US2015203589A1PendingUtilityA1

Fusion proteins and methods thereof

59
Assignee: UNIV COLUMBIAPriority: Jul 24, 2012Filed: Jan 23, 2015Published: Jul 23, 2015
Est. expiryJul 24, 2032(~6 yrs left)· nominal 20-yr term from priority
Inventors:Antonio IavaroneAnna LasorellaRaul Rabadan
G01N 33/5759C07K 16/18C07K 2319/00C07K 14/47C07K 14/71C12N 9/12C12Q 1/6886G01N 2333/91205C07K 16/2863C07K 14/82C12Q 2600/158C07K 2319/73C07K 2317/76C07K 16/40C12Y 207/10001C07K 14/435G01N 33/57492C12N 15/62
59
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Claims

Abstract

The invention discloses oncogenic fusion proteins. The invention provides methods for treating gene-fusion based cancers.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . An antibody or antigen-binding fragment thereof, that specifically binds to a purified fusion protein comprising a tyrosine kinase domain of an FGFR protein fused to the TACC domain of a transforming acidic coiled-coil-containing (TACC) protein. 
     
     
         2 . The antibody or antigen-binding fragment of  claim 1 , wherein the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. 
     
     
         3 . The antibody or antigen-binding fragment of  claim 1 , wherein the TACC protein is TACC1, TACC2, or TACC3. 
     
     
         4 . The antibody or antigen-binding fragment of  claim 1 , wherein the fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 
     
     
         5 . The antibody or antigen-binding fragment of  claim 4 , wherein the FGFR1-TACC1 fusion protein comprises the amino acid sequence of SEQ ID NO: 150. 
     
     
         6 . The antibody or antigen-binding fragment of  claim 4 , wherein the FGFR3-TACC3 fusion protein comprises the amino acid sequence of SEQ ID NO: 79, 158, 159, 160, or 161. 
     
     
         7 . A composition for decreasing in a subject the expression level or activity of a fusion protein comprising the tyrosine kinase domain of an FGFR protein fused to the TACC domain of a TACC protein, the composition in an admixture of a pharmaceutically acceptable carrier comprising an inhibitor of the fusion protein. 
     
     
         8 . The composition of  claim 7 , wherein the TACC protein is TACC1, TACC2, or TACC3. 
     
     
         9 . The composition of  claim 7 , wherein the inhibitor comprises an antibody that specifically binds to a FGFR-TACC fusion protein or a fragment thereof; a small molecule that specifically binds to a FGFR protein; a small molecule that specifically binds to a TACC protein; an antisense RNA or antisense DNA that decreases expression of a FGFR-TACC fusion polypeptide; a siRNA that specifically targets a FGFR-TACC fusion gene; or a combination thereof. 
     
     
         10 . The composition of  claim 7 , wherein the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. 
     
     
         11 . The composition of  claim 7 , wherein the FGFR-TACC fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 
     
     
         12 . The composition of  claim 9 , wherein the small molecule that specifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080, LY2874455, or a combination thereof. 
     
     
         13 . A method for decreasing in a subject in need thereof the expression level or activity of a fusion protein comprising the tyrosine kinase domain of an FGFR protein fused to the TACC domain of a TACC protein, the method comprising:
 (a) administering to the subject a therapeutic amount of a composition of  claim 7 ; and   (b) determining whether the fusion protein expression level or activity is decreased compared to fusion protein expression level or activity prior to administration of the composition,   thereby decreasing the expression level or activity of the fusion protein.   
     
     
         14 . A method of decreasing growth of a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of a FGFR fusion molecule inhibitor, wherein the inhibitor decreases the size of the solid tumor and, wherein the FGFR fusion comprises the tyrosine kinase domain of FGFR fused to the TACC domain of TACC. 
     
     
         15 . The method of  claim 14 , wherein the solid tumor comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma. 
     
     
         16 . The method of  claim 14 , wherein the inhibitor comprises an antibody that specifically binds to a FGFR-TACC fusion protein or a fragment thereof; a small molecule that specifically binds to a FGFR protein; a small molecule that specifically binds to a TACC protein; an antisense RNA or antisense DNA that decreases expression of a FGFR-TACC fusion polypeptide; a siRNA that specifically targets a FGFR-TACC fusion gene; or a combination thereof. 
     
     
         17 . The method of  claim 13  or  14 , wherein the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. 
     
     
         18 . The method of  claim 13  or  16 , wherein the fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 
     
     
         19 . The method of  claim 16 , wherein the small molecule that specifically binds to a FGFR protein comprises AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080, LY2874455, or a combination thereof. 
     
     
         20 . A diagnostic kit for determining whether a sample from a subject exhibits a presence of a FGFR fusion, the kit comprising at least one oligonucleotide that specifically hybridizes to a FGFR fusion, or a portion thereof, and wherein the FGFR fusion comprises the tyrosine kinase domain of FGFR fused to the TACC domain of TACC. 
     
     
         21 . The kit of  claim 20 , wherein the oligonucleotides comprise a set of nucleic acid primers or in situ hybridization probes. 
     
     
         22 . The kit of  claim 20 , wherein the oligonucleotide comprises SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, 169, or a combination thereof. 
     
     
         23 . The kit of  claim 21 , wherein the primers prime a polymerase reaction only when a FGFR fusion is present. 
     
     
         24 . The kit of  claim 20 , wherein the determining comprises gene sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. 
     
     
         25 . A diagnostic kit for determining whether a sample from a subject exhibits a presence of a FGFR fusion protein, the kit comprising an antibody that specifically binds to a FGFR fusion protein comprising SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160, or 161, wherein the antibody will recognize the protein only when a FGFR fusion protein is present, and wherein the FGFR fusion protein comprises a tyrosine kinase domain of an FGFR protein fused to the TACC domain of a transforming acidic coiled-coil-containing (TACC) protein. 
     
     
         26 . The kit of  claim 25 , wherein the antibody is directed to an FGFR fusion protein comprising SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160, or 161. 
     
     
         27 . The kit of  claim 20  or  25 , wherein the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. 
     
     
         28 . The kit of  claim 20  or  25 , wherein the FGFR fusion is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 
     
     
         29 . A method for detecting the presence of a FGFR fusion in a human subject, wherein the FGFR fusion comprises the tyrosine kinase domain of FGFR fused to the TACC domain of TACC, the method comprising:
 (a) obtaining a biological sample from the human subject; and   (b) detecting whether or not there is a FGFR fusion present in the subject.   
     
     
         30 . The method of  claim 29 , wherein the detecting comprises measuring FGFR fusion protein levels by ELISA using an antibody directed to SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160, or 161; western blot using an antibody directed to SEQ ID NO: 79, 85, 86, 87, 88, 89, 150, 158, 159, 160, or 161; mass spectroscopy, isoelectric focusing, or a combination thereof. 
     
     
         31 . The method of  claim 29 , wherein the detecting of step (b) comprises detecting whether or not there is a nucleic acid sequence encoding a FGFR fusion protein in the subject. 
     
     
         32 . The method of  claim 31 , wherein the nucleic acid sequence comprises any one of SEQ ID NOS: 1-77, 80-84, or 95-145. 
     
     
         33 . The method of  claim 31 , wherein the detecting comprises using hybridization, amplification, or sequencing techniques to detect a FGFR fusion. 
     
     
         34 . The method of  claim 33 , wherein the amplification uses primers comprising SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, or 169. 
     
     
         35 . The method of  claim 29  or  31 , wherein the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. 
     
     
         36 . The method of  claim 29  or  31 , wherein the FGFR fusion is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 
     
     
         37 . A method of identifying a compound that decreases the oncogenic activity of a FGFR-TACC fusion, the method comprising:
 a) transducing a cell cultured in vitro with FGFR-TACC DNA;   b) contacting a cell with a ligand source for an effective period of time; and   c) determining whether the cells acquire the ability to grow in anchorage-independent conditions, form multi-layered foci, or a combination thereof, compared to cells cultured in the absence of the test compound.   
     
     
         38 . A purified fusion protein comprising the tyrosine kinase domain of an FGFR protein fused to the TACC domain of a transforming acidic coiled-coil-containing (TACC) protein. 
     
     
         39 . The purified fusion protein of  claim 38 , wherein the FGFR protein is FGFR1, FGFR2, FGFR3, or FGFR4. 
     
     
         40 . The purified fusion protein of  claim 38 , wherein the TACC protein is TACC1, TACC2, or TACC3. 
     
     
         41 . The purified fusion protein of  claim 38 , wherein the fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 
     
     
         42 . The purified fusion protein of  claim 38 , wherein the fusion protein comprises SEQ ID NO: 79, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, or SEQ ID NO: 161. 
     
     
         43 . The purified fusion protein of  claim 38 , wherein the fusion protein has a breakpoint comprising at least 3 consecutive amino acids from amino acids 730-758 of SEQ ID NO: 90 and comprising at least 3 consecutive amino acids from amino acids 549-838 of SEQ ID NO: 92. 
     
     
         44 . The purified fusion protein of  claim 38 , wherein the fusion protein has a breakpoint comprising SEQ ID NO: 78, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, or SEQ ID NO:89. 
     
     
         45 . The purified fusion protein of  claim 38 , wherein the fusion protein comprises SEQ ID NO: 150. 
     
     
         46 . The purified fusion protein of  claim 38 , wherein the fusion protein has a breakpoint comprising at least 3 consecutive amino acids from amino acids 746-762 of SEQ ID NO: 146 and comprising at least 3 consecutive amino acids from amino acids 572-590 of SEQ ID NO: 148. 
     
     
         47 . The purified fusion protein of  claim 38 , wherein the fusion protein has a breakpoint comprising SEQ ID NO: 88. 
     
     
         48 . A cDNA encoding a fusion protein comprising the tyrosine kinase domain of FGFR fused to the TACC domain of TACC. 
     
     
         49 . The cDNA of  claim 48 , wherein the FGFR is FGFR1, FGFR2, FGFR3, or FGFR4. 
     
     
         50 . The cDNA of  claim 48 , wherein the TACC is TACC1, TACC2, or TACC3. 
     
     
         51 . The cDNA of  claim 48 , wherein the fusion protein is FGFR1-TACC1, FGFR2-TACC2, or FGFR3-TACC3. 
     
     
         52 . The cDNA of  claim 48 , wherein the cDNA comprises SEQ ID NO: 94. 
     
     
         53 . The cDNA of  claim 48 , where in the cDNA has a breakpoint comprising at least 9 consecutive in-frame nucleotides from nucleotides 2443-2530 of SEQ ID NO: 91 and comprising at least 9 consecutive in-frame nucleotides from nucleotides 1800-2847 of SEQ ID NO: 93. 
     
     
         54 . The cDNA of  claim 48 , where in the cDNA has a breakpoint comprising any one of SEQ ID NOs: 1-77. 
     
     
         55 . The cDNA of  claim 48 , wherein the cDNA comprises SEQ ID NO: 151. 
     
     
         56 . The cDNA of  claim 48 , where in the cDNA has a breakpoint comprising at least 9 consecutive in-frame nucleotides from nucleotides 3178-3228 of SEQ ID NO: 147 and comprising at least 9 consecutive in-frame nucleotides from nucleotides 2092-2794 of SEQ ID NO: 149. 
     
     
         57 . The cDNA of  claim 48 , where in the cDNA has a breakpoint comprising SEQ ID NO: 83. 
     
     
         58 . The cDNA of  claim 48 , comprising a combination of exons 1-16 of FGFR3 spliced 5′ to a combination of exons 8-16 of TACC3, wherein a breakpoint occurs in:
 a) any one of exons 1-16 of FGFR3 and any one of exons 8-16 of TACC3; 
 b) any one of introns 1-16 of FGFR3 and any one of exons 8-16 of TACC3; 
 c) any one of exons 1-16 of FGFR3 and any one of introns 7-16 of TACC3; or 
 d) any one of introns 1-16 of FGFR3 and any one of introns 7-16 of TACC3. 
 
     
     
         59 . The cDNA of  claim 48 , comprising a combination of exons 1-17 of FGFR1 spliced 5′ to a combination of exons 7-13 of TACC1, wherein a breakpoint occurs in any one of exons 1-17 of FGFR3 and any one of exons 7-13 of TACC3. 
     
     
         60 . The cDNA of  claim 48 , comprising a combination of exons 1-18 of FGFR2 spliced 5′ to a combination of exons 1-23 of TACC2.

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