cyanine moiety is Cy5.5. 
     
     
         68 . The method of  claim 62 , wherein the chlorotoxin conjugate comprises from one to three red or near-infrared emitting fluorescent moieties covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         69 . The method of  claim 62 , wherein the chlorotoxin, variant, or fragment is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, and recombinant chlorotoxin. 
     
     
         70 . The method of  claim 69 , wherein the chlorotoxin or variant comprises 36 amino acids. 
     
     
         71 . The method of  claim 62 , wherein the method further comprises differentiating foci of cancers that express chlorotoxin binding sites from non-neoplastic tissue. 
     
     
         72 . The method of  claim 62 , wherein the cancer that expresses chlorotoxin binding sites is selected from the group consisting of gliomas, astrocytomas medulloblastomas, choroids plexus carcinomas, ependymomas, brain tumors, neuroblastomas, head and neck cancers, lung cancers, breast cancers, intestinal cancers, pancreatic cancers, liver cancers, kidney cancers, sarcomas, osteosarcomas, rhabdomyosarcomas, Ewing's sarcoma, carcinomas, melanomas, ovarian cancers, cervical cancers, lymphomas, thyroid cancers, anal cancers, colo-rectal cancers, endometrial cancers, germ cell tumors, laryngeal cancers, multiple myelomas, prostate cancers, retinoblastomas, gastric cancers, testicular cancers, and Wilm's tumor. 
     
     
         73 . The method of  claim 62 , wherein the cancer that expresses chlorotoxin binding sites is selected from the group consisting of gliomas, medulloblastomas, sarcomas, prostate cancer, and intestinal cancer. 
     
     
         74 . The method of  claim 62 , wherein the chlorotoxin conjugate is combined with a pharmaceutically acceptable carrier prior to contacting a tissue of interest. 
     
     
         75 . A method for detecting the presence of a cancer cell in a sample comprising:
 (a) contacting the sample with a chlorotoxin conjugate, wherein the chlorotoxin conjugate comprises a fluorescent moiety and a chlorotoxin or variant or fragment thereof, wherein the conjugate has the binding activity of chlorotoxin and is capable of passing across the blood-brain barrier; and   (b) detecting binding of the chlorotoxin conjugate in the sample, wherein an elevated level of binding indicates the presence of a cancer cell.   
     
     
         76 . The method of  claim 75 , wherein the level of binding of the chlorotoxin conjugate is detected by fluorescence imaging. 
     
     
         77 . The method of  claim 75 , wherein the fluorescent moiety is a red or near-infrared emitting fluorescent moiety covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         78 . The method of  claim 75 , wherein the red or near-infrared emitting fluorescent moiety is a cyanine moiety. 
     
     
         79 . The method of  claim 78 , wherein the cyanine moiety is Cy5.5. 
     
     
         80 . The method of  claim 75 , wherein the chlorotoxin conjugate comprises from one to three red or near-infrared emitting fluorescent moieties covalently coupled to the chlorotoxin or variant or fragment thereof. 
     
     
         81 . The method of  claim 75 , wherein the chlorotoxin, variant, or fragment is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, and recombinant chlorotoxin. 
     
     
         82 . The method of  claim 81 , wherein the chlorotoxin or variant comprises 36 amino acids. 
     
     
         83 . The method of  claim 75 , wherein the method further comprises differentiating foci of cancers that express chlorotoxin binding sites from non-neoplastic tissue. 
     
     
         84 . The method of  claim 75 , wherein the cancer that expresses chlorotoxin binding sites is selected from the group consisting of gliomas, astrocytomas medulloblastomas, choroids plexus carcinomas, ependymomas, brain tumors, neuroblastomas, head and neck cancers, lung cancers, breast cancers, intestinal cancers, pancreatic cancers, liver cancers, kidney cancers, sarcomas, osteosarcomas, rhabdomyosarcomas, Ewing's sarcoma, carcinomas, melanomas, ovarian cancers, cervical cancers, lymphomas, thyroid cancers, anal cancers, colo-rectal cancers, endometrial cancers, germ cell tumors, laryngeal cancers, multiple myelomas, prostate cancers, retinoblastomas, gastric cancers, testicular cancers, and Wilm's tumor. 
     
     
         85 . The method of  claim 75 , wherein the cancer that expresses chlorotoxin binding sites is selected from the group consisting of gliomas, medulloblastomas, sarcomas, prostate cancer, and intestinal cancer. 
     
     
         86 . The method of  claim 75 , wherein the chlorotoxin conjugate is combined with a pharmaceutically acceptable carrier prior to contacting a tissue of interest. 
     
     
         87 . A method for detecting cells expressing a matrix metalloproteinase (MMP-2) protein complex, the method comprising:
 (a) contacting a tissue of interest with a chlorotoxin conjugate having affinity and specificity for matrix metalloproteinase (MMP-2) protein complex, wherein the chlorotoxin conjugate comprises one or more red or near infrared emitting fluorescent moieties covalently coupled to a chlorotoxin; and   (b) measuring the level of binding of the chlorotoxin conjugate, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of a tumor expressing matrix metalloproteinase (MMP-2) protein complex.   
     
     
         88 . A method for treating a cancer that expresses chlorotoxin binding sites, the method comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising the chlorotoxin conjugate and a pharmaceutically acceptable carrier, wherein the chlorotoxin conjugate comprises one or more red or near infrared emitting fluorescent moieties covalently coupled to a chlorotoxin. 
     
     
         89 . A method for treating a tumor expressing a matrix metalloproteinase (MMP-2) protein complex, the method comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising the chlorotoxin conjugate and a pharmaceutically acceptable carrier, wherein the chlorotoxin conjugate comprises one or more red or near infrared emitting fluorescent moieties covalently coupled to a chlorotoxin. 
     
     
         90 . A method for inhibiting invasive activity of cells that express chlorotoxin binding sites, the method comprising administering to cells that express chlorotoxin binding sites an effective amount of a pharmaceutical composition comprising the chlorotoxin conjugate and a pharmaceutically acceptable carrier, wherein the chlorotoxin conjugate comprises one or more red or near infrared emitting fluorescent moieties covalently coupled to a chlorotoxin.