Gene defects and mutant alk kinase in human solid tumors

Patent metadata

Field	Value
Publication number	US-2016186268-A1
Application number	US-201514870154-A
Country	US
Kind code	A1
Filing date	Sep 30, 2015
Priority date	Apr 14, 2006
Publication date	Jun 30, 2016
Grant date	—

Abstract

Novel gene deletions and translocations involving chromosome 2 resulting in fusion proteins combining part of Anaplastic Lymphoma Kinase (ALK) kinase with part of a secondary protein have been identified herein in human solid tumors, e.g. non-small cell lung carcinoma (NSCLC). Secondary proteins include Echinoderm Microtubule-Associated Protein-Like 4 (EML-4) and TRK-Fusion Gene (TFG). The EML4-ALK fusion protein, which retains ALK tyrosine kinase activity, was confirmed to drive the proliferation and survival of NSCLC characterized by this mutation. The invention therefore provides, in part, isolated polynucleotides and vectors encoding the disclosed mutant ALK kinase polypeptides, probes for detecting it, isolated mutant polypeptides, recombinant polypeptides, and reagents for detecting the fusion and truncated polypeptides. The disclosed identification of this new fusion protein enables methods for screening for compounds that inhibit the proteins, and methods for inhibiting the progression of a cancer characterized by the mutant polynucleotides or polypeptides.

First claim

1 .- 20 . (canceled)

21 . A composition, comprising a nucleic acid reagent, a DNA polymerase, and a biological sample from a human having lung cancer, wherein the nucleic acid reagent comprises a primer pair that hybridizes to a polynucleotide encoding a polypeptide with Anaplastic Lymphoma Kinase (ALK) kinase activity, and wherein the polypeptide with ALK kinase activity comprises amino acid residues 1116-1383 of SEQ ID NO: 5.

22 . The composition of claim 21 , wherein said lung cancer is non-small cell lung cancer (NSCLC).

23 . The composition of claim 21 , wherein said biological sample is a serum, pleural effusion, or tissue biopsy sample.

24 . The composition of claim 21 , wherein the polynucleotide is mRNA.

25 . The composition of claim 24 , wherein the mRNA encodes an Echinoderm Microtubule-Associated Protein-Like 4 (EML4)-ALK fusion polypeptide, or a TRK-fused gene (TFG)-ALK fusion polypeptide.

26 . The composition of claim 21 , wherein said polynucleotide is a fusion polynucleotide encoding an ALK fusion polypeptide, wherein the ALK fusion polypeptide comprises at least a portion of a fusion partner and at least a portion of a wild type ALK polypeptide, said portion of a wild type ALK polypeptide comprising amino acid residues 1116-1383 of SEQ ID NO: 5, and wherein the nucleic acid reagent comprises a primer pair that amplifies at least a fragment of said fusion polynucleotide.

27 . The composition of claim 26 , wherein one primer of the primer pair hybridizes to the part of the polynucleotide encoding said portion of the fusion partner, and the other primer of the primer pair hybridizes to the part of the polynucleotide encoding said portion of a wild type ALK.

28 . The composition of claim 27 , wherein the ALK fusion polynucleotide is an EML4-ALK fusion polynucleotide.

29 . The composition of claim 28 , wherein the primer that hybridizes to the EML4 portion of the EML4-ALK fusion polynucleotide hybridizes to exon 6 of the EML4 gene.

30 . The composition of claim 28 , wherein the primer that hybridizes to the EML4 portion of the EML4-ALK fusion polynucleotide hybridizes to exon 13 of the EML4 gene.

31 . The composition of claim 27 , wherein the ALK fusion polynucleotide is a TFG-ALK fusion polynucleotide.

32 . The composition of claim 31 , wherein the primer that hybridizes to the TFG portion of said TFG-ALK fusion polynucleotide hybridizes to exon 3 of the TFG gene.

33 . The composition of claim 27 , wherein the primer that hybridizes to the ALK portion of said ALK fusion polynucleotide hybridizes to exon 20 of the ALK gene.

34 . The composition of claim 26 , wherein said polynucleotide is a fusion polynucleotide encoding an ALK fusion polypeptide, wherein the ALK fusion polypeptide comprises at least a portion of a fusion partner and at least a portion of a wild type ALK polypeptide, said portion of a wild type ALK polypeptide comprising amino acid residues 1116-1383 of SEQ ID NO: 5, and wherein the nucleic acid reagent comprises a nucleic acid probe which hybridizes to the fusion junction of the ALK fusion polynucleotide.

35 . The composition of claim 34 , wherein the ALK fusion polynucleotide is an ALK fusion mRNA.

36 . The composition of claim 35 , wherein the ALK fusion mRNA is an EML4-ALK fusion mRNA.

37 . The composition of claim 36 , wherein the fusion junction is between exon 6 of the EML4 gene and exon 20 of the ALK gene.

38 . The composition of claim 36 , wherein the fusion junction is between exon 13 of the EML4 gene and exon 20 of the ALK gene.

39 . The composition of claim 35 , wherein the ALK fusion polynucleotide is a TFG-ALK fusion mRNA.

40 . The composition of claim 39 , wherein the fusion junction is between exon 3 of the TFG gene and exon 20 of the ALK gene.

41 . A composition comprising fluorescently labeled break-apart probes that are specific to the ALK locus for use in a fluorescence in situ hybridization (FISH) assay, and a biological sample from a human having lung cancer.

42 . The composition of claim 41 , wherein the break apart probes hybridize to genomic DNAs on opposite sides of the breakpoint of the ALK gene.

43 . The composition of claim 41 , wherein the sample is a formalin-fixed, paraffin-embedded lung tissue sample comprising tumor cells.

44 . The composition of claim 41 , wherein said lung cancer is non-small cell lung cancer (NSCLC).

45 . The composition of claim 41 , wherein said biological sample is a serum, pleural effusion, or tissue biopsy sample.

46 . A composition, comprising a nucleic acid reagent and a biological sample from a human having cancer, wherein the nucleic acid reagent comprises a primer pair, wherein the primer pair hybridizes to a polynucleotide encoding an EML4-ALK fusion polypeptide that has ALK kinase activity and comprises amino acid residues 1116-1383 of SEQ ID NO: 5.

47 . A method, comprising detecting an Anaplastic Lymphoma Kinase (ALK) fusion polynucleotide or an ALK gene rearrangement in a biological sample from a human subject having lung cancer, wherein said ALK fusion polynucleotide encodes an ALK fusion polypeptide which comprises a fragment of the wild type ALK of SEQ ID NO: 5.

Assignees

• Cell Signaling Technology Inc

Inventors

• Rikova Klarisa
• Haack Herbert
• Sullivan Laura
• Guo Ailan
• Possemato Anthony
• Macneill Joan
• Gu Ting-Lei
• Yu Jian

Classifications

• A61K31/713

  Double-stranded nucleic acids or oligonucleotides · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• G01N33/6893

  related to diseases not provided for elsewhere · CPC title

• C12Y207/10001

  Receptor protein-tyrosine kinase (2.7.10.1) · CPC title

• G01N2333/912

  transferring phosphorus containing groups, e.g. kinases (2.7) · CPC title