JP2020156481A - Gene defects and mutant alk kinase in human solid tumors - Google Patents

Abstract

To provide a method of detecting the presence of a mutant anaplastic lymphoma kinase (ALK) polynucleotide in a biological sample obtained from a human non-small cell lung carcinoma (NSCLC) patient.SOLUTION: A method comprises detecting the presence, in a sample, of an ALK fusion polynucleotide encoding an ALK fusion polypeptide having ALK kinase activity and having a specific amino acid sequence, where the detection includes performing the polymerase chain reaction (PCR) using a pair of oligonucleotides hybridizing to the ALK portion of the ALK fusion polynucleotide.SELECTED DRAWING: Figure 1A

Description

(Related application)
This application claims the priority of U.S. Patent Application No. (USSN) No. 60 / 792,364, which is currently under review and filed on April 14, 2006, with reference to all of its disclosures herein. Incorporate into.

The present invention generally relates to proteins and genes contained in cancer, and the detection, diagnosis and treatment of cancer.

Many cancers are characterized by abnormal regulation of cellular processes or impaired cell signaling pathways that lead to uncontrolled growth and proliferation of cells. These disorders are often due to altered activity of certain signaling proteins, such as kinases. Typical of these cancers are solid tumors such as non-small cell lung cancer (NSCLC). NSCLC is the leading cause of cancer death in the United States and accounts for about 87% of all lung cancers. There are about 151,000 new cases of NSCLC annually in the United States, and it is estimated that the disease alone will kill more than 120,000 patients annually in the United States alone. See "Cancer Facts and Figures 2005" by the American Association for Cancer Research. Mortality within 2 years of diagnosis is 75% because NSCLC contains three different subtypes and is often detected only after metastasis.

It is well known that deletions and translocations of genes that cause aberrant signaling activity in kinase fusion proteins can directly lead to some cancers. For example, the tyrosine kinase fusion protein, BCR-ABL cancer protein, has been directly demonstrated to be the causative agent of human chronic myelogenous leukemia (CML). The BCR-ABL cancer protein found in at least 90-95% of CML cases is the gene sequence from the c-ABL protein tyrosine kinase on chromosome 9 to the BCR sequence on chromosome 22 that produces the so-called Philadelphia chromosome. Produced by the translocation of. See, for example, Kurzock et al., N. Engl. J. Med. 319: 990-998 (1988). This translocation is also observed in cases of acute lymphocytic leukemia and NSCLC.

Translocations and deletions of genes that result in mutants and fusion proteins associated with various other cancers have been described. For example, Falini et al ,. Blood 99 (2): 409-426 (2002) results in hematologic cancer, including the NPM-ALK fusion found in ALCL (Anaplastic Large Cell Lymphoma). It outlines the known translocations. To date, only gene translocations, deletions and mutated proteins that result in a limited number of lung cancers have been described, including the t (15; 19) translocation associated with Notch 3. See Dang et al., J. Natl. Can. Instit. 92 (16): 1355-1357 (2000). Deletions of expression and / or activity of RNA-binding protein-6 (EML-4) have been found in small and non-small cell lung cancer. See Drabkin et al., Oncogene 8 (16): 2589-97 (1999). However, to date, no translocation or deletion of protein kinases in human NSCLC cancer has been described.

Deletions in ALK kinase expression due to fusion of NPM to ALK in anaplastic large cell lymphoma have been described. See Morris et al., 1994; Shiota et al., 1994. Moesin, non-muscle myosin heavy chain 9 (Tort et al., 2001), clathrin heavy chain (Touriol et al., 2000; Bridge et al., 2001), tropomyosin 3 (TPM3) (Lamant et al., 2000; Bridge et al., 2001), TRK-fusion gene (TGF) (Hernandez et al., Am. J. Path. 160 (4): 1487-1493 (2002)) and description of fusion of ALK to other genes Has been done. In particular, the TGF-ALK fusion has been reported for non-solid lymphoma, but to date this fusion in solid tumors has not been described. The general role of ALK in cancer has been described. See Pulford et al., J. Cell Physiol. 199 (3): 330-358 (2004). However, to date, no description has been made of deletions in EML-4 expression and / or activation.

Identifying mutations in human cancer is the development of novel therapies that target such fusion or mutated proteins, and novel diagnostic methods to identify patients with such gene mutations. Is highly desirable as it can bring about. For example, BCR-ABL has become a therapeutic target for treating leukemia. Recently, a small molecule inhibitor of ABL kinase, Gleevec® (Imatinib mesylate; STI-571), has been approved for the treatment of CML. This drug is the first in a new class of antiproliferative agents designed to interfere with signaling pathways that promote the growth of cancer cells. The development of this drug is the traditional treatment of CML and ALL, chemotherapy and irradiation therapy, which are plagued by well-known side effects and can specifically target the root cause of malignancies. It shows significant progress beyond (often having only limited effects because it cannot). Similarly, reagents and methods for specifically detecting BCR-ABL fusion proteins in patients are described to identify patients who are most likely to respond to targeted inhibitors such as Gleevec®. Has been done.

Thus, identification of novel gene mutants, such as translocations or deletions that result in fusion or mutation proteins involved in the development of solid tumors, including human cancers, especially lung cancers such as NSCLC, and such. There is a need for reagents and methods for the study and detection of fusion proteins. Among other things, the identification of such fusion proteins makes it desirable to enable new methods for selecting patients for targeted therapy and for screening new agents that further inhibit such mutations / fusion proteins. Will do.

(Summary of invention)
According to the present invention, a novel gene deletion mutation that results in a fusion protein in which the anaplastic lymphoma kinase (ALK) portion is bound to a secondary protein that occurs on human chromosome 2 is a human solid tumor, non-small cell lung cancer. Identified in (NSCLC). Secondary proteins involved in ALK fusion include microtubule-associated protein-like 4 (EML-4) and TRK-fusion gene (TFG). Mutant / fused ALK kinase is currently being observed in samples from patients with non-small cell lung cancer.

Thus, the invention is, in part, isolated polynucleotides and vectors encoding the disclosed mutant / fused ALK polypeptides, probes and assays to detect them, isolated mutant / fused ALK polypeptides. , Recombinant mutant polypeptide, and mutant ALK polynucleotides and reagents for detecting the polypeptide. The disclosure of these novel mutant ALK kinases and translocations / deletions is a novel method for determining the presence of mutant ALK polynucleotides or polypeptides in biological samples, screening for compounds that inhibit the mutant kinase protein. These are also provided by the present invention, enabling methods and methods of inhibiting the development of cancer characterized by the expression of mutant ALK polynucleotides or polypeptides.
That is, the present invention relates to the following (1) to (43).
(1) (a) Echinoderm Microtubule-Associated Protein-Like 4 / Anaplastic Lymphoma Kinase (EML4-ALK) containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18. A nucleotide sequence encoding a fusion polypeptide;
(B) A nucleotide sequence encoding an EML4-ALK fusion polypeptide, the nucleotide sequence containing the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 19;
(C) The N-terminal amino acid sequence of EML-4 (residue 1-233 of SEQ ID NO: 3 or residue 1-495 of SEQ ID NO: 3) and the kinase domain of ALK (residue 1116-1383 of SEQ ID NO: 5). Nucleotide sequence encoding the EML4-ALK fusion polypeptide contained;
(D) Contains the N-terminal nucleotide sequence of EML-4 (1-700 nucleotides of SEQ ID NO: 4 or 1-1486 nucleotides of SEQ ID NO: 4) and the kinase domain nucleotide sequence of ALK (3348-4149 nucleotides of SEQ ID NO: 6). Nucleotide sequence;
(E) A nucleotide sequence containing at least 6 adjacent nucleotides comprising a fusion junction of an EML4-ALK fusion polynucleotide (700-701 nucleotides of SEQ ID NO: 2 or 1486-1487 nucleotides of SEQ ID NO: 19);
(F) A polypeptide containing at least 6 adjacent amino acids comprising a fusion junction of an EML4-ALK fusion polypeptide (residue 233-234 of SEQ ID NO: 1 or residue 495-996 of SEQ ID NO: 18). Encoding nucleotide sequence; and nucleotide sequence homologous to any of the nucleotide sequences of (g) (a) to (f):
An isolated polynucleotide containing a nucleotide sequence having at least 95% homology to a sequence selected from the group consisting of.
(2) An isolated polynucleotide that hybridizes to the polynucleotide according to (1) above under stringent hybridization conditions, and the hybridizing isolated polynucleotide is stringent hybridization. An isolated polynucleotide that does not hybridize to a nucleotide having a nucleotide sequence containing only A or T residues under conditions.
(3) The isolated polynucleotide according to (2) above, wherein the polynucleotide further contains a detectable label.
(4) A method for forming a recombinant vector, which comprises inserting the isolated nucleic acid molecule according to (1) above into a vector.
(5) A recombinant vector prepared by the method according to (4) above.
(6) A method for producing a recombinant host cell, which comprises introducing the recombinant vector according to (5) above into a host cell.
(7) A recombinant host cell formed by the method according to (6) above.
(8) The method comprises culturing the recombinant host cell according to (7) above and recovering the polypeptide under conditions suitable for expression of the EML4-ALK fusion polypeptide. A method for making a recombinant EML4-ALK fusion polypeptide or a cleaved active ALK polypeptide.
(9) (a) Amino acid sequence encoding an EML4-ALK fusion polypeptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18;
(B) The N-terminal amino acid sequence of EML-4 (residue 1-233 of SEQ ID NO: 3 or residue 1-495 of SEQ ID NO: 3) and the kinase domain of ALK (residue 1116-1383 of SEQ ID NO: 5). The amino acid sequence encoding the EML4-ALK fusion polypeptide contained; and (c) the fusion junction of the EML4-ALK fusion polypeptide (residue 233-234 of SEQ ID NO: 1 or residue 495 of SEQ ID NO: 18). Amino acid sequence encoding a polypeptide containing at least 6 adjacent amino acids, including 496):
An isolated polypeptide containing an amino acid sequence having at least 95% homology to a sequence selected from the group consisting of.
(10) A recombinant EML4-ALK fusion polypeptide or a cleaved active ALK polypeptide prepared using the recombinant vector according to (5) above or the recombinant host cell according to (7) above.
(11) It specifically binds to or detects the EML4-ALK fusion polypeptide according to (9) above, but does not bind to or binds to either wild-type EML-4 or wild-type ALK. An isolated reagent that is not detected.
(12) The isolated reagent according to (11) above, wherein the reagent is an antibody or a heavy isotope-labeled (AQUA) peptide.
(13) The isolated reagent according to (11) above, wherein the reagent is a polymerase chain reaction (PCR) probe or a fluorescence in situ hybridization (FISH) probe.
(14) The heavy isotope-labeled (AQUA) peptide according to (12) above, wherein the peptide contains an amino acid sequence at a fusion junction of an EML4-ALK fusion polypeptide or a cut point of a wild-type ALK.
(15) The method is a process:
(A) Obtaining a biological sample from mammalian cancer; and (b) at least one detecting a fused polynucleotide or its encoded fusion polypeptide containing a portion of ALK and a portion of secondary protein. Using a reagent to determine if an ALK mutant polynucleotide and / or its encoded mutant ALK polypeptide is present in the biological sample:
A method for detecting the presence of a mutated ALK polynucleotide or a mutated ALK polypeptide encoded by the mutant ALK polynucleotide in a biological sample obtained from a mammalian cancer.
(16) The method according to (15) above, wherein the cancer is a solid tumor, sarcoma or carcinoma.
(17) The method according to (16) above, wherein the carcinoma is lung cancer.
(18) The method according to (17) above, wherein the lung cancer is non-small cell lung cancer (NSCLC).
(19) The method according to (15) above, wherein the mutant ALK polypeptide is a fusion polypeptide containing residues 11116-1383 of ALK (SEQ ID NO: 5) and a portion of the secondary protein.
(20) The method according to (15) or (16) above, wherein the secondary protein is selected from the group consisting of EML-4 (SEQ ID NO: 3) and TRK-fusion gene (TFG) protein (SEQ ID NO: 22). ..
(21) The fusion polypeptide contains residue 1-233 or residue 1-495 of EML-4 (SEQ ID NO: 3), or residue 1-138 of TFG (SEQ ID NO: 22). 20) The method according to.
(22) The method according to (15) above, wherein the fusion polynucleotide contains an EML4-ALK fusion polynucleotide (SEQ ID NO: 2 or 19) or a TFG-ALK fusion polynucleotide (SEQ ID NO: 21).
(23) The method according to (15) above, wherein the fusion polypeptide contains an EML4-ALK fusion polypeptide (SEQ ID NO: 1 or 18) or a TFG-ALK fusion polypeptide (SEQ ID NO: 20).
(24) The method according to (15) above, wherein the fusion polynucleotide is the fusion polynucleotide according to (1) above.
(25) The method according to (15) above, wherein the fusion polypept is the fusion polypept according to (9) above.
(26) The method according to (15) above, wherein the reagent contains the polynucleotide according to (1) above and / or at least one reagent according to (11) above.
(27) The reagent specifically binds to or detects a TFG-ALK fusion polypeptide (SEQ ID NO: 20) or TFG-ALK fusion polynucleotide (SEQ ID NO: 21), but wild-type TFG or wild. The method according to (15) above, which contains an isolated reagent that does not bind to or detect any of the types of ALK.
(28) The method according to (27) above, wherein the reagent is an antibody or a heavy isotope labeled (AQUA) peptide.
(29) The method according to (27) above, wherein the reagent is a polymerase chain reaction (PCR) probe or a fluorescence in situ hybridization (FISH) probe.
(30) The method according to (28) above, wherein the heavy isotope-labeled (AQUA) peptide contains an amino acid sequence at a fusion junction of a TGF-ALK fusion polypeptide or a cut point of a wild-type ALK.
(31) The method according to (15) above, wherein the method is performed in an assay format of flow cytometry (FC), immunohistochemical examination (IHC), or immunofluorescence (IF).
(32) The method according to (15) above, wherein the method is carried out in the assay format of fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR).
(33) The method according to (15) above, which detects the activity of the ALK fusion polypeptide.
(34) The method comprises the step of determining whether the compound inhibits the expression and / or activity of the ALK fusion polypeptide in the cancer, wherein the compound is characterized by the expression of the ALK fusion polypeptide. A method of determining whether or not to inhibit the development of solid tumors in mammals.
(35) The method according to (34) above, wherein the ALK fusion polypeptide contains residues 11116-1383 of ALK (SEQ ID NO: 5) and a portion of the secondary protein.
(36) The method according to (34) above, wherein the secondary protein is selected from the group consisting of EML-4 (SEQ ID NO: 3) and TRK-fusion gene (TFG) protein (SEQ ID NO: 22).
(37) The fusion polypeptide contains residues 1-233 or 1-495 of EML-4 (SEQ ID NO: 3), or residues 1-138 of TFG (SEQ ID NO: 22). 36).
(38) Inhibition of expression and / or activity of the ALK fusion polypeptide is at least one reagent and / or at least one reagent according to (11) above that detects the polynucleotide according to (1) above. Or the method according to (34) above, which is determined using at least one reagent for detecting a TFG-ALK fusion polynucleotide or polypeptide.
(39) A method for inhibiting the progression of a cancer expressing an EML4-ALK fusion polypeptide, wherein the method comprises a step of inhibiting the expression and / or activity of the EML4-ALK fusion polypeptide in the cancer.
(40) A method for inhibiting the development of a solid tumor expressing a TFG-ALK fusion polypeptide, wherein the method comprises a step of inhibiting the expression and / or activity of the TFG-ALK fusion polypeptide in the cancer. ..
(41) The method according to (39) or (40) above, wherein the cancer or the solid tumor is lung cancer.
(42) The method according to (41) above, wherein the lung cancer is non-small cell lung cancer (NSCLC).
(43) The expression and / or activity of the EML4-ALK fusion polypeptide or the TFG-ALK fusion polypeptide is inhibited by a composition containing WHI-131 and / or WHI-154 or an analog thereof. The method according to (39) or (40) above.
Aspects and embodiments of the present invention will be described in more detail below.

FIG. 1A shows the positions of the EML-4 and ALK genes on chromosome 2 (above), the full-length EML-4 and ALK protein domain positions, and the EML4-ALK fusion protein (short variant) domain position. (Figure below); a fusion junction occurs at amino acids 233-234, and the fusion protein contains the kinase domain of ALK (transmembrane and non-cellular domains). The DNA (and protein) sequences of the EML4 exon 6 / intron 6 / ALK exon 20 fusion junction region (SEQ ID NO: 7 and SEQ ID NO: 8, respectively) are also shown (figure below). FIG. 1B shows the positions of the EML-4 and ALK genes on chromosome 2 (above), the full-length EML-4 and ALK protein domain positions, and the EML4-ALK fusion protein (long variant) domain position. (Figure below); fusion junctions occur at amino acids 495-496, and fusion proteins contain the kinase domain of ALK (transmembrane and non-cellular domains). The DNA (and protein) sequences of the EML4 exon 13 / ALK exon 20 fusion junction region (SEQ ID NO: 24 and SEQ ID NO: 25, respectively) are also shown (figure below). FIG. 1C shows the positions of the TFG gene on chromosome 6 and the ALK gene on chromosome 2 (upper figure), the domain positions of the full-length TFG and ALK proteins, and the domain positions of the TFG-ALK fusion protein (lower figure). The fusion junction occurs at amino acids 138-139, and the fusion protein contains the kinase domain of ALK (transmembrane and non-cellular domains). The DNA (and protein) sequences of the TFG exon 3 / ALK exon 20 fusion junction region (SEQ ID NO: 26 and SEQ ID NO: 27, respectively) are also shown (figure below). FIG. 2A is an amino acid sequence (1 character code) (SEQ ID NO: 1) (upper figure) of a human EML4-ALK fusion protein (short mutant), which encodes a DNA sequence (SEQ ID NO: 2) (lower figure). ) Is also shown. The residue of the EML-4 moiety is italic, while the kinase domain of ALK is bold. FIG. 2B shows the amino acid sequence (1 character code) (SEQ ID NO: 18) (upper figure) of the human EML4-ALK fusion protein (long mutant), and the encoding DNA sequence (SEQ ID NO: 19) (lower figure). ) Is also shown. The residue of the EML-4 moiety is italic, while the kinase domain of ALK is bold. FIG. 2B is an amino acid sequence (1 character code) (SEQ ID NO: 18) (upper figure) of a human EML4-ALK fusion protein (long mutant), which encodes a DNA sequence (SEQ ID NO: 19) (lower figure). ) Is also shown. The residue of the EML-4 moiety is italic, while the kinase domain of ALK is bold. FIG. 2C shows the amino acid sequence (1 character code) (SEQ ID NO: 20) (upper figure) of the human TFG-ALK fusion protein, and also shows the encoding DNA sequence (SEQ ID NO: 21) (lower figure). .. The residue of the TFG moiety is italic, while the kinase domain of ALK is bold. FIG. 3A-3B is an amino acid sequence (1 character code) (SEQ ID NO: 3) (SwissProt Accession No.061936) of the human EML-4 protein, which encodes a DNA sequence (SEQ ID NO: 4) (GeneBank Accession No. NM019063). ) Is also shown. The residues retained in the short mutant of the deleted mutant are underlined, while the residues conserved in the long mutant are italic. 3A-3B is the amino acid sequence (1 character code) (SEQ ID NO: 3) (SwissProt Accession No.061936) of the human EML-4 protein, which encodes the DNA sequence (SEQ ID NO: 4) (GeneBank Accession No. NM019063). ) Is also shown. The residues retained in the short mutant of the deleted mutant are underlined, while the residues conserved in the long mutant are italic. 3A-3B is the amino acid sequence (1 character code) (SEQ ID NO: 3) (SwissProt Accession No.061936) of the human EML-4 protein, which encodes the DNA sequence (SEQ ID NO: 4) (GeneBank Accession No. NM019063). ) Is also shown. The residues retained in the short mutant of the deleted mutant are underlined, while the residues conserved in the long mutant are italic. FIG. 4A-4B shows the amino acid sequence (1 character code) (SEQ ID NO: 5) (SwissProt Accession No. Q9UM73) of human ALK kinase, which also encodes the DNA sequence (SEQ ID NO: 6) (GeneBank Accession No. HSU66559). Shown. The residues retained in the deletion mutant are underlined, while the residues in the kinase domain are bold. FIG. 4A-4B shows the amino acid sequence (1 character code) (SEQ ID NO: 5) (SwissProt Accession No. Q9UM73) of human ALK kinase, which also encodes the DNA sequence (SEQ ID NO: 6) (GeneBank Accession No. HSU66559). Shown. The residues retained in the deletion mutant are underlined, while the residues in the kinase domain are bold. FIG. 4A-4B shows the amino acid sequence (1 character code) (SEQ ID NO: 5) (SwissProt Accession No. Q9UM73) of human ALK kinase, which also encodes the DNA sequence (SEQ ID NO: 6) (GeneBank Accession No. HSU66559). Shown. The residues retained in the deletion mutant are underlined, while the residues in the kinase domain are bold. FIG. 4C-4D shows the amino acid sequence (1 character code) (SEQ ID NO: 22) (SwissProt Accession No. Q92734) of the human TFG protein, and also the encoding DNA sequence (SEQ ID NO: 23) (GeneBank Accession No. NM006070). Shown. The residues retained in the deletion mutant are underlined. FIG. 4C-4D shows the amino acid sequence (1 character code) (SEQ ID NO: 22) (SwissProt Accession No. Q92734) of the human TFG protein, and also the encoding DNA sequence (SEQ ID NO: 23) (GeneBank Accession No. NM006070). Shown. The residues retained in the deletion mutant are underlined. FIG. 5 shows (A) detection of ALK by 5'RACE generation with ALK primers after the second round of PCR; UAP shows Universal Amplification Primer and GSP is Gene Specific. Primer), which represents (B) detection of fusion genes formed by EML-4 and ALK-deficient variants by RT-PCR. FIG. 5 shows (C) detection of the EML4-ALK fusion gene (short and long mutants) in a human NSCLC tumor sample by 5'RACE, and (D) TFG- in a human NSCLC tumor sample by 5'RACE. A gel representing the detection of an ALK fusion gene. FIG. 6 was formed by translocation of EML-4 and ALK in H2228 cells by FISH assay using bicolor (orange / green) degradation probes containing probes on both sides of the ALK gene cleavage point 2p23. An image showing the detection of a fusion gene; the size and position of the probe is shown in the figure above.

(Detailed description of the invention)
According to the present invention, deletions and translocations of previously unknown genes that result in mutated fusion proteins in which the anaplastic lymphoma kinase (ALK) portion is bound to the secondary protein portion are non-human solid tumors. It was identified in small cell lung cancer (NSCLC). The discovered secondary proteins involved in ALK fusion include microtubule-associated protein-like 4 (EML-4) and TRK-fusion gene (TFG).

The two deletions of the present disclosure that occur between EML4 and the ALK gene on chromosome 2 are those in which the N-terminus of EML-4, a microtubule binding protein of 401 amino acids, is a membrane tyrosine kinase of 1620 amino acids. It produces a fusion protein that binds to the domain and the C-terminus of ALK. The resulting EML4-ALK fusion protein, which is 796 amino acids (short mutants) and 1059 amino acids (long mutants), respectively and retains ALK kinase activity, proliferates a human solid tumor subset, including NSCLC. And is thought to promote survival.

The translocations of the present disclosure that occur between the TFG gene on chromosome 6 and the ALK gene on chromosome 2 are the kinase domain and ALK in which the N-terminus of TFG, a 400 amino acid protein, is a membrane tyrosine kinase of 1620 amino acids. Produces a fusion protein that binds to the C-terminus of. The resulting TFG-ALK fusion protein is an amino acid of 701 and has been previously observed in non-solid human lymphoma (Hernadez et al. (2002), supra.), But has not yet been described for solid tumors. The TFG-ALK fusion protein retains ALK kinase activity and is thought to promote the growth and survival of a subset of human solid tumors, including NSCLC.

Several gene translocations or deletions that result in aberrant fusion proteins, including the t (15; 19) translocation for Notch 3 (Dang et al., Supra.), Have been described in NSCLC, but are disclosed herein. EML4-ALK deletion mutants and fusion proteins are novel. Similarly, translocation variants and fusion proteins of TFG-ALK are known in non-solid tumors such as lymphoma, but novel in the solid tumor NSCL. EML-4 is a microtubule-related protein expressed in most human tissues. To date, no defects in EML-4 expression and / or activity have been reported. ALK is a membrane tyrosine kinase that is expressed in humans in the brain and CNS tissues, as well as in the small intestine and testis, but not in normal lymphocytes. It plays an important role in the normal development and function of the nervous system (Iwahara et al., 1997).

Abnormal ALK expression and / or activity have been found in large cell anaplastic lymphomas and neuroblastomas (see Morris et al., 1994, Osajima-Hakomori et al., 2005). Fusion of ALK to moesin, non-muscle myosin heavy chain 9, clathrin heavy chain, tropomicin 3 (TPM3), TRK fusion gene (TFG), and other genes has been described. See Tort et al .; Tourio et al., Hernadez et al., Supra. Interestingly, the disclosed fusion of EML-4 to ALK (short variant) is exactly as wild-type ALK (1058 amino acids), as already described for other ALK fusion mutants. Occurs in the same position.

As described in more detail below, the EML4-ALK-deficient mutant and the expressed fusion protein are isolated, sequenced, and a cDNA expressing the fusion protein is produced. Thus, the invention, in part, produces isolated polynucleotides encoding EML4-ALK fusion polypeptides, nucleic acid probes that hybridize to such polynucleotides, and recombinant mutant ALK polypeptides. Methods, vectors and host cells for utilizing such polynucleotides for use are provided. The present invention is, in part, specific to an isolated polypeptide comprising an amino acid sequence encoding an EML4-ALK fusion polypeptide, a recombinant mutant polypeptide, and an EML4-ALK fusion polypeptide. Also provided is an isolated reagent that binds to and / or detects it, but does not bind to or detect either wild-type EML-4 or wild-type ALK. These aspects of the invention, described in more detail below, are among other things, further studies of cancer mechanisms promoted by the expression / activity of mutant ALK kinase, solid tumors (eg, carcinomas including lung cancers and cancers and cancers). Sarcoma) and other cancers characterized by ALK deletions and translocation mutations and / or fusion proteins in the disclosure, and in the practice of the methods of the invention as further described below.

Deletions and gene deletions and translocation mutations of novel ALK kinase are important in the potential diagnosis and treatment of solid tumors, such as NSCLC, characterized by one or more of these fusion proteins. It has meaning. For example, NSCLC is often detected only after it has metastasized, so the mortality rate within 2 years of diagnosis is 75%. Therefore, it is highly desirable to identify patients with gene mutations that cause NSCLC as soon as possible.

Therefore, the discovery of EML4-ALK fusion proteins (short or long variants) resulting from gene deletion and TFG-ALK fusion proteins resulting from gene translocations, which are thought to promote the growth and survival of solid tumors (NSCLC), is An important novel method for accurately identifying solid mammalian solid tumors, including lung cancer (such as NSCLC), and other cancers in which ALK fusion proteins (such as EML4-ALK or TFG-ALK) are expressed. To enable. These cancers are most likely to respond to inhibitors of the kinase activity of the mutant ALK protein, such as WHI-131 or WHI-154. Identifying cancers promoted by mutant ALK protein kinases as soon as possible can be very helpful in clinically determining which therapy or combination therapy is best suited for a particular patient, and in fact cancers. It will help avoid the formulation of inhibitors that target other kinases that are not the major signaling molecules that promote.

Accordingly, the invention provides, in part, a method of detecting the presence of ALK mutant polynucleotides and / or fusion polypeptides in cancer using the fusion-specific and mutant-specific reagents of the invention. To do. Such methods can be performed to identify solid tumors, such as NSCLC, which appear to respond, for example, to inhibitors of the protein's ALK kinase activity. The invention also provides, in part, a method of determining whether a compound inhibits the progression of cancer characterized by an EML4-ALK fusion polypeptide. Furthermore, the present invention provides a method of inhibiting the development of a solid tumor expressing an EML4-ALK fusion polypeptide or a TFG-ALK fusion polypeptide by inhibiting the expression and / or activity of the mutant polypeptide. Such a method is described in detail below.

Further aspects, benefits and specific embodiments of the present invention are described in more detail below. All references cited herein are incorporated herein by reference in their entirety.

(Definition)
In the present specification, the following terms have the meaning of notation.
"Antibodies" (s) include all types of immunoglobulins, including IgG, IgM, IgA, and IgE, including _Fabs or antigen recognition fragments thereof, including chimeric, polyclonal and monoclonal antibodies. Shown. As used herein, the term "humanized antibody" refers to an antibody in which the amino acids in the non-antigen binding region have been replaced so as to closely resemble a human antibody while retaining its original binding capacity.

The term "biologically active" refers to a protein that has a structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, "immunologically active" provides an appropriate animal or cell-specific immune response of a natural, recombinant, or synthetic EML4-ALK or TFG-ALK fusion polypeptide, or oligopeptides thereof. Shows the ability to induce and bind to specific antibodies.

The term "biological sample" is used in its broad sense and contains ALK fusion polynucleotides or polypeptides or fragments thereof (including fusion polynucleotides and polypeptides of EML4-ALK and TFG-ALK). Means any biological sample that is presumed to be, and in cells, chromosomes isolated from cells (eg, nucleic acids of mid-mitotic chromosomes), genomic DNA (in solution or on solid supports for Southern analysis, etc.) (Binding), RNA (binding to solid support in solution or for Northern analysis), cDNA (binding to solid support in solution or), extract from cells, blood, urine, bone marrow , Or tissue or the like may be contained.

"Characterized by" for cancer and mutant ALK polynucleotides and polypeptides is a gene deletion or translocation and / or a polypeptide for expressed ALK such a gene deletion and / or fusion polypeptide. Indicates a cancer that is present as compared to a cancer that is not present. The presence of mutant polypeptides, in whole or in part, may promote the growth or survival of such cancers.

The "consensus sequence" has been rearranged to remove unwanted bases or stretched in the 5'and / or 3'directions using XL-PCR (registered trademark, Perkin Elmer, Norwalk, Conn.). Rearranged or constructed with duplicate sequences of two or more Insight clones using the GELVIEW® fragment assembly system (GCG, Madison, Wis.), Or stretched and constructed. The nucleic acid sequence is shown.

An "ALK kinase inhibitor" is an expression or activity of wild or truncated ALK kinase alone and / or as part of a fusion protein (such as an EML4-ALK fusion protein or a TFG-ALK fusion protein). , A composition that contains one or more chemical or biological compounds that inhibit either directly or indirectly.

"Derivatization" refers to a nucleic acid sequence encoding a disclosed fusion polynucleotide or a chemical modification of the encoded polypeptide itself. An example of such a modification would be the substitution of hydrogen with an alkyl group, an acyl group, or an amino group. Nucleic acid derivatives will encode polypeptides that retain the essential biological properties of the natural molecule.

"Detectable labels" for a polypeptide, polynucleotide, or reagent disclosed herein include, but are not limited to, fluorescence, mass, residues, stains, radioisotopes, labels, or tag modifications. It means a chemical, biological or other modification that can include and thereby detect the presence of the molecule of interest.

"Expression" or "expressed" for an ALK fusion polypeptide in a biological sample means that the fusion polypeptide is significantly expressed as compared to a control sample in which it is not significantly expressed.

"High isotope-labeled peptide" (used synonymously with AQUA peptide) means a peptide containing at least one heavy isotope label, which will be discussed further below, WO 03. / 016661, "Absolute Quantification of Proteins and Modified Forms by Multi-Step Mass Spectrometry" (Gygi et al.), Suitable for absolute quantification or detection of proteins. With respect to such AQUA peptides, the term "specifically detected" is a polypeptide that does not contain an AQUA peptide sequence by detecting and quantifying only polypeptides and proteins in which the peptide contains an AQUA peptide sequence. And means that the protein will not be detected substantially.

"Isolated" (or "substantially purified") refers to a nucleic acid or amino acid sequence taken from their natural environment and isolated or isolated. They preferably do not contain at least 60%, more preferably 75%, and most preferably 90% or more of the other components they were naturally bound to.

"Mimicking" refers to a molecule whose structure results from the structural recognition of the ALK fusion polypeptide or protein thereof and can affect some or all of the translocation effects on the protein-like molecule.

A "mutated ALK" or "fusion" polynucleotide or polypeptide is a fusion polynucleotide or polypeptide containing an ALK and a secondary protein (eg, EML-4 or TFG), as described herein. Means a polypeptide.

A "polynucleotide" (or "nucleotide sequence") is an oligonucleotide, a nucleotide or a polynucleotide, and a fragment or portion thereof, and a single or double strand, which may represent a sense or antisense strand, a genome. Alternatively, it indicates synthetically derived DNA or RNA.

A "polypeptide" (or "amino acid sequence") refers to an oligopeptide, peptide, polypeptide, or protein sequence, and a fragment or portion thereof, and indicates a natural or synthetic molecule. Here, when "amino acid sequence" is described herein to indicate the amino acid sequence of a natural protein molecule, "amino acid sequence" and similar terms such as "polypeptide" or "protein". Does not mean limiting the amino acid sequence to the complete, original amino acid sequence associated with the protein molecule described.

The "EML4-ALK fusion polynucleotide" is of either natural, synthetic, semi-synthetic, or recombinant origin from any species, particularly mammals including cows, sheep, pigs, rats, horses and preferably humans. The nucleic acid sequences of substantially purified EML4-ALK deletion mutant genes or fusion polynucleotides (short or long variants), as described herein, are shown.

The "EML4-ALK fusion polypeptide" is of either natural, synthetic, semi-synthetic, or recombinant origin from any species, particularly mammals, including bovine, sheep, pig, rat, horse and preferably human. The amino acid sequence of the substantially purified EML4-ALK fusion polypeptide (short or long variant) described herein, obtained from.

A "TFG-ALK fusion polynucleotide" is of either natural, synthetic, semi-synthetic, or recombinant origin from any species, particularly mammals, including bovines, sheep, pigs, rats, horses and preferably humans. The nucleic acid sequence of the product or fusion polynucleotide of the translocation mutant gene of TFG-ALK that is substantially purified, as described herein, is shown.

The "TFG-ALK fusion polypeptide" is of either natural, synthetic, semi-synthetic, or recombinant origin from any species, particularly mammals including bovine, sheep, pig, rat, horse and preferably human. The amino acid sequence of the substantially purified TFG-ALK fusion polypeptide described herein, obtained from.

The term "specifically binds" (or "specific binding" or "specific binding") to the interaction of an antibody with a protein or peptide means that the interaction has a specific structure on the protein (ie, an antigenic determinant). Or an epitope); in other words, it means that an antibody recognizes and binds to a specific protein structure rather than a general protein. The term "non-binding" with respect to the binding of an antibody to a sequence or antigenic determinant other than the specific one means that the antibody does not react substantially as compared to the binding of the antibody to a specific antigenic determinant or sequence. Means.

The term "stringent condition" for sequence or probe hybridization conditions is about T _m minus 5 ° C (5 ° C lower than the melting temperature (T _m ) of the probe or sequence) to about 20 ° C to 25 ° C lower than T _m. It is the "severity" that occurs within the range of. Typical stringent conditions are: 50% formamide, 5-fold SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5-fold denaturation. After culturing overnight at 42 ° C. in a solution containing Denhardt's solution, 10% dextran sulfate, and 20 micrograms / ml of denatured, sheared salmon sperm DNA, the filtrate was 0. Wash in 1x SSC. As will be appreciated by those skilled in the art, stringent conditions of hybridization can be modified to identify or detect homologous or related polynucleotide sequences.

A "mutant" of a mutant ALK polypeptide indicates an amino acid sequence in which one or more amino acids have been modified. The variants have "conservative" changes and the substituted amino acids have similar structural or chemical properties (eg, replace leucine with isoleucine). More rarely, the mutant has "non-conservative" changes, such as the replacement of glycine with tryptophan. Similar minor mutations include amino acid deletions and / or insertions. Guidance on which amino acid to replace, insert or delete without losing biological or immunological activity can be found using computer programs known in the art, such as DNASTAR software. right.

A. Identification of Mutant ALK Kinases in Human Solid Tumors Disclosed herein, which results in the expression of two fusion protein variants that occur on chromosome 2 and bind the N-terminus of EML-4 to the kinase domain and the C-terminus of ALK. A novel human gene deletion has been surprisingly identified during extensive phosphopeptide profile experiments in non-small cell lung cancer (NSCLC) cell lines (including H2228) and solid tumor extracts of patients. It was. A solid tumor, NSCLC is a subtype of lung cancer. The proteins contained in these deletion fusions are shown in the figure above Figure 1A-1B.

Phosphorylation profiles of H2228 cell lines are characterized by a recently described technique for isolating modified peptides from complex mixtures and mass spectrometric analysis (Rush et al., US Patent Application Publication No. 20030044848, "From Complex Mixtures". Using immunoaffinity isolation of modified peptides (see "IAP" technique)), it was revealed for the first time as further described in Example 1 below. The application of IAP technology using a phosphotyrosine-specific antibody (CELL SIGNALING TECHNOLOGY, INC., Beverly, MA, 2003/04 Cat. # 9411) is that the H2228 cell line expresses ALK kinase, but this protein is clearly not cleaved. Was clarified. This screen identified many other activated kinases in cell lines, including some known to be activated in lung cancer. Analysis of sequence 5'to ALK by 5'RACE then revealed that this kinase was fused to the N-terminus of EML-4 (see Figure 6).

Subsequent experiments with 154 cancer samples from NSCLC patients using similar extensive phosphoprofiling means not only confirmed the presence of EML4-ALK (short mutant) mutations in these patient populations, but also others. The presence of a second EML4-ALK (long mutant) and TFG-ALK mutation in the patient population was also revealed (see Examples 1B and 1C).

It can be confirmed that the mutant ALK protein promotes cell proliferation and survival in these NSCLC cancers by inhibiting the cells using siRNA silencing (see Example 3).

The EML4-ALK fusion gene (short and long variants) and the TFG-ALK fusion gene were amplified by PCR, isolated and sequenced (see Example 3). As is apparent from the figure below FIG. 1A-1B, this deletion of EML4-ALK is due to the N-terminus of wild-type EML-4 (short mutant amino acids 1-123, or long mutant amino acids 1-. Either 495) is bound to the wild-type ALK kinase domain and C-terminus (amino acids 1057-1620) (see also SEQ ID NOs: 3 and 5). The fusion junction occurs exactly from the C-terminus of the wild-type ALK to the transmembrane domain (see Figure 1A-1B). The EML4-ALK fusion polypeptide carries the N-terminal amino acid 233 or 495 of EML-4, respectively, which contains the coiled coil domain of this protein. The resulting EML4-ALK fusion protein consisting of 796 amino acids (short variants) or 1059 amino acids (long variants) retains the kinase activity of ALK. The relevant exons and fusion joints are shown in the figure below Figure 1A-1B. The fusion junction comprises an exon 6 followed by an EML-4 intron 6 (short variant) or an EML-4 exon 13 (long variant).

As shown in the figure below FIG. 1C, the TFG-ALK translocation mate the N-terminus of wild-type TFG (amino acids 1-138) with the kinase domain and C-terminus of wild-type ALK (amino acids 1057-1620). Combine (see also SEQ ID NOs: 22 and 5; and the figures below FIG. 1C and FIG. 4C (SEQ ID NOs: 20 and 1)). The fusion junction occurs exactly from the C-terminus of the wild-type ALK to the transmembrane domain (see Figure 1C) and retains the kinase activity of ALK. The relevant exons and fusion joints are shown in Figure 1C (below). The fusion junction comprises exon 3 of TFG and exon 20 of ALK.

A FISH probe was used to detect the presence of an EML4-ALK (short variant) fusion protein in a group of 400 paraffin-embedded human NSCLC samples (Examples 6 and 7; see FIG. 6). The incidence of mutations in this short variant at this sample size was very low. However, in another group of 154 patient-derived frozen human NSCLC cancer samples, TFG-ALK fusion proteins were detected at higher expression rates using IAP techniques that test a broader phosphorylation profile (Example 1B). Please refer to).

B. An isolated polynucleotide.
The present invention, in part, to produce an isolated polynucleotide encoding an EML4-ALK fusion polypeptide, a nucleotide probe that hybridizes to such a polynucleotide, and a recombinant fusion polypeptide. Methods, vectors, and host cells for using the same polynucleotide are provided.

Unless otherwise indicated, all nucleotide sequences determined by sequencing of DNA molecules herein are determined using an automated DNA sequencer (such as Applied Biosystems Inc.'s model 373) and are herein determined. All amino acid sequences of the polypeptide encoded by the DNA molecule determined in are determined using an automated peptide sequencer (see Example 2). As is known in the art for DNA sequences determined by this automated means, any nucleotide sequence determined herein may also contain some error. Nucleotide sequences determined by automation generally have at least about 90% homology to the actual nucleotide sequence of the sequenced DNA molecule, and more typically at least about 95% to at least about. It has 99.9% homology. The actual sequence can be more accurately determined by other means, including a manual of DNA sequencing methods known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence was sequenced as a predicted amino acid sequence encoded by the determined nucleotide sequence. In the translation of the nucleotide sequence, it will result in a frame shift starting from such an insertion or deletion position, as is completely different from the amino acid sequence actually encoded by the DNA molecule.

Unless otherwise indicated, each nucleotide sequence shown herein is represented as a sequence of deoxyribonucleotides (abbreviated as A, G, C, T). However, if each thymidine deoxyribonucleotide (T) in a particular deoxyribonucleotide sequence is replaced with ribonucleotide uridine (U), then for the DNA molecule or polynucleotide, the deoxyribonucleotide sequence, and the RNA molecule or polynucleotide. , Nucleotide molecule, or "nucleotide sequence" of a polynucleotide is intended the corresponding sequence of ribonucleotides (A, G, C and U). For example, a reference to an RNA molecule having the sequence of SEQ ID NO: 2 shown using the abbreviation of deoxyribonucleotide is such that each deoxyribonucleotide A, G or C of SEQ ID NO: 2 has a corresponding ribonucleotide A, G or It is intended to indicate an RNA molecule that is substituted with C and has a sequence in which each deoxyribonucleotide T is substituted with ribonucleotide U.

In one aspect, the invention
(A) Microtubule-associated protein-Associated Protein-LIke 4 / Anaplastic Lymphoma Kinase (EML4-ALK) fusion polypeptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18. The nucleotide sequence encoding
(B) A nucleotide sequence encoding an EML4-ALK fusion polypeptide, the nucleotide sequence containing the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 19;
(C) The N-terminal amino acid sequence of EML-4 (residue 1-233 of SEQ ID NO: 3 or residue 1-495 of SEQ ID NO: 19) and the kinase domain of ALK (residue 1116-1383 of SEQ ID NO: 5). Nucleotide sequence encoding the EML4-ALK fusion polypeptide contained;
(D) Contains the N-terminal nucleotide sequence of EML-4 (1-700 nucleotides of SEQ ID NO: 4 or 1-1486 nucleotides of SEQ ID NO: 4) and the kinase domain nucleotide sequence of ALK (3348-4149 nucleotides of SEQ ID NO: 6). Nucleotide sequence;
(E) A nucleotide sequence containing at least 6 adjacent nucleotides comprising a fusion junction of an EML4-ALK fusion polynucleotide (700-701 nucleotides of SEQ ID NO: 2 or 1486-1487 nucleotides of SEQ ID NO: 19);
(F) A polypeptide containing at least 6 adjacent amino acids comprising a fusion junction of an EML4-ALK fusion polypeptide (residue 233-234 of SEQ ID NO: 1 or residue 495-996 of SEQ ID NO: 18). Encoding nucleotide sequence; and nucleotide sequence having homology with any of the nucleotide sequences of (g) (a) to (f):
Provided are isolated polynucleotides containing a nucleotide sequence having at least 95% homology to a sequence selected from the group consisting of.

Using the information provided herein, such as the nucleotide sequence of FIG. 2 (SEQ ID NO: 2), the nucleic acid molecule of the invention encoding the mutant ALK polypeptide of the invention uses mRNA as a starting material. It can be obtained using standard cloning and screening methods, such as cloning of the cDNA used. As an example of the present invention, the EML4-ALK fusion polynucleotide (short mutant) shown in FIG. 2 (SEQ ID NO: 2) was isolated from genomic DNA derived from a human NSCLC cell line (Example 2 below). Further described in). The fusion gene can also be identified in genomic DNA or cDNA libraries in other cancers, including solid tumors, where the disclosed EML4-ALK gene deletion (chromosome 2) occurs.

The nucleotide sequence of the determined EML4-ALK fusion gene encodes a kinase fusion protein of 796 amino acids (short variants) and 1059 amino acids (long variants), respectively (FIGS. 2AB (SEQ ID NOs: 1 and 18)). And see Figures 1A-B). The EML4-ALK fusion polynucleotide is a nucleotide sequence of wild EML-4 encoding the N-terminus of the protein (amino acid 1-233 (short variant) or amino acid 1-495 (long variant) (FIG. 3 (sequence)). The portion of (see number 4)) is included along with the portion of the nucleotide sequence of the wild-type ALK encoding the kinase domain and C-terminus of the protein (see Figure 4 (reference number 6)). See Figures 1A-B. The kinase domain is the residue 292-568 of a short mutagenesis protein (encoded by nucleotides 874-568 of a short mutagen polynucleotide), or a long mutagenesis protein (long mutagenoid poly). Consists of residues 558-831 (encoded by nucleotides nucleotides 1663-2494), see Figure 2A-2B.

As shown, the invention provides, in part, a mature form of the EML4-ALK fusion protein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence that is cleaved from the mature protein upon initiation of translocation of the growing protein chain across the rough vesicles. Most mammalian and even insect cells cleave secretory proteins with similar specificity. However, in some cases, the opening of secreted proteins is not perfectly uniform, resulting in two or more mature species of protein. Furthermore, it has long been known that the cleavage specificity of secreted proteins is ultimately determined by the primary structure of the complete protein. That is, it is unique to the amino acid sequence of the polypeptide.

For example, a mature EML4-ALK polypeptide having an amino acid sequence encoded by a deposited cDNA clone is a deposit in mammalian cells (eg, 3T3 cells as described below). It means the mature form of the fusion protein produced by the expression of the complete loading frame encoded by the human DNA sequence of the clone being cloned or another clone encoding the mature fusion polypeptide.

As shown, the polynucleotides of the invention may be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA obtained, for example, by cloning or synthetically. The DNA may be double-stranded or single-stranded. The single-stranded DNA or RNA may be a coding strand, also known as the sense strand, or may be a non-coding strand, also known as the antisense strand.

The isolated polynucleotides of the present invention are nucleic acid molecules, DNA or RNA extracted from their natural environment. For example, the recombinant DNA molecule contained in the vector is believed to have been isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules in heterologous host cells, or purified (partially or substantially) DNA molecules in solution. The isolated RNA molecules include RNA transcripts of the DNA molecules of the invention in vivo or in vitro. The isolated nucleic acid molecules according to the invention further include molecules such as those produced by synthesis.

The isolated polynucleotide of the present invention encodes the DNA molecule shown in FIGS. 2AB (SEQ ID NOs: 2 and 19) and the mature EML4-ALK fusion protein shown in FIGS. 1AB (SEQ ID NOs: 1 and 18). A DNA molecule containing a sequence to be used, and a DNA that contains a sequence substantially different from that described above, but still encodes the ALK mutant polypeptide of the present invention due to the degeneracy of the genetic code. Includes molecules. This genetic code is known in the art and therefore it would be normal for one of ordinary skill in the art to make such a degenerate variant.

In another aspect, the invention provides an isolated polynucleotide encoding an EML4-ALK fusion polypeptide containing the EML4-ALK fusion nucleotide sequence contained in the deposited cDNA clone described above. .. Preferably, such a nucleic acid molecule is a mature fusion polypeptide encoded by the deposited cDNA clone described above, or another clone expressing the full length of the EML4-ALK fusion protein described herein. Will code.
In another aspect, the invention presents the N-terminal amino acid sequence of EML-4 (residue 1-233 of SEQ ID NO: 3 or residue 1-495 of SEQ ID NO: 3) and the kinase domain of ALK (of SEQ ID NO: 5). Provided is an isolated nucleotide sequence encoding an EML4-ALK fusion polypeptide containing residues 1116-1183).
In one aspect, the polypeptide containing the kinase domain of ALK comprises residues 1057-1620 of SEQ ID NO: 5 (see figure below FIG. 1C).
In other embodiments, the N-terminal amino acid sequence of EML-4 and the kinase domain of ALK are nucleotide sequences containing nucleotide 1-700 of SEQ ID NO: 4 or nucleotides of SEQ ID NO: 4 1-1486 and SEQ ID NO: 6 It is encoded by the nucleotides of 3171-4860, respectively.

The present invention further provides an isolated polynucleotide comprising a nucleotide sequence having a sequence complementary to one of the mutant ALK polynucleotides of the invention. Such isolated molecules, especially DNA molecules, are humans as probes for gene mapping by in situ hybridization on chromosomes and, for example, by Northern blot analysis as further described in column F below. It is useful for detecting the expression of the EML4-ALK fusion protein in tissues.

The present invention further relates to fragments of isolated nucleic acid molecules described herein. Fragments of the isolated EML4-ALK polynucleotides of the invention are nucleotides of at least about 15 length, and more preferably at least about, which are useful as diagnostic probes and primers considered herein. A fragment of 20 nucleotides, more preferably at least about 30 nucleotides, even more preferably at least about 40 nucleotides is intended. Of course, larger fragments of nucleotides about 50-1500 in length are also deposited cDNA, as shown in FIG. 2 (SEQ ID NO: 2), or FIG. 2AB (SEQ ID NO: 2 or It is useful by the present invention as a fragment corresponding to most mutant ALK nucleotide sequences, but not all of the other clones expressing the fusion polynucleotide as shown in 19). A fragment having a length of at least 20 nucleotides is intended, for example, a fragment containing 20 or more contiguous bases from each nucleotide sequence from which the fragment is derived. Generation of such DNA fragments is common to those of skill in the art, for example, restriction endonuclease cleavage, or synthesis according to a sequence available from a deposited cDNA clone or disclosed herein. It can be carried out by shearing the DNA by sonication. Also, such fragments can be produced by direct synthesis.

Preferred nucleic acid fragments or probes of the invention include nucleic acid molecules encoding fusion junctions of the fusion gene product of EML4-ALK (FIGS. 1AB, see figure below). For example, in one preferred embodiment, the isolated polynucleotide of the invention comprises a fusion junction of EML4-ALK fusion polynucleotides (nucleotides 700-701 of SEQ ID NO: 2, or nucleotides 1486-1487 of SEQ ID NO: 19). Contains a nucleotide sequence / fragment consisting of at least 6 adjacent nucleotides (see Figures 1AB, below (SEQ ID NOs: 8 and 25)).
In another preferred embodiment, the isolated polynucleotide of the invention comprises a fusion junction of an EML4-ALK fusion polypeptide (residue 233-234 of SEQ ID NO: 1 or residue 495-996 of SEQ ID NO: 18). Contains a nucleotide sequence / fragment encoding a polypeptide containing at least 6 adjacent amino acids (see FIGS. 1AB, bottom view (SEQ ID NOs: 7 and 24)).

In another aspect, the invention comprises isolated polynucleotides that hybridize to some of the mutant ALK polynucleotides of the invention as described herein under stringent hybridization conditions. provide. "Stringent hybridization conditions" are 50% formamide, 5 times SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 times denatured solution (5 times Denhaz solution). Denhardt's solution), cultivated overnight at 42 ° C. in a solution containing 10% dextran sulfate and 20 micrograms / ml denatured, sheared salmon sperm DNA, and then 0.1-fold the filtrate at about 65 ° C. Intended to be washed in SSC.

A polynucleotide that hybridizes to a "part" of a polynucleotide is at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt. And even more preferably, a polynucleotide (either DNA or RNA) that hybridizes to about 30-70 nt is intended. These are useful as diagnostic probes or primers as discussed in more detail above and below.

Of course, a larger portion of the reference polynucleotide (eg, the mature EML4-ALK fusion polynucleotide set forth in FIG. 2 (SEQ ID NO: 2)) (eg, nt in length 50-750, or the total length of the reference polynucleotide). The polynucleotides that hybridize to (up to) are also shown in the nucleotide sequence of the deposited cDNA or in FIGS. 2AB (SEQ ID NO: 2 or 19) or FIG. 1AB (below) (SEQ ID NOs: 7 and 24). It is useful as a probe according to the present invention because it is a polynucleotide corresponding to most, if not all, of the nucleotide sequences shown.

A portion of a "at least 20" length polynucleotide is intended, for example, 20 or more adjacent nucleotides from the nucleotide sequence of the reference polynucleotide. As shown, such parts are, for example, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed., Sambrook, J., Fritsch, EF and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. , NY (1989) (incorporated herein by reference in its entirety), either a probe by conventional DNA hybridization techniques or a primer that amplifies the target sequence by polymerase chain reaction (PCR). It is diagnostically useful as either. Of course, a polynucleotide that hybridizes only to a poly A sequence (such as the 3'terminal poly (A) tract of the EML-4-ALK sequence shown in FIG. 2 (SEQ ID NO: 2)) is such a polynucleotide. This is because it will hybridize to any of the nucleic acid molecules that contain poly (A) stretches or their complements (eg, substantially any of the double-stranded cDNA clones). It will not be included in the polynucleotides of the invention used to hybridize to some of the nucleic acids of the invention.

As shown, the nucleic acid molecule of the invention encoding the mutant polypeptide of the invention is such as, but not limited to, encoding the amino acid sequence of a mature polypeptide alone; encoding a mature polypeptide. Sequences and additional sequences, such as those encoding leader or secretory sequences such as pre- or pro-, pre-pro-protein sequences; coding sequences of mature polypeptides, such as, but not limited to. Includes intron and non-coding 5'and 3'sequences, such as transcriptional, untranslated sequences, that serve as transcriptional, mRNA processing, including splicing and polyadenylation signals, including ribosome binding and mRNA stabilization. Along with the additional and non-encoding sequences, those having or not having the above additional coding sequence; may include additional coding sequences encoding additional amino acids that provide additional function.

Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as the sequence encoding the peptide that facilitates purification of the fusion protein.
In one preferred embodiment of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the label provided on the pQE vector (Qiagen, Inc.), many of which are commercially available. .. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for example hexa-histidine provides convenient purification of fusion proteins. The "HA" label is another peptide useful for purification that corresponds to an antigenic determinant from an influenza hemagglutinin protein and has been described by Wilson et al., Cell 37: 767 (1984). Other such fusion proteins, as discussed below, include EML4-ALK fusion polypeptides that themselves are fused to N- or C-terminal Fc.

The present invention further relates to variants of the nucleic acid molecules of the invention that encode parts, analogs or derivatives of the EML4-ALK fusion polypeptides disclosed herein. Mutants may occur spontaneously, such as natural allelic variants. An "allelic variant" is intended as one of several alternative forms of a gene that occupies a given gene conformation on the chromosome of an organism. See, for example, GEENS II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-natural variants can be produced using known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions, deletions or additions. This substitution, deletion or addition comprises one or more nucleotides. The mutant can modify the coding region, the non-coding region, or both. Modifications of the coding region can produce conservative or non-conservative amino acid substitutions, deletions or additions. Of these, particularly preferred are mild substitutions, additions and deletions that do not alter the properties and activities (eg, kinase activity) of the mutant ALK polypeptides disclosed in the present invention. Also particularly preferred in this regard is a conservative substitution.

A further aspect of the invention is a polynucleotide sequence encoding an EML4-ALK fusion polypeptide having the complete amino acid sequence set forth in FIG. 2 (SEQ ID NO: 1) of the mutant ALK polynucleotide of the invention; or EML-. With nucleotide sequences encoding the N-terminal of 4 and the kinase domain of ALK (see Figures 1AB, below; and FIGS. 3 and 4); or nucleotides complementary to such exemplary sequences). Have at least 90% homology, and more preferably at least 95%, 96%, 97%, 98% or 99% homology.

A nucleotide having a reference nucleotide sequence encoding a mutated ALK polypeptide and at least, for example, 95% "homologous" nucleotide sequence is a reference nucleotide in which the nucleotide sequence of the polynucleotide encodes a mutated ALK polypeptide. It is intended to be homologous to the reference sequence, except that each 100 nucleotides of the sequence may contain up to 5 mutations. That is, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% homologous to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or in the reference sequence. A number of up to 5% of the total nucleotides in the nucleotide may be inserted into the reference sequence. These mutations in the reference sequence are individually integrated or referenced between the nucleotides in the reference sequence at the 5'- or 3'-terminal positions of the reference nucleotide sequence, or somewhere between these ends. It can be triggered by either incorporating one or more contiguous groups in the sequence.

In fact, any particular nucleic acid molecule is at least 90%, 95%, with, for example, the nucleotide sequence shown in FIGS. 2AB (SEQ ID NOs: 2 and 19), or the nucleotide sequence of the deposited cDNA clone described above. For 96%, 97%, 98% or 99% homology, the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive) , Madison, Wis. 53711), and can be determined in the usual way using known computer programs. Bestfit uses Smith and Waterman's Local Homology Algorithm (Advances in Applied Mathematics 2: 482-489 (1981)) to find the best homology between the two sequences. Best fit or other sequence to determine if a particular sequence has, for example, 95% homology with the reference EML4-ALK fusion polynucleotide sequence or the truncated ALK polynucleotide sequence according to the invention. When using any of the placement programs, of course, the percentage of homology to the total length of the reference nucleotide sequence is calculated, and a deviation of up to 5% of the total number of nucleotides in the reference sequence is allowed. The parameters are set so that they are.

The present invention includes at least the nucleic acid sequence shown in FIG. 2 (SEQ ID NO: 2) or the nucleic acid sequence of the deposited cDNA, whether or not they encode polypeptides having ALK kinase activity. It contains nucleic acid molecules having 90%, 95%, 96%, 97%, 98% or 99% homology. It is used by those skilled in the art as to how to use this nucleic acid molecule, eg, as a hybridization probe or as a primer for the polymerase chain reaction (PCR), even if the particular nucleic acid molecule does not encode a fusion polypeptide having ALK kinase activity. Because I already know. The uses of the nucleic acid molecules of the invention that do not encode a polypeptide having a kinase are, among other things, (1) an EML4-ALK deleted gene, or a cleaved ALK gene, or its allelic variant in a cDNA library. (2) In situ hybridization, as described in HUMAN CHROSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988) by Verma et al., For example, "FISH") to provide the exact chromosomal location of the EML4-ALK deleted gene or the cleaved ALK gene; and the expression of the EML4-ALK fusion protein or cleaved ALK kinase mRNA in a particular tissue. Includes Northern Blot Analysis for Detection.

However, a nucleic acid molecule having a sequence that is at least 95% homologous to the nucleic acid sequence of the mutant ALK polypeptide of the invention or the deposited cDNA, which actually encodes a fusion polypeptide having ALK kinase activity, is preferred. Such activity is measured by a specific biological assay and is either an EML4-ALK fusion protein disclosed herein (a full-length protein, a mature protein, or a protein fragment that retains kinase activity. ), It does not have to be the same. For example, the kinase activity of ALK is to determine the ability to phosphorylate one or more tyrosine-containing peptide substrates, eg, insulin receptor substrates 1 or 2 (IRS1, IRS2), which are substrates for ALK kinase. Can be tested.

To reduce the genetic code, at least 90%, 95%, 96%, 97%, 98% or the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in FIGS. 2AB (SEQ ID NOs: 2 and 19). Those skilled in the art will immediately recognize that many nucleic acid molecules with 99% homologous sequences will encode mutant polypeptides with ALK activity. In fact, all of the degenerate variants of these nucleotide sequences encode similar peptides, which will be apparent to those of skill in the art without the above comparative assay. Furthermore, it will be recognized in the art that for such nucleic acid molecules that are not degenerate variants, a significant number will encode polypeptides that retain ALK activity. It is well recognized by those skilled in the art for amino acid substitutions (eg, replacing one aliphatic amino acid with a second aliphatic amino acid) that have little or no significant effect on protein function. Because it is.

For example, Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306-1310 (1990), provides advice on how to make phenotypically minor amino acid substitutions. Published in, which describes two major approaches to assessing the tolerance of the amino acid sequence to be modified. The first method relies on an evolutionary process in which mutations are either accepted or rejected by natural selection. The second approach utilizes genetic recombination techniques to introduce amino acid changes at specific sites of cloned genes, and selection or screening to identify sequences that retain functionality. These studies have clarified that proteins are surprisingly tolerant of amino acid substitutions. Those skilled in the art who are familiar with such techniques will also understand which amino acid changes are acceptable at certain positions in the protein. For example, most embedded amino acid residues require non-polar side chains, whereas most surface side chain properties are generally unprotected. Minor substitutions on other such phenotypes are described in Bowie et al., Supra. And the references cited therein.

Methods known and generally available in the art for determining DNA sequences can be used to carry out any of the aspects of the polynucleotides of the invention. This method involves Klenow fragmentation of DNA polymerase 1, SEQENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or Enzymes such as a combination of recombinant polymerase with a calibrated exonuclease such as the ELONGASE Amplification System commercially available by Gibco BRL (Gaithersburg, Md.) Can be used. This process should be automated using instruments such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) And the ABI 377 DNA sequencers (Perkin Elmer). Is preferable.

The polynucleotide sequence encoding the mutant ALK polypeptide of the invention uses a portion of the nucleotide sequence to utilize a number of methods known in the art for detecting upstream sequences such as promoters and control elements. Can be extended. For example, one available method, "restricted site" PCR, uses universal primers to search for unknown sequences flanking known loci (Sarkar, G., PCR Methods Applic. 2: 318- 322 (1993)). In particular, genomic DNA is first amplified in the presence of primers for the linker sequence and primers specific for known regions. Typical primers are as described in Example 2 herein. The amplified sequence is then subjected to a second PCR with the same linker and different specific primers inside the first DNA. The products of each PCR are transcribed with the appropriate RNA polymerase and sequenced with reverse transcriptase.

Reverse PCR can also be used to amplify or extend the sequence with branched primers based on known regions (Triglia et al., Nucleic Acids Res. 16: 8186 (1988)). Primers have a GC content of 50% or more in a nucleotide length of 22-30, using OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), Or another suitable program. And can be designed to anneal to the target sequence at a temperature of about 68-72 ° C. This method uses several restriction enzymes to produce suitable fragments in known regions of the gene. This fragment is then cyclized by intramolecular ligation and used as a PCR template.

Another method available is capture PCR involving PCR amplification of DNA fragments flanking known sequences in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1: 111-119 (1991)). ). In this method, digestion and ligation with multiple restriction enzymes can also be used to insert the modified double-stranded sequence into an unknown portion of the DNA molecule prior to PCR. Another method that can be used to search for unknown sequences is that described in Parker et al., Nucleic Acids Res. 19: 3055-3060 (1991). In addition, PROMOTER FINDER® libraries for PCR, nested primers, and genomic DNA can be used (Clontech, Palo Alto, Calif.). This process does not require a screen library and is useful for detecting intron / exon connections.

When screening full-length cDNAs, it is preferable to use a size-selected library to include larger cDNAs. Random prime libraries are also preferred as they contain more sequences containing the 5'region of the gene. Random prime libraries may be particularly useful in situations where oligo d (T) libraries do not yield full-length cDNA. Genomic libraries may be useful for extending sequences to 5'and 3'non-transcriptional regulatory regions.

A commercially available capillary electrophoresis system can be used to analyze the size of sequencing or PCR products or to confirm nucleotide sequences. In particular, capillary sequencing utilizes fluid polymers for electrophoretic separation, four different optical brighteners activated by laser (one for each nucleotide), and emission wavelength detection with a charge-binding element camera. To do. Light output intensity can be converted to electrical signals using appropriate software (eg, GENOTYPER® and SEQUENCE NAVIGATOR®, Perkin Elmer), everything from sample insertion to computer analysis and display of electrical data. The process can be computer controlled. Capillary electrophoresis is particularly preferred for sequencing small pieces of DNA that may be present in limited amounts in a particular sample.

C. Vectors and Host Cells The invention also provides the production of recombinant vectors containing the isolated polynucleotides of the invention, host cells genetically engineered with recombinant vectors, and recombinant EML4-ALK polypeptides or fragments thereof by recombinant techniques. To do.

Recombinant constructs can be introduced into host cells using well-known techniques such as infection, transduction, transfection, transfection, electroporation and transformation. The vector may be, for example, a phage, plasmid, viral or retrovirus vector. The retrovirus vector may be replicable or replication-deficient. In the latter case, viral growth will generally occur only in complementary host cells.

The polynucleotide can be attached to a vector containing markers that are selectable for growth in the host. Generally, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using the appropriate packaging cell line and then transduced into host cells.

A vector containing a cis action control region with respect to the desired polynucleotide is preferable. Appropriate trans-acting factors can be supplied by the host, by the complementary vector, or by the vector itself by introduction into the host. In one preferred embodiment in this regard, the vector exhibits inducible and / or cell type specific, specific expression. Particularly preferred of such vectors are those that can be induced by easily manipulated environmental factors such as temperature and nutrient additives.

Expression vectors useful in the present invention include vectors derived from chromosomes, episomes, and viruses, such as bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, baculoviruses, papovaviruses, waxinaviruses, adenoviruses, poultry diphtheriaviruses, pseudomad dog diseases. Includes vectors from these combinations, such as viruses and retroviruses, and cosmid and phagemid.

DNA inserts containing the EML4-ALK polynucleotide or the polynucleotide of the invention include the lambda phage PL promoter, the Escherichia coli lac, trp and tac promoters, the SV40 early and late promoters and the promoters of the retrovirus LTRs, to name a few. Must operably bind to the appropriate promoter, such as. Other suitable promoters are known to those of skill in the art. The expression construct will further contain a site for initiating and terminating transcription, and a ribosome binding site for translation in the transcription region. The coding portion of the mature transcript expressed by this construct may include a translation initiation site at the start and stop codons (UAA, UGA or UAG) located exactly at the end of the polypeptide being translated. Would be preferred.

As shown, it would be preferable that the expression vector contains at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, and tetracycline or ampicillin resistance genes in E. coli and other bacterial cultures. Representative examples of suitable hosts are, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces and murine typhoid cells; fungal cells such as yeast cells; Drosophila S2 and Spodoptera Sf9 cells. Insect cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Suitable culture media and conditions for the host cells are known in the art.

Preferred vectors used in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, available from Stratagene, Includes pNH46A; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be apparent to those of skill in the art.

Among the known bacterial promoters, those suitable for use in the present invention include the E. coli lac1 and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters are CMV pre-early promoter, HSV thymidine kinase promoter, late and early SV40 promoters, retrovirus LTRs promoters such as Rous sarcoma virus (RSV), and metallothioneine promoters such as mouse metallotionein-1 promoter. Including.

In yeast, Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For a review, see Ausubel et al. (1989) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y. and Grant et al., Methods Enzymol. 153: 516-544 (1997).

Introduction of this construct into host cells can be achieved by calcium phosphate transfection, DEAE dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard experimental manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986).

Transcription of the DNA encoding the EML4-ALK fusion polypeptide of the invention by higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act to increase the transcriptional activity of promoters in a given host cell. Examples of enhancers include SV40 enhancers located at the late site of the copy origin of base pairs 100-270, cytomegalovirus early promoter enhancers, polyoma enhancers at the late site of the copy origin, and adenovirus enhancers.

Appropriate secretory signals can be incorporated into the expressed polypeptide for secretion of the translated protein into the endoplasmic reticulum lumen, into the pericellular space, or into the extracellular environment. This signal may be endogenous to the polypeptide, or they may be heterologous signals.

The polypeptide may be expressed in a modified form such as a fusion protein (eg, GST-fusion) and may contain additional heterologous functional regions as well as secretory signals. For example, the addition of additional amino acids, especially regions of charged amino acids, to the N-terminus of the polypeptide during purification or subsequent processing or storage to improve stability and retention in the host cell. Can be done. Peptide residues may also be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. In particular, addition of peptide residues to a polypeptide to induce secretion or excretion, to improve stability, and to facilitate purification is a well-known and conventional procedure in the art. .. Preferred fusion proteins contain heterologous regions derived from immunoglobulins that are useful for solubilizing the protein.

EML4-ALK fusion polypeptide, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography It can be recovered and purified from the recombinant cell culture by a known method including. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification. The polypeptides of the invention include naturally purified products, products of chemical synthesis processes, and products produced by recombination techniques from prokaryotic and eukaryotic hosts, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. To do. Depending on the host used in the recombinant production process, the polypeptides of the invention may or may not be glycosylated. In addition, the polypeptides of the invention may contain initially modified methionine residues, in some cases as a result of host-mediated processes.

Thus, in some embodiments, the invention is a recombinant EML4-ALK fusion polypeptide by culturing recombinant host cells (as described above) under conditions suitable for expression of the fusion polypeptide and recovering the polypeptide. Provide a method of producing. Culture conditions suitable for the growth of host cells and the expression of recombinant polypeptides from such cells are known to those of skill in the art. See, for example, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausbel FM et al., Eds., Volume 2, Chapter 16, Wiley Interscience.

D. An isolated polypeptide.
The present invention, in part, provides an isolated mutant ALK kinase polypeptide and fragments thereof. In one aspect, the invention
(A) Amino acid sequence encoding an EML4-ALK fusion polypeptide containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 18;
(B) The N-terminal amino acid sequence of EML-4 (residue 1-123 of SEQ ID NO: 3 or residue 1-495 of SEQ ID NO: 3) and the kinase domain of ALK (residue 1116-1383 of SEQ ID NO: 5). Amino acid sequence encoding an EML4-ALK fusion polypeptide containing: and (c) the fusion junction of the EML4-ALK fusion polypeptide (residue 233-234 of SEQ ID NO: 1 or residue 495 of SEQ ID NO: 18). Amino acid sequence encoding a polypeptide containing at least 6 adjacent amino acids, including -496):
Provided are an isolated polypeptide containing an amino acid sequence having at least 95% homology to a sequence selected from the group consisting of.

In one preferred embodiment, there is provided a recombinant mutant ALK polypeptide of the invention that can be produced using a recombinant vector or recombinant host cell as described above.

It has been recognized in the art that some of the amino acid sequences of the EML4-ALK fusion polypeptide or cleaved active ALK kinase polypeptide can be modified without significantly affecting the structure or function of the mutant protein. Will be. When considering such differences in the sequence, care should be taken to ensure that the protein has important sites that determine activity (eg, the kinase domain of ALK). In general, residues that exhibit similar function can be used to replace the residues that form tertiary structure. In another example, the type of residue does not have to be of any importance if the modification is made in a non-critical region of the protein.

Accordingly, the invention further retains substantial ALK kinase activity or contains EML-4 or other regions of the ALK protein, such as protein moieties as discussed below, EML4-. It contains variants of the ALK fusion polypeptide. Such mutants include deletions, insertions, inversions, repetitions, and type substitutions (eg, one that replaces one hydrophilic residue with another, but generally one that is highly hydrophilic and one that is highly hydrophobic. Does not replace). Something like minor modifications or "neutral" amino acid substitutions will generally have little effect on activity.

Typical conservative substitutions are mutual substitutions between the aliphatic amino acids Ala, Val, Leu and Ile; exchange of hydroxy residues Ser and Thr, exchange of acidic residues ASP and Glu, amide residue. Substitution between groups Asn and Gln, exchange of base residues Lys and Arg, and substitution between aromatic residues Ph and Tyr. Examples of conservative amino acid substitutions known to those of skill in the art are; aromatics: phenylalanine, tryptophan, tyrosine; hydrophobic: leucine, isoleucine, valine; polar: glutamine, aspartin; basic: arginine, lysine, histidine; acidic: Aspartic acid, glutamine; small molecules: alanine, serine, threonine, methionine, glycine; As detailed above, Bowie provides further guidance on which amino acid exchanges appear to be phenotypically minor (ie, do not appear to have a significant adverse effect on function). It can be found in et al., Science 247, supra.

The polypeptides of the invention are preferably provided in isolated form, preferably in substantially purified form. The recombinant product of the EML4-ALK fusion polypeptide of the present invention can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988).

The polypeptide of the present invention is the N-terminal amino acid sequence (SEQ ID NO:) of EML4-ALK fusion polypeptide, EML-4, of FIGS. 2A-B (SEQ ID NOs: 1 and 18) (with or without a leader sequence). Amino acid encoding an EML4-ALK fusion polypeptide containing residue 1-123 of 3, or residue 1-495 of SEQ ID NO: 3) and the kinase domain of ALK (residue 1116-1383 of SEQ ID NO: 5). A polypeptide containing the sequence and at least 6 adjacent amino acids comprising the fusion junction of the EML4-ALK fusion polypeptide (residue 233-234 of SEQ ID NO: 1 or residue 495-49 of SEQ ID NO: 18). The amino acid sequence encoding the above (FIGS. 1AB, see also bottom figure) further comprises at least 90% homology, preferably at least 95% homology, and even more preferably at least 96%. It also includes polypeptides having 97%, 98% or 99% homology.

The "%" homology to two polypeptides is the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and It is intended as a homology score created by comparing the amino acid sequences of two polypeptides using the default settings for determining homology. Bestfit uses Smith and Waterman's Local Homology Algorithm (Advances in Applied Mathematics 2: 482-489 (1981)) to find the best part of homology between two sequences.

When a polypeptide has, for example, at least 95% "homologous" amino acid sequence with the reference amino acid sequence of the EML4-ALK fusion polypeptide of the invention, the amino acid sequence of this polypeptide is a mutant ALK polypeptide. It is intended to be homologous to the reference sequence, except that the amino acid sequence may contain up to 5 amino acid modifications per 100 amino acids of each of the reference amino acid sequences of. That is, in order to obtain a polypeptide having an amino acid sequence that is at least 95% homologous to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or replaced with other amino acids, or the reference sequence. The number of amino acids up to 5% of the total amino acid residues in may be inserted into the reference sequence. These modifications of the reference sequence can be made at either the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or between these terminal positions, individually between the residues in the reference sequence, or in one of the reference sequences. It can be generated by interspersing it with or more adjacent groups.

Of course, when using either Best Fit or any other sequencing program to determine if a particular sequence has 95% homology with a reference sequence according to the invention, the reference amino acid The parameters are set so that the percentage of homology is calculated relative to the total length of the sequence and that a deviation of up to 5% of the total number of amino acid residues in the reference sequence is allowed.

The EML4-ALK fusion polypeptide of the present invention can be used as a molecular weight marker on an SDS-PAGE gel or molecular sieve gel filtration column using, for example, a method known to those skilled in the art.

As described in more detail below, the polypeptides of the invention can be used in assays to detect the expression of mutant ALK polypeptides as described below, or to enhance or inhibit the function / activity of mutant ALK proteins. It can also be used to create a fusion polypeptide-specific reagent, such as a polyclonal and monoclonal antibody, or a cleaved polypeptide-specific reagent that is useful as an agonist or antagonist capable of it can. In addition, such polypeptides are used in yeast 2-hybrid systems to "capture" EML4-ALK fusion polypeptides, or fragment ALK kinase polypeptide-binding proteins that are also candidate agonists and antagonists according to the invention. be able to. The yeast two hybrid system is described in Fields and Song, Nature 340: 245-246 (1989).

In another aspect, the invention comprises an epitope containing an epitope of the polypeptide of the invention, eg, an epitope containing a fusion junction of an EML4-ALK fusion polypeptide (FIG. 1AB, see bottom figure). Provided are peptides or polypeptides containing. The epitope portion of this polypeptide is an immunogenic or antigenic epitope of the polypeptide of the invention. An "immunogenic epitope" is defined as part of a protein that elicits an antibody response when all proteins are the source of immunity. These immunogenic epitopes are thought to be confined to several loci on the molecule. On the other hand, the region of the protein molecule to which the antibody can bind is defined as an "antigen epitope". The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983). The production of antibodies specific for the fusion polypeptide of the invention is described in more detail below.

Antibodies produced with peptides or polypeptides containing antigenic epitopes are useful in detecting mimicking proteins, and antibodies against different peptides track the fate of various regions of the protein precursor undergoing post-translation treatment. Can be used to. Peptides and anti-peptide antibodies have been shown, for example, in immunoprecipitation assays to be able to bind and replace even shorter peptides (eg, about 9 amino acids) to larger peptides, and thus for mimetic proteins. It can be used in qualitative and quantitative assays, such as competitive assays. See, for example, Wilson et al., Cell 37: 767-778 (1984) at 777. The anti-peptide antibody of the present invention is useful, for example, for purifying mimic proteins by adsorption chromatography using methods known in the art. The method of immunoassay is described in more detail below.

The recombinant mutant ALK polypeptide is also within the scope of the present invention and can be produced using the polynucleotide of the present invention as described in column B above. For example, the present invention is, in part, a recombinant EML4-ALK fusion poly by culturing recombinant host cells (as described above) under conditions suitable for expressing the fusion polypeptide and then recovering the polypeptide. A method for producing a peptide is provided. Suitable conditions for the growth of host cells and the expression of recombinant polypeptides from such cells are known to those of skill in the art.

E. Mutant-Specific Reagents Mutant ALK polypeptide-specific reagents useful for practicing the disclosed methods include, among other things, fusion polypeptide-specific antibodies and corresponding AQUA peptides (heavy isotope labeling). Peptides) and are suitable for detecting and quantifying the expression of EML4-ALK fusion polypeptides in biological samples derived from cancer, such as mammalian solid sarcoma or cancer tumors. Cleavage-specific reagents such as antibodies, AQUA peptides or nucleic acid probes suitable for detecting the presence or absence of a cleaved ALK kinase polynucleotide or polypeptide of the invention are also useful. Reagents specific for the fusion polypeptide are biological or chemical capable of specifically binding to the EML4-ALK fusion polypeptide expressed in a biological sample to detect and / or quantify its presence / level. It is one of the reagents. The term includes, but is not limited to, preferred antibodies and AQUA peptide reagents discussed below, and equivalent reagents are within the scope of the present invention.

Antibodies Suitable reagents for use in the practice of the methods of the invention include antibodies specific for the EML4-ALK fusion polypeptide and antibodies specific for the TFG-ALK fusion polypeptide. Antibodies specific for the fusions of the invention specifically bind to the EML4-ALK fusion polypeptide of the invention (eg, SEQ ID NO: 1) but are substantially either wild EML-4 or wild ALK. Does not bind, or specifically binds to the TFG-ALK fusion polypeptide described herein (eg, SEQ ID NO: 20) but does not substantially bind to either wild-type TFG or wild-type ALK. , An isolated antibody (including multiple). Because other suitable reagents specifically bind to epitopes in the extracellular domain of the wild-type ALK protein sequence, which domain is not present in the cleaved active ALK kinase disclosed herein. , Includes epitope-specific antibodies capable of detecting the presence (or absence) of wild-type ALK in samples.

Antibodies specific for human EML4-ALK or TFG-ALK fusion polypeptides may bind to highly homologous and homologous epitope peptide sequences in other mammalian species, such as mice or rabbits, and vice versa. Is the same. Antibodies useful for carrying out the methods of the present invention are (a) monoclonal antibodies, (b) fusion junctions of target polypeptides (eg, EML4-ALK fusion polypeptides (Fig. 1AB, see bottom figure). Purified polyclonal antibody that specifically binds to (see) or the fusion junction of the TFG-ALK fusion polypeptide (Fig. 1C, see bottom figure), (c) other non-human species (eg, mice). , Equal and highly homologous epitopes in (rats), or antibodies as described above (a)-(b) that bind to phosphorylation sites, and (d) disclosed herein. Includes fragments of (a)-(c) above that bind to an antigen (or more preferably an epitope) to which a typical antibody is bound.

As used herein, the term "antibody" refers to all types of immunoglobulins, including IgG, IgA, IgD, and IgE. The antibody may be monoclonal or polyclonal, may be of any species origin, including (eg) mouse, rat, rabbit, horse or human, or may be a chimeric antibody. For example, M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrison et al., Proc. Natl. Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature 312: See 604 (1984). The antibody may be a recombinant monoclonal antibody produced by the method disclosed in US Pat. No. 4,474,893 (Reading) or US Pat. No. 4,816,567 (Cabilly et al.). The antibody may be a chemically constructed specific antibody produced by the method disclosed in US Pat. No. 4,676,980 (Segel et al.).

The preferred epitope site of the antibody specific for the EML4-ALK fusion polypeptide of the invention is a peptide consisting essentially of about 11-17 amino acids of the human EML4-ALK fusion polypeptide sequences (SEQ ID NOs: 1 and 18). Fragments, which occur at fusion junctions (short mutant fusion protein residues 233-234 and long mutant fusion protein residues 495-436 (see Figure 1AB (bottom)). It is understood that an antibody that specifically binds to a shorter or longer peptide / epitope comprising a fusion junction of an EML4-ALK fusion polypeptide is within the scope of the invention. Let's go.

Similarly, preferred epitope sites for antibodies specific for the TFG-ALK fusion polypeptide useful in practicing the disclosed methods are essentially about 11-17 of the human TFG-ALK fusion polypeptide sequence (SEQ ID NO: 20). A peptide fragment consisting of the amino acids of, which contains the fusion junction (which occurs at residues 137-138 (see Figure 1C (bottom figure)).

The invention is not limited to the use of antibodies, but fusion proteins or cleavages into essentially the same epitopes to which an antibody specific for an EML4-ALK or TFG-ALK fusion polypeptide useful in the methods of the invention binds. It also includes the use of equivalent molecules, such as protein binding domains or nucleic acid epitopes, that bind in a protein-specific manner. See, for example, Neuberger et al., Nature 312: 604 (1984). Such uniform non-antibody reagents can be adequately utilized in the methods of the invention described further below.

Polyclonal antibodies useful for carrying out the methods of the invention are described herein in accordance with known techniques, with suitable animals (eg, rabbits, sheep, etc.) and epitopes specific for the desired fusion protein (eg, herein). By immunizing with an antigen containing (the fusion junction of the ALK fusion protein), collecting immune serum from the animal, and separating the polypeptide antibody from the immune serum, and purifying the polyclonal antibody having the desired specificity. It can be produced according to standard techniques. The antigen may be a synthetic peptide antigen containing the desired epitope sequence, selected and constructed according to known techniques. For example, ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p.75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods in Enzymology, 201: 264-283 (1991); Merrifield, J. See Am. Chem. Soc. 85: 21-49 (1962). Polyclonal antibodies produced as described herein can be screened and isolated as further described below.

Monoclonal antibodies can also be conveniently utilized in the methods of the invention and can be produced in hybridoma cell lines according to Kohler and Milstein's known techniques. See also Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6:511 (1976); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausbel et al. Eds. (1989). The monoclonal antibody thus produced is highly specific and enhances the selectivity and specificity of the assay methods provided in the present invention. For example, a solution containing a suitable antigen (eg, a synthetic peptide containing a fusion junction of an EML4-ALK fusion polypeptide) is injected into mice and retained after a sufficient period of time (conventional techniques). Then, the mice can be killed to obtain spleen cells. The spleen cells are then fused and immortalized with myeloma cells, generally in the presence of polyethylene glycol, to produce hybridoma cells. Rabbit fusion hybridomas can be produced, for example, as described in US Pat. No. 5,675,063 (K. Knight), registered October 7, 1997. Hybridoma cells are then grown in a suitable selective medium, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant is prepared for monoclonal antibodies with the desired specificity, as described below. Screen. The secreted antibody can be recovered from the tissue culture supernatant by conventional methods such as precipitation, ion exchange, affinity chromatography and the like.

Monoclonal Fab fragments can also be produced in E. coli by recombinant techniques known in the art. See, for example, W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Natl. Acad. Sci. 87: 8095 (1990). If a monoclonal antibody of one isotype is preferred for a particular application, a particular isotype is selected from the first fusion and made directly, or a sibling selection technique (Steplewski) for isolating class switch variants. , et al., Proc. Natl. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)) to secrete monoclonal antibodies of different isotypes. It can be created secondarily from the parent hybridoma. The antigen binding site of a monoclonal antibody can be cloned by PCR and a single chain antibody produced such as a phage display recombinant antibody or an E. coli soluble antibody (see, eg, ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor). ).

In addition, US Pat. No. 5,194,392 (Geysen, 1990) provides a monomer (amino acid) that is homeomorphic of an epitope (ie, "mimotope") complementary to a particular paratope (antigen binding site) of the antibody of interest. Or other compounds) describe general methods for detecting or determining the sequence. More generally, this method involves the detection or determination of a sequence of monomers that are locally isomorphic of a ligand that is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Patent No. 5,480,971 (Houghten et al., 1996 ) is a library of linear _C 1 -C- alkyl peralkylated oligopeptides and sets and such peptides, further such Also disclosed is a method of sequencing a hyperalkylated oligopeptide that preferentially binds to a receptor molecule of interest using a unique peptide set and library. Non-peptide analogs of peptides containing the epitopes of the invention can also be commonly made by these methods.

Antibodies useful in the methods of the invention, whether polyclonal or monoclonal, can be screened for epitope and fusion protein specificity by standard techniques. See, for example, Czernik et al., Methods in Enzymology, 201: 264-283 (1991). For example, the specificity of the antibody to the desired antibody and, if desired, the specificity of reacting only with, for example, the EML4-ALK fusion polypeptide of the invention and not with wild-type EML-4 or wild-type ALK. To confirm both, the peptide library can be screened by ELISA. To cell preparations containing the target protein, to confirm reactivity of the antibody with only the desired target and to ensure that it does not sensibly bind to other fusion proteins, including ALK. It can also be tested by Western blotting. The production, screening, and use of antibodies specific for the fusion protein are known and described by those of skill in the art. See, for example, US Patent Application Publication No. 20050214301 (Wetzel et al., September 29, 2005).

Antibodies specific for fusion polypeptides useful in the methods of the invention are epitopes of wild-type EML-4, wild-type TFG, and wild-type ALK that form fusion junctions with similar fusion epitopes of other fusion proteins. May show some limited cross-reactivity. This is unexpected, as most antibodies will show some degree of cross-reactivity and anti-peptide antibodies will often cross-react with epitopes that have high homology or identity with immune peptides. is not. See, for example, Czernic, supra. Cross-reactivity with other fusion proteins can be easily clarified by Western blotting performed in parallel with markers of known molecular weight. The amino acid sequence of the cross-reacting protein can be tested to identify sites with high homology or identity to the EML4-ALK or TFG-ALK fusion polypeptide sequence to which the antibody binds. Undesirable cross-reactivity can be eliminated by negative selection using antibody purification on a peptide column (eg, selecting an antibody that binds to either wild-type EML-4 and / or wild-type ALK).

The EML4-ALK fusion polypeptide-specific antibodies (and TFG-ALK fusion polypeptide-specific antibodies) of the invention that are useful for carrying out the methods disclosed herein are ideally. It is specific for human fusion polypeptides, but is not limited to binding only to the human species itself. The present invention includes the production and use of antibodies that also bind to conserved and highly homologous or identical epitopes found in other mammalian species (eg, mice, rats, monkeys). Highly homologous or identical sequences in other species are the human EML4-ALK fusion polypeptide sequences (SEQ ID NOs: 1 and 18) disclosed herein or the human TFG-ALK disclosed herein. It can be easily identified by standard sequence comparison with the fusion polypeptide sequence (SEQ ID NO: 20), such as using BLAST.

Antibodies used in the methods of the invention are further characterized by specific assay formats, such as use in flow cytometry (FC), immunohistochemistry (IHC), and / or immunocytochemistry (ICC). , Can be enabled for these. The use of antibodies specific for ALK fusion polypeptides in such a manner is further described in column F below. Optical brighteners for use in multi-parameter analysis with other signal conversions (phospho-AKT, phospho-Erk 1/2) and / or cellular marker (cytokeratin) antibodies, as described in column F. It can also be advantageously combined with labels such as (eg, Alexa488, PE), or quantum dots.

In the practice of the methods of the invention, the expression and / or activity of wild-type EML-4, wild-type TFG, and / or wild-type ALK in a given biological sample is also antibody (phosphate-specific) to these wild-type proteins. Or can be tested advantageously with any) specific to all. For example, antibodies specific for all ALK and phosphorylation sites are commercially available (see CELL SIGNALING TECHNOLOGY, INC., Beverly MA. 2005/06 Catalog, # s 3341, 3342). Such antibodies can also be produced by standard methods such as those described above. The amino acid sequences of human EML-4, TFG, and ALK are published, as are the sequences of these proteins from other species (see Figures 3A and 4A-4C, and SwissProt Accession Nos.).

Detecting the expression and / or activity of wild-type EML-4, TFG, and wild-type ALK along with the expression of EML4-ALK and / or TFG-ALK fusion polypeptides in biological samples (eg, tumor samples) , Provides information on whether only fusion proteins promote tumors, or whether wild-type ALK also activates and promotes tumors. Such information is clinically useful in assessing whether to target fusion proteins, wild-type proteins, or both, or to inhibit tumor progression, and appropriate treatments or combinations thereof. Most useful for choosing a match. Antibodies specific for the wild-type ALK kinase extracellular domain that are not present in the truncated active ALK kinase disclosed herein are particularly useful for determining the presence / absence of mutant ALK kinase. is there.

It will be appreciated that one or more antibodies can be used to carry out the above method. For example, one or more EML4-ALK fusion polypeptide-specific antibodies are suspected to be activated in cancers expressing another kinase, receptor, or EML4-ALK fusion polypeptide. Such other in biological samples containing cells of such cancer origin, either at the same time as one or more antibodies specific for the kinase substrate that are potentially activated. The activity of signaling molecules can be detected.

Those skilled in the art can combine EML4-ALK fusion polypeptides of the invention and fragments thereof, including the fusion junction epitopes described above, with parts of the constructive domain of immunoglobulin (IgG) to result in chimeric polypeptides. Will be understood. These fusion proteins promote purification and exhibit an increased half-life in vivo. This has been shown for chimeric proteins containing, for example, the first two domains of human CD4-polypeptides and multiple domains of the heavy or light chain constructing regions of mammalian immune grubulin (EPA 394). , 827; Traunecker et al., Nature 331: 84-86 (1988)). A fusion protein having a disulfide bond dimer structure with an IgG moiety can more effectively bind and neutralize with other molecules than the monomeric EML4-ALK fusion polypeptide alone. (Fountoulakis et al., J. Biochem 270: 3958-3964 (1995)).

Heavy isotope labeled peptide (AQUA peptide).
EML4-ALK or TFG-ALK fusion polypeptide-specific reagents useful in carrying out the disclosed methods are absolute quantifications of the expressed ALK fusion polypeptide or cleaved ALK kinase polypeptide in a biological sample. It may contain a polyisotope-labeled peptide suitable for. The production and use of AQUA peptides for the absolute quantification of protein (AQUA) in complex mixtures has been described. WO 03/016681, "Absolute Quantization of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry", Gygi et al. And Gerber et al. Proc. Natl. Acad. Sci. USA 100: 6940-5 (2003) ) Also; all of these teachings are incorporated herein by reference).

The AQUA procedure involves digested biological samples to detect known amounts of at least one polyisotope-labeled peptide standard, which has a unique code detectable by LC-SRM chromatography. Utilize the absolute quantification of peptides with the same sequence and protein modification in biological samples by introduction and comparison with peptide standards. That is, the AQUA procedure has two steps: selection and verification of the peptide internal standard and preparation of the method; and implementation for detecting and quantifying the target protein in the sample using the verified peptide internal standard. are doing. This method is a powerful technique for detecting and quantifying certain peptides / proteins in complex biological mixtures, such as cell lysates, eg, changes in protein phosphorylation as a result of drug treatment. It can be used to quantify or to quantify differences in protein concentration in different biological states.

In general, a particular peptide (or modified peptide) in a target protein sequence is selected based on its amino acid sequence and the particular protease used for digestion in order to create a suitable internal standard. Peptides are then produced by solid phase peptide synthesis so that one residue is replaced by the same residue containing a stable isotope ( ¹³ C, ¹⁵ N). The resulting peptide is chemically identified as its natural counterpart formed by proteolysis, but is readily distinguishable by MS through a 7-Da mass shift. The newly synthesized AQUA internal standard peptide is then evaluated by LC-MS / MS. This process provides quantitative information about peptide retention by reverse phase chromatography, ionization efficiency, and fragmentation by collision-induced dissociation. Chromatograph retention function to select beneficial and abundant fragment ions for a set of natural and internal standard peptides, and then to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard. Is continuously measured as.

The second step of the AQUA procedure is its implementation to measure the amount of protein or modified protein from the complex mixture. Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis to remove regions of the gel that are consistent with protein transfer. This step is followed by Ingel proteolysis and LC-SRM analysis in the presence of AQUA peptide. (See Gerber et al. Supra). The AQUA peptide is placed in a complex peptide mixture obtained by digesting a whole cell degradation product with a proteolytic enzyme to purify the immunocompatibility as described above. Since the retention time and fragmentation pattern of the native peptide formed by digestion (eg, trypsin treatment) is consistent with that of the AQUA internal standard peptide measured earlier, LC-MS / MS analysis using the SRM experiment It provides measurements with high specificity and sensitivity for both internal standards and specimens directly derived from highly complex peptide mixtures.

Since the absolute amount of AQUA peptide is added (eg, 250 fmol), the area ratio below the curve can be used to determine the exact expression level of the phosphorylated form of the protein in the protein or the original cytolytic product. it can. Also, peptide extraction efficiency from gel sections, absolute loss during sample processing (including vacuum centrifugation), and variability during introduction into the LC-MS system affect the ratio of natural and AQUA peptide abundances. An internal standard is present during Ingel digestion to form the natural peptide so as not to give.

AQUA peptide standards are prepared for known sequences in the target protein previously identified by IAP-LC-MS / MS. If the site has been modified, one AQUA peptide may be prepared that includes a modified form of a particular residue within the site, with a second containing the residue in the unmodified form. Create an AQUA peptide. In this way, the two standards can be used to detect and quantify both modified and unmodified forms of sites in the biological sample.

Peptide internal standards can also be produced by testing the primary amino acid sequence of a protein to determine the boundaries of the peptide produced by cleavage with a protease. Alternatively, the protein can actually be digested with a protease and then the specific peptide fragment produced can be sequenced. Suitable proteases include, but are not limited to, serine proteases (eg trypsin, hepsin), metalloproteases (eg PUMP1), chymotrypsin, cathepsins, pepsin, thermolysine, carboxypeptidase and the like.

The peptide sequence within the target protein is selected according to one or more criteria that optimize the use of the peptide as an internal standard. It is preferable to choose the size of the peptide so as to minimize the chance that the peptide sequence will be replicated somewhere in other non-target proteins. Therefore, peptides having at least about 6 amino acids are preferred. The size of the peptide is also optimized to maximize the ionization frequency. Therefore, peptides longer than about 20 amino acids are not preferred. The preferred range is about 7 to 15 amino acids. Sequences containing cysteine, tryptophan, or methionine are avoided because the peptide sequences are also selected during mass spectrometry so that they do not become highly reactive.

Peptide sequences that do not contain modified regions of the target region can be selected so that peptide internal standards can be used to quantify all morphologies of the protein. Alternatively, a peptide internal standard containing the modified amino acid may be desirable for detecting and quantifying only the modified form of the target protein. Peptide standards for both modified and unmodified regions to detect the extent of modification in a particular sample (ie, determine how much of the total amount of protein is represented by the modified form). , Can be used together. For example, peptide standards for both phosphorylated and non-phosphorylated forms of proteins known to be phosphorylated at specific sites can be used to quantify the amount of phosphorylated form in a sample. it can.

Label the peptide with one or more labeled amino acids (ie, the label is the actual portion of the peptide), or, less preferably, add the label after synthesis according to standard methods. You can also do it. Preferably, the label is a mass-altering label chosen based on the following considerations: the mass must be specific to the mass-shifted fragment mass produced by MS spectrometry, which shifts to a spectral region with low background; The characteristic component of mass is the portion of the labeled site that preferably exhibits distinctive ionic mass characteristics in MS spectrometry; the total mass of the constituent atoms of the label is uniquely different from all possible amino acid fragments. Is preferable. As a result, labeled amino acids and peptides are readily distinguished from unlabeled by the resulting mass spectrometric ion / mass pattern. It is preferred that the characteristic component of the ionic mass imparts mass to protein fragments that do not correspond to the residual mass for any of the 20 natural amino acids.

The label can withstand the fragmentation conditions of MS and must not undergo unfavorable fragmentation. The labeling chemistry must be efficient within conditions, especially denaturing conditions, and the labeled tag should preferably remain soluble in the buffer system of choice in MS. It is preferred that the label does not suppress the ionizing effect of the protein and is not chemically reactive. A mixture of two or more isotopically distinguishable species can be included such that each labeled fragment position produces a unique mass spectrometric pattern. Stable isotopes such as ² H, ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, or ³⁴ S are particularly preferred labels. Pairs of peptide internal standards incorporating different isotope labels can also be created. Preferred amino acid residues that can incorporate heavy isotope labeling include leucine, proline, valine and phenylalanine.

Peptide internal standards are characterized by their mass-to-charge ratio (m / z) and preferably by retention time on a chromatography column (eg, HPLC column). An internal standard that elutes with an unlabeled peptide of a sequence having homology is selected as the optimal internal standard. The internal standard is then analyzed by fragmenting the peptide by any suitable method, such as using argon or helium as the collision gas, such as collision-induced dissociation (CID). Fragments are then analyzed, for example, by multi-step mass spectrometry (MS ⁿ ) to obtain fragment ion spectra to obtain the characteristics of peptide fragmentation. It is preferred that the peptide fragments have significantly different m / z ratios so that the peaks corresponding to each fragment can be well separated to obtain characteristics specific to the target protein. If the first step does not provide the appropriate fragment features, then an additional step of MS is performed until the unique features are obtained.

Fragment ions in the MS / MS and MS ³ spectra are generally very specific for the peptide of interest and, when combined with the LC method, contain thousands or tens of thousands of proteins, cell lysates. It enables highly selective methods for detecting and quantifying target peptides / proteins in complex protein mixtures, such as. Any biological sample that potentially contains the target protein / peptide can be assayed. It is preferred to use crude or partially purified cell extracts. Generally, the sample has at least 0.01 mg of protein, typically 0.1-10 mg / mL, which can be adjusted to the desired buffer concentration and pH.

A known amount of a labeled peptide internal standard, preferably about 10 femtomols, corresponding to the target protein to be detected / quantified is then added to the biological sample, such as cell lysate. The added sample is then digested with one or more proteases for an appropriate amount of time before digestion. Separation is then performed to isolate the labeled internal standard and the corresponding target peptide from the other peptides in the sample (eg, by HPLC, reverse phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.). Microcapillary LC is the preferred method.

Each isolated peptide is then tested by monitoring the selective response in MS. This uses the prior knowledge gained from the characterization of the peptide internal standard and then continuously observes specific ions in the MS / MS or MS ⁿ spectrum for both the peptide of interest and the internal standard. It embraces the need for MS for. After elution, the area under the curve (AUC) of the peaks of both the peptide standard and the target peptide is calculated. The ratio of the two areas provides an absolute quantification that can standardize the number of cells and the molecular weight of the protein used in the analysis, resulting in the exact number of protein copies per cell. Further details of the AQUA approach are described in Gygi et al., And Gerber et al. Supra.

The AQUAQ internal peptide standard (heavy isotope-labeled peptide) detects any specific site within the mutant ALK polypeptide of the invention (eg, the fusion junction within the disclosed EML4-ALK fusion polypeptide). And for quantification, it can be preferably produced, as described above. For example, an AQUA phosphopeptide corresponding to a fusion junction sequence of an EML4-ALK fusion polypeptide (see Figure 1AB (bottom)) or a cleavage position of either EML4, TFG, or ALK. Can be created. Peptide standards can be produced for EML4-ALK or TFG-ALK fusion junctions, and such standards can be used for fusion junctions in biological samples (ie, the presence of EML4-ALK fusion polypeptide). It can be used in the AQUA method for detection and quantification.

For example, a representative AQUA peptide of the invention contains the amino acid sequence INQVYR (FIGS. 1A-C, see bottom), which is a fusion junction within the EML4-ALK fusion polypeptide (SEQ ID NO: 7). Corresponds to the three amino acids located directly on the side of each site (see). It will be appreciated that larger AQUA peptides containing the fusion junction sequence (and additional residues downstream or upstream thereof) can also be constructed. Similarly, smaller AQUA peptides containing less than all residues of such sequences (but still containing the fusion junction itself) may also be constructed. Such larger or smaller AQUA peptides are within the scope of the invention, and the selection and production of preferred AQUA peptides can be carried out as described above (Gygi et al., Gerber et al. Supra). Please refer to).

Nucleic Acid Probes The fusion-specific reagents provided by the present invention include nucleic acid probes suitable for the detection of EML4-ALK fusion polypeptides or cleaved ALK kinase polynucleotides, as detailed in column B above. It also includes primers. Such probes preferably include, among others, the cleavage point probes corresponding to both sides of the cleavage point in the wild-type EML4 and / or wild-type ALK genes that produce the fusion. Specific use of such probes in assays such as fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) amplification is described in column F below. A similar cleavage point probe can be made to detect the presence of a TFG-ALK fusion polynucleotide (see FIG. 1C (SEQ ID NO: 21)).

F. Diagnostic application and assay format The methods of the invention can be performed in a wide variety of different assays known in the art.

Immunoassay An immunoassay useful for performing the methods of the invention may be an allogeneic or heterologous immunoassay. In allogeneic assays, the immunological response is generally a reagent specific for a mutant ALK-kinase polypeptide (eg, an antibody specific for an EML4-ALK fusion polypeptide), a labeled sample, and the organism of interest. Contains the sample. When the antibody binds to the labeled specimen, signal generation from the label is directly or indirectly modified. Both the immunological reaction and the detection of its extent are carried out in homologous solutions. Available immunochemical labels include free radicals, radioisotopes, optical brighteners, enzymes, bacteriophages, coenzymes, and the like. Semiconductor nanocrystal labels, or "quantum dots," can also be used advantageously, and their creation and use are well documented. In general, see K. Barovsky, Nanotech. Law & Bus. 1 (2): Article 14 (2004) and the patents cited therein.

In heterologous assay techniques, reagents are typically biological samples, reagents specific for mutant ALK kinase polypeptides (eg, antibodies specific for EML4-ALK fusion), and suitable means for producing detectable signals. Is. Biological samples as further described below can be used. Antibodies are generally immobilized on a support such as beads, plates or slides and contacted in liquid phase with a sample that appears to contain the antigen. The support is separated from the liquid phase and either the support phase or the liquid phase is tested for detectable signals using means for generating such signals. This signal is related to the presence of the specimen in the biological sample. Methods of generating detectable signals include the use of radioactive labels, fluorescent labels, enzyme labels, quantum dots, and the like. For example, if the antigen to be detected contains a second binding site, the antibody that binds to that site can be attached to a detectable group and added to the liquid phase reaction solution prior to the separation step. The presence of a detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays include radioimmunoassays, immunofluorescence, enzyme-bound immunoassays, and the like.

Immunoassay formats and variants thereof that are useful in carrying out the methods disclosed herein are known in the art. In general, see E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.). For example, US Pat. No. 4,727,022 (Skold et al., "Methods for Modulating Ligand-Receptor Interactions and their Application"); US Pat. No. 4,659,678 (Forrest et al., "Immunoassay of Antigens"). See also US Pat. No. 4,376,110 (David et al., "Immunometric Assays Using Monoclonal Antibodies"). Suitable conditions for the formation of reagent-antibody complexes are known to those of skill in the art. Please refer to the same document. Monoclonal antibodies specific for the EML4-ALK fusion polypeptide can be used in "two-site" or "sandwich" assays to provide a single hybridoma cell line that serves as a source of both labeled and bound monoclonal antibodies. Have. Such an assay is described in US Pat. No. 4,376,110. The concentration of the detectable reagent must be sufficient to allow the binding of the EML4-ALK or TFG-ALK fusion polypeptide to be detectable relative to the background.

Antibodies useful in practicing the methods disclosed herein are solid supports suitable for diagnostic assays, such as beads made from materials such as latex or polystyrene, by known techniques such as precipitation methods. It can be attached to a plate, slide or well). Reagents that bind to antibodies or other ALK fusion polypeptide or cleaved ALK kinase polypeptide are further subjected to known techniques such as radiolabeling (eg, ³⁵ S, ¹²⁵ I, ¹³¹ I), enzyme labeling (eg, horseradish). It can be attached to detectable groups such as peroxidase, alkaline phosphatase) and fluorescent labels (eg, fluorescein).

Cell-based assays, such as flow cytometry (FC), immunohistochemistry (IHC), or immunofluorescence (IF), are clinically suitable for such assays and are mutated in vivo. Particularly in the practice of the methods of the invention, as it allows detection of expression of ALK kinase polypeptides and avoids the risk of artificial activity alterations that result in manipulation of cells obtained, for example, from tumor samples, to obtain extracts. desirable. Therefore, in some preferred embodiments, the methods of the invention are performed in the form of flow cytometry (FC), immunohistochemistry (IHC), or immunofluorescence (IF) assays.

Flow cytometry (FC) can be used to determine the expression of a mutant ALK kinase polypeptide in mammalian tumors before, during, and after treatment with agents that target inhibition of ALK kinase activity. .. For example, tumor cells derived from bone marrow samples may be analyzed by flow cytometry for the expression and / or activity of the EML4-ALK or TFG-ALK fusion polypeptide, and, if desired, for markers that identify cancer cell types and the like. Can be done. Flow cytometry can be performed by standard methods. See, for example, Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). That is, for example, the following protocol for cytometric analysis can be utilized; cell immobilization with 2% paraformaldehyde for 10 minutes at 37 ° C., followed by permeation in 90% methanol for 30 minutes on ice. Conversion. The cells are then stained with a primary antibody specific for the EML4-ALK or TFG-ALK fusion polypeptide, washed and labeled with a fluorescently labeled secondary antibody. The cells will then be analyzed on a flow cytometer (eg, Beckman Counter FC500) according to the specific protocol of the instrument used. Such an analysis would be able to identify the level of EML4-ALK or TFG-ALK fusion polypeptide expressed in the tumor. Similar analysis of tumors after treatment with ALK inhibitor therapeutics will reveal the responsiveness of tumors expressing ALK fusion polypeptides to targeted inhibitors of ALK kinase.

Immunohistochemistry (IHC) staining is also a mutant ALK kinase poly in mammalian cancers (eg, solid tumors such as NSCLC) before, during, and after treatment with agents that target inhibition of ALK kinase activity. It can be used to determine the state of expression and / or activity of a peptide. IHC can be performed according to known techniques. See, for example, ANTIBODIES: A LABOLATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). That is, for example, paraffin-embedded tissue (eg, tumor tissue taken by biopsy) is deparaffinized with xylene and then ethanol to deparaffinize tissue sections; water and then hydrated in PBS; citrate buffer Heat the slides in liquid to demask the antigen; incubate the sections in hydrogen peroxide; block in blocking solution; anti-EML4-ALK or anti-TFG-ALK fusion polypeptide primary antibody and secondary The slides are cultured in the antibody; and finally prepared for immunohistochemical staining by detection with ABC avidin / biotin according to the manufacturer's instructions.

An immunofluorescence (IF) assay also determines the state of expression and / or activity of a mutant ALK kinase polypeptide in mammalian cancer before, during, and after treatment with a drug that targets inhibition of ALK kinase activity. Can be used to. IF can be performed according to known techniques. See, for example, J.M. Polak and S. Van Noorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed .; ROYAL MICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37, BioScientific / Springer-Verlag. That is, for example, a patient's sample is immobilized in paraformaldehyde, then in methanol, blocked in a blocking solution such as horse serum, the primary antibody to the EML4-ALK or TFG-ALK fusion polypeptide, followed by Alexa488 It is cultured with a second antibody labeled with such a fluorescent dye and analyzed by epifluorescence microscopy.

The antibodies used in the above assays are for use in multi-parameter analysis with other signal conversion (eg, EGFR, phospho-AKT, phospho-Erk1 / 2) and / or cellular marker (eg, cytokeratin) antibodies. , Fluorescent dyes (eg, Alexa488, PE), or other labels such as quantum dots can be advantageously combined.

A wide variety of other protocols for measuring mutant ALK kinase polypeptide, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS), are in the art. Known in Japan, it provides the basis for diagnosing altered expression and abnormal levels of EML4-ALK or TFG-ALK fusion polypeptide. Normal or standard values of expression of these fusion polypeptides are EML4-ALK or TFG-ALK fusions of normal mammalian subjects, preferably human-derived body fluids or cell extracts, under conditions suitable for complex formation. It is confirmed by binding to an antibody against the polypeptide. The amount of standard complex formation can be quantified by a variety of methods, but photometric methods are preferred. The amount of EML4-ALK or TFG-ALK fusion polypeptide expressed in subjects, controls, and sick samples from biopsy tissue is compared to standard values. The deviation between the standard value and the subject's value establishes the parameters for diagnosing the disease.

Similar to peptide and nucleic acid assays , AQUA peptides for detecting / quantifying mutant ALK kinase polypeptides expressed in biological samples containing tumor-derived cells were prepared as detailed in column E above. It can be used in standard AQUA assays. Therefore, in some preferred embodiments of the methods of the invention, the ALK fusion polypeptide-specific reagent is a fusion of an EML4-ALK fusion polypeptide or a TFG-ALK fusion polypeptide, as described in column E above. It contains a heavy isotope-labeled phosphopeptide (AQUA peptide) corresponding to the peptide sequence consisting of the junction.

A reagent specific for a mutated ALK polypeptide useful for carrying out the method of the present invention can be detected by directly hybridizing to a transcript expressing a polypeptide fused or cleaved in a biological sample. It may be an oligonucleotide or a DNA probe. Such probes are discussed in detail in column B above. That is, for example, a sample of a patient immobilized with formalin and embedded in paraffin can be searched with a fluorescently labeled RNA probe, washed with formamide, SSC and PBS, and analyzed with a fluorescence microscope. .. FISH probes containing cut-point probes capable of fluorescent detection of gene rearrangements, such as EML4-ALK deletion mutations on chromosome 2, are also preferred (see Example 6).

A polynucleotide encoding a mutated ALK kinase polypeptide can also be used for diagnostic purposes. Polynucleotides that can be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNA. This polynucleotide presents gene expression in biopsy-collected solid tumor tissue where expression of the EML4-ALK or TFG-ALK fusion polypeptide or truncated active ALK kinase polypeptide appears to be associated with the disease. It can be used for detection and quantification. For example, this diagnostic assay is used to identify the presence, absence, and overexpression of EML4-ALK or TFG-ALK fusion polypeptides, and to observe control of levels of ALK fusion polypeptides during therapeutic intervention. Can be used for.

In one preferred embodiment, hybridization with a PCR probe capable of detecting a polynucleotide sequence containing a genomic sequence and encoding an ALK fusion polypeptide or a cleaved ALK kinase polypeptide or a molecule similar thereto is a mutant ALK polypeptide. Can be used to identify the nucleic acid sequence encoding. The construction and use of such probes is described in column B above. The specificity of the probe, i.e. whether it was created from a region of high specificity, eg, 10 unique nucleotides of the fusion junction, or region of low specificity, eg, a 3'coding region, and hybridization or The severity of amplification (highest, highest, moderate or low) determines whether this probe identifies only the native sequence, allele, or related sequence encoding the mutant ALK polypeptide. Will.

The probe can also be used to detect the relevant sequence and preferably contains at least 50% of nucleotides from any of the mutant ALK polypeptides encoding the sequence. The hybridization probe of the present invention may be derived from DNA or RNA and the nucleotide sequences of SEQ ID NOs: 2, 19, and 21, most preferably fusion junctions (FIGS. 1AC, bottom figure and SEQ ID NO: 7, 24, and 26), or derived from genomic sequences that include promoters, enhancer components, and introns of native EML-4, TFG, and ALK polypeptides, as further described in column B above. Includes.

For example, the EML4-ALK fusion polynucleotides of the invention use a body fluid or tissue taken from a patient's bioassay to detect the expression of a modified ALK polypeptide, Southern or Northern analysis, dot blot, or other. It can be used in membrane-based techniques; in PCR techniques; or in dipstick, pin, ELISA or chip assays. Such qualitative or quantitative methods are known in the art. In certain embodiments, the nucleotide sequences encoding the mutant ALK polypeptides of the invention will be useful in assays that detect activation and induction of a wide variety of cancers, including lung cancer. Mutant ALK polynucleotides can be labeled by standard methods and added to patient-derived body fluids or tissues under conditions suitable for the formation of hybridization complexes. After an appropriate culture period, the sample is washed, the signal is weighed and compared to standard values. If the amount of signal from the biopsy-derived or extracted sample was significantly different from that of the comparable control sample, then this nucleotide sequence hybridized with the nucleotide sequence in the sample and the EML4-ALK fusion in the sample. The presence of altered levels of the nucleotide sequence encoding the polypeptide or the cleaved ALK kinase polypeptide indicates the presence of the associated disease. Such assays can also be used to assess the effectiveness of specific treatment treatment schemes in animal experiments, clinical trials, or observations of treatment for individual patients.

Establish a normal or standard profile for expression to provide the basis for the diagnosis of diseases characterized by the expression of a mutant ALK kinase polypeptide. It is suitable for hybridization or amplification of a body fluid or cell extract from a normal subject, either animal or human, with a sequence encoding an EML4-ALK or TFG-ALK fusion polypeptide, or a fragment thereof. It can be accomplished by mixing under certain conditions. Standard hybridization can be quantified by comparing values obtained from normal subjects with those obtained in experiments with known amounts of substantially purified polynucleotides. Standard values obtained from a sample of a normal person can be compared with values obtained from a sample of a patient showing a medical condition. The deviation between the standard value and the subject's value is used to establish the presence of the disease.

Once the disease has been demonstrated and the treatment protocol has begun, the hybridization assay can be repeated regularly to assess whether the level of expression in the patient has begun to approach the level obtained from normal patients. The results obtained from the series of assays can be used to show the effect of treatment over a period ranging from days to months.

Further diagnostic uses of the mutant ALK polynucleotides of the invention may relate to the use of the polymerase chain reaction (PCR), a standard and preferred assay format for those skilled in the art. See, for example, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition, Sambrook, J., Fritsch, E.F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). PCR oligomers can be chemically synthesized, enzymatically produced, or produced from recombinant sources. Oligomers consist of two nucleotide sequences, one in the sense direction (5'to 3') and the other in the antisense direction (3'to 5'), making them ideal for identifying specific genes or conditions. It would be preferable to use it under the conditions that have been modified. Two identical oligomers, nested aggregates of oligomers, or even degenerate pools of oligomers can be used to detect and / or quantify similar DNA or RNA sequences under non-stringent conditions.

Methods that can also be used to quantify the expression of ALK fusion polypeptides or cleaved ALK kinase polypeptides include radiolabeled or biotinylated nucleotides, co-amplification of control nucleic acids, and a standard curve into which experimental results are inserted. (Melby et al., J. Immunol. Methods, 159: 235-244 (1993); Duplaa et al. Anal.Biochem. 229-236 (1993)). The rate of quantification of multiple samples can be accelerated by assaying in an ELISA format in which the oligomer of interest is present at various dilutions, and the spectrophotometric or colorimetric response results in fast quantification.

In another aspect of the invention, the mutant ALK polynucleotides of the invention, and also adjacent genomic regions proximal and distal to them, generate hybridization probes useful for mapping native genomic sequences. Can be used to This sequence can be mapped to a particular gene or a specific region of a gene using known techniques. Such techniques are fluorescent in situ hybridization as outlined in Price, CM, Blood Rev. 7: 127-134 (1993) and Trask, BJ, Trends Genet. 7: 149-154 (1991). (FISH), FACS, or artificial chromosome constructs such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructs or single chromosome cDNA libraries.

In one preferred embodiment, the FISH method is used (as described in Verma et al. HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York, NY (1988)) and other physical chromosomal mappings. Can be associated with technology and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 1981f). What is the correlation between the location of a gene encoding an EML4-ALK or TFG-ALK fusion polypeptide or a cleaved active ALK kinase polypeptide on a physical chromosomal map and a particular disease or predisposition to a particular disease? , Will help distinguish regions of DNA associated with genetic disorders. Such nucleotide sequences of the present invention can be used to detect differences in gene sequences between normal, carrier, or affected individuals. A bicolor cut-point FISH probe can be used, for example, to detect the presence of mutant EML-4, TFG, and / or ALK genes in a sample.

Physical mapping techniques such as in situ hybridization of chromosomal preparations and linkage analysis with established chromosomal markers can be used to extend the genetic map. In many cases, gene substitutions on the chromosomes of other mammalian species, such as mice, can reveal relevant markers, even if the number and arm of a particular human chromosome is unknown. The new sequence can be assigned to the arm or part of the chromosome by physical mapping. This provides valuable information to researchers examining disease genes using positional cloning or other gene discovery techniques. When a disease or syndrome is loosely localized by genetic linkage from a particular genetic region, eg, AT to 11q22-23 (Gatti et al., Nature 336: 577-580 (1988)), it is mapped to that region. Any sequence can indicate a related or regulatory gene for further study. The nucleotide sequences of the present invention can also be used to detect differences in chromosomal position due to translocation, inversion, etc. between normal, carrier, or affected individuals.

Other methods suitable for the detection of nucleic acids, such as subgroove-bound conjugated oligonucleotide probes (see, eg, US Pat. No. 6,951,930, "Hybridization-Triggered Fluorescent Detection of Nucleic Acids") , Known to those skilled in the art.

Biological Samples Biological samples useful in the practice of the methods of the invention can be obtained from either mammals in the presence or development of cancer characterized by the expression of EML4-ALK or TFG-ALK fusion polypeptides. In some embodiments, the mammal may be a human, who may be a candidate for ALK inhibitory therapy for the treatment of cancer, eg, NSCLC. The human candidate may be a patient who has recently been treated with or is considered to be treated with an ALK kinase inhibitor such as WHI-131 and / or WHI-154. In another aspect, the mammal is a large animal, such as a horse or cow, while in another aspect, the mammal is a small animal, such as a dog or cat, all of which express cancer, including lung cancer. It is known to do.

Any biological sample containing cells (or cell extracts) from mammalian cancer is suitable for use in the methods of the invention. In the case of the EML4-ALK fusion polypeptide, any cancer, solid or non-solid, will be suitable. In the case of TFG-ALK, solid tumors are within the method of the invention. For example, the biological sample may contain cells obtained from an exudate such as pleural effusion. Pleural effusion (a fluid that forms in the abdominal cavity and outside the lungs and contains cancer cells) is known to form in many patients with advanced lung cancer (including NSCLC). Thus, the presence of such exudates heralds poor outcomes and short survival. Standard techniques for obtaining pleural effusion samples have been described and are known in the art (see Sahn, Clin Chest Med. 3 (2): 443-52 (1982)). Circulating tumor cells can also be referred to as tumor markers, cytokeratin protein markers or other methods of negative selection as described (Ma et al., Anticancer Res. 23 (1A): 49-62 (2003)). Can be obtained from serum using. Serum and bone marrow samples are particularly preferred for leukemia patients. For cancers, including solid tumors, such as sarcomas and carcinomas, biological samples can contain cells obtained from tumor biopsies, which can be obtained by standard clinical techniques. Abnormal expression of ALK is observed in a variety of cancers, including, for example, neuroblastoma and neuroectoderm cancer. See, for example, Pulford et al., Supra. However, the TFG-ALK translocation mutant has been described to be found only in lymphomas and has not been observed in solid tumors to date.

The biological sample can contain cancer-derived cells (or cell extracts) that express and / or activate the ALK fusion polypeptide but do not express and / or activate the wild-type ALK kinase. The sample also expresses and / or activates both the mutant ALK polypeptide and the wild-type ALK kinase, or expresses and / or activates the wild-type ALK kinase and / or EML-4 and / or TFG. It can also contain cancer-derived cells that are kinased but do not express and / or activate the mutant ALK polypeptide.

The cell extracts of the biological samples described above can be prepared according to standard techniques into either crude or partially (or completely) purified and used in the methods of the invention. In addition, biological samples containing whole cells include immunohistochemistry (IHC), flow cytometry (FC), immunofluorescence (IF) and fluorescence in situ hybridization (FISH) as described above. Can be used in preferred assay formats such as. Such whole-cell assays minimize the manipulation of tumor cell samples, thereby reducing the risk of changes in cellular signaling / activation status and / or the introduction of anthropogenic signals in vivo. It is advantageous in that. Whole-cell assays are also advantageous in that they are expressed and signaled and characterized only in tumor cells, not in a mixture of tumor cells and normal cells.

A mammalian bone marrow transplant model or xenotransplantation in the practice of the disclosed method for determining whether a compound inhibits tumor progression characterized by an EML4-ALK or TFG-ALK fusion gene and / or fusion polypeptide. Biological samples containing hemi-derived cells can also be used advantageously. A preferred xenograft (or transplant recipient) is that of a small mammal, such as a mouse, transplanted with a human tumor expressing a mutant ALK kinase polypeptide. Xenografts transplanted with human tumors are known in the art (see Kal, Cancer Treat Res. 72: 155-69 (1995)) and xenografts transplanted with human tumors. The production of pieces is well described (see Winograd et al., In Vivo. 1 (1) 1-13 (1987)). Similarly, the generation and use of bone marrow transplantation models is well documented (eg, Schwaller, et al., EMBO J. 17: 5321-333 (1998); Kelly et al., Blood 99: 310-318. (2002)). "Cancers characterized by EML4-ALK or TFG-ALK fusion polynucleotides and / or fusion polypeptides" are such as compared to cancers in which such fusion genes and / or fusion polypeptides are not present. Indicates a cancer in which a mutant ALK gene and / or an expressed polypeptide is present.

Control samples showing cells that do not produce such translocations and / or fusion proteins in assessing the presence of ALK polynucleotides or expression of polypeptides in biological samples containing cells from mammalian cancer tumors are for comparison purposes. Can be preferably used in. Ideally, the control sample is a subset of a particular cancer (eg, NSCLC) that is symbolic of a subset that does not develop mutations (eg, EML4-ALK deletion mutations) and / or does not express the fusion polypeptide. Contains cells derived from. The presence or absence of mutant ALK polynucleotides and / or polypeptides is identified by comparing the levels in the control sample with those in the test biological sample. Alternatively, since EML4-ALK and / or TFG-ALK fusion polynucleotides and / or polypeptides are absent in most cancers, they also express (or mutated polynucleotides) such mutant ALK polypeptides. Any of the non-supporting tissues can be used as a control.

The methods below will have valuable diagnostic uses for determining cancers characterized by mutant ALK polynucleotides and / or polypeptides, and treatment methods for them. For example, a biological sample has never been previously diagnosed with a cancer characterized by an EML4-ALK deletion mutation and / or fusion polypeptide and has never been treated for such a cancer. A method can be obtained from a subject, and the method is considered diagnostically for a tumor of interest that is considered a subset of the tumor in which the EML4-ALK fusion polynucleotide and / or polypeptide is present and / or expressed (eg, NSCLC). Used to identify. The method of the invention is to observe the progression or inhibition of a cancer expressing a mutant ALK kinase polypeptide after treating a subject with a therapeutic agent or composition containing a therapeutic agent combination that inhibits ALK kinase. Can also be used.

Such diagnostic assays can be performed after or before preliminary evaluation or surgical diagnostic procedures. The identification methods of the present invention are advantageous for diagnosis to identify patients with cancer, such as NSCLC, promoted by EML4-ALK and / or TFG-ALK fusion proteins or cleaved ALK kinases. Such patients can be used and are most likely to respond to therapeutic agents that target to inhibit ALK kinase activity, such as WHI-131 and / or WHI-154 or their relatives. right. The ability to select such patients will be useful in clinically assessing the efficacy of next-generation ALK-targeting agents, as well as in future prescribing of such agents to patients.

The ability to selectively identify cancers in which a diagnostic EML4-ALK and / or TFG-ALK fusion polynucleotide and / or fusion polypeptide is present is important for the accurate identification of such cancers for diagnostic purposes. Inhibitions targeting different kinases when new methods are also applied, and when such cancers are likely to respond to therapeutic compositions that inhibit ALK, or when administered as a single agent to treat cancer. It also makes it possible to obtain useful information in determining whether a drug is unlikely to respond partially or at all.

Thus, in some embodiments, the invention provides a method of detecting the presence of a mutated ALK polynucleotide and / or a mutated ALK polypeptide encoded by it in a biological sample derived from mammalian cancer, the method of which is step:
(A) Obtaining a biological sample from mammalian cancer; and (b) At least one that contains a fusion polynucleotide, or portion of ALK and a portion of secondary protein, and detects a fusion polypeptide encoding it. Using a reagent to determine if an ALK mutant polynucleotide and / or a mutant ALK polypeptide encoding it is present in the biological sample:
Includes.

In one preferred embodiment, the cancer is solid sarcoma or carcinoma, and in one embodiment, the carcinoma is lung cancer, such as NSCLC. In another preferred embodiment, the mutated ALK polypeptide is a fusion polypeptide comprising residue 1116-1383 of ALK (SEQ ID NO: 5) with a portion of the secondary protein. In another preferred embodiment, the secondary protein is selected from the group consisting of EML-4 (SEQ ID NO: 3) and TRK-fusion gene (TFG) protein (SEQ ID NO: 22). In yet another preferred embodiment, the fusion polypeptide comprises residue 1-233 or residue 1-495 of EML-4 (SEQ ID NO: 3) or residue 1-138 of TFG (SEQ ID NO: 22).

In another preferred embodiment, the fusion polynucleotide comprises an EML4-ALK fusion polynucleotide (SEQ ID NO: 2 or 19) or a TFG-ALK fusion polynucleotide (SEQ ID NO: 21), and in yet another embodiment, the fusion polypeptide. Peptides include the EML4-ALK fusion polypeptide (SEQ ID NO: 1 or 18) or the TFG-ALK fusion polypeptide (SEQ ID NO: 20). In yet another preferred embodiment, the fusion polynucleotide is the EML4-ALK fusion polynucleotide or polypeptide described above.

In a more preferred embodiment, the method uses a reagent comprising an EML4-ALK fusion polynucleotide and / or at least one EML4-ALK fusion polypeptide-specific reagent (antibody or AQUA peptide) as described above. In some preferred embodiments, the reagent specifically binds to or detects a TFG-ALK fusion polypeptide (SEQ ID NO: 20) or TFG-ALK fusion polynucleotide (SEQ ID NO: 21), but in the wild type. Includes isolated reagents that do not bind to or detect either TFG or wild-type ALK. In another preferred embodiment, the reagent is a polymerase chain reaction (PCR) probe or a fluorescence in situ hybridization (FISH) probe. In one preferred embodiment, a heavy isotope-labeled (AQUA) peptide comprising the amino acid sequence of a fusion junction of a TFG-ALK fusion polypeptide or a cut point within a wild-type ALK is used.

In some preferred embodiments, the diagnostic methods of the invention are incorporated into the assay form of flow cytometry (FC), immunohistochemistry (IHC), or immunofluorescence (IF), as described above. In another preferred embodiment, the activity of the EML4-ALK or TFG-ALK fusion polypeptide is detected. In another preferred embodiment. The diagnostic methods of the present invention are incorporated into the fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) assay format as described above.

The invention further provides a method of determining whether a compound inhibits the progression of cancer characterized by an EML4-ALK or TFG-ALK fusion polynucleotide and / or fusion polypeptide, which method is said to be relevant. It comprises the step of determining whether the compound inhibits the expression and / or activity of the EML4-ALK or TFG-ALK fusion polypeptide in the cancer. In certain preferred embodiments, inhibition of expression and / or activity of the ALK fusion polypeptide is an EML4-ALK fusion polynucleotide or polypeptide of the invention and / or the TFG-ALK fusion polynucleotide or poly described herein. Judgment is made using at least one reagent that detects the peptide. Compounds suitable for inhibiting ALK kinase activity are investigated in more detail in column G below.

Mutant ALK polynucleotide probes and polypeptide-specific reagents useful for practicing the methods of the invention are described in more detail in columns B and D above. In one preferred embodiment, the reagent specific for the ALK fusion polypeptide comprises an antibody specific for the fusion polypeptide. In another preferred embodiment, the reagent specific for the fusion polypeptide is a heavy isotope-labeled phosphopeptide corresponding to the fusion junction of the ALK fusion polypeptide (see FIGS. 1AC (bottom)). AQUA peptide) is contained. In yet another preferred embodiment, the fusion polynucleotide-specific reagent comprises a fusion junction of the ALK fusion gene and / or a FISH probe corresponding to the wild-type EML4, TFG, or cleavage point of the ALK gene.

The method of the invention described above optionally comprises determining the level of expression or activation of other kinases, such as wild-type ALK and EGFR, or other downstream signaling molecules in the biological sample. You may. Elucidation of the expression / activation of ALK fusion polypeptides and the expression / activation of other kinases and pathways in a given biological sample is most beneficial in which kinases and pathways promote the disease and which treatment regimen Can provide valuable information about what is possible.

Screening of Compounds The discovery of novel EML4-ALK fusion polypeptides described herein also allows the development of novel compounds that inhibit the activity of this mutant ALK protein, especially its ALK kinase activity. Accordingly, the present invention provides, in one aspect, a method of determining whether a compound inhibits the progression of a cancer characterized by an EML4-ALK fusion polynucleotide and / or a fusion polypeptide, which method is said to be relevant. It comprises the step of determining whether the compound inhibits the expression and / or activity of the EML4-ALK fusion polypeptide in the cancer. In certain preferred embodiments, inhibition of expression and / or activity of the EML4-ALK fusion polypeptide is determined using at least one reagent that detects the fusion polynucleotide and / or fusion polypeptide of the invention. The preferred reagents of the present invention are described above. Compounds suitable for inhibiting ALK kinase activity are described in more detail in column G below.

The compound may be a kinase inhibitor, such as, for example, a small molecule or antibody inhibitor. It may be a pan-kinase, or a kinase-specific inhibitor, that has activity against several different kinases. Compounds that inhibit ALK kinase are described in more detail in column G below. Biological samples of the patient are taken before and after treatment with the inhibitor, and then the biological effects of the inhibitor on ALK kinase activity, including phosphorylation of downstream substrate proteins, are analyzed using the methods described above. Such a pharmacodynamic assay would be useful in determining the bioactive dose of the drug, which is preferred as the maximum acceptable dose. Such information may also be useful in clarifying the mechanism of drug action and applying for a drug license. The identification of compounds with such desirable inhibitory properties is further described in column G below.

G. Therapeutic Inhibition of Cancer According to the present invention, the progression of mammalian solid cancer tumors (NSCLC) in which the EML4-ALK fusion protein is expressed, by inhibiting the activity of ALK kinase in such cancers in vivo. , It was shown that it can be stopped. Similarly, as described herein, the activity of mammalian solid tumors in which the TFG-ALK fusion protein is expressed by inhibiting the activity of ALK kinase in such cancers in vivo. Can be inhibited. Cancer characterized by the expression of a mutant ALK polypeptide by contacting the cancer (eg, tumor) with a therapeutic agent that inhibits ALK kinase, such as a small molecule kinase inhibitor such as WHI-131 or WHI-154. Can inhibit ALK activity in. As further described in Example 2 below, inhibition of growth of tumors expressing ALK fusion proteins is achieved, for example, by inhibiting fusion kinases with siRNA, a typical ALK-inhibiting therapeutic agent. Can be achieved. Therefore, the present invention, in one aspect, is a solid expressing a cancer or TFG-ALK fusion polypeptide expressing an EML4-ALK fusion polypeptide by inhibiting the expression and / or activity of a mutant ALK kinase in the cancer. Provided is a method of inhibiting the progression of a tumor.

Therapeutic agents that inhibit ALK kinase directly or indirectly inhibit the expression and / or activity of ALK kinase in vivo, including the following exemplary compounds, biologically or chemically. It may be any of the compositions containing at least one of the compounds. Such compounds include therapeutic agents that act directly on ALK kinase itself, or on proteins or molecules that alter the activity of ALK, or act indirectly by inhibiting the expression of ALK. Such compositions include compositions containing only a single ALK kinase inhibitor compound, as well as compositions containing multiple therapeutic agents, including those for other RTKs. It may contain non-specific therapeutic agents, such as chemotherapeutic agents or general transcriptional inhibitors.

Small Molecule Inhibitors In some preferred embodiments, ALK-inhibiting therapeutic agents useful in carrying out the methods of the invention include small molecule inhibitors such as WHI-131 and WHI-154, or analogs thereof. I'm targeting. WHI-131 and WHI-154 are quinazoline-type small molecules that target inhibitors of ALK and their properties have been described. See Marzec et al., Lab. Invest. 85 (12): 1544-54 (2005). These compounds have been shown to induce apoptosis of lymphoma cells and suppress their growth. Other small molecules targeting kinase inhibitors are known in the art. For example, Gleevec®, which specifically binds and blocks the ATP-binding site of BCR-ABL fusion kinases (as well as other kinases) and thereby inhibits phosphorylation and activation of this enzyme, STI-571. , Imatinib) is commercially available and its properties are well known. See, for example, Dewar et al., Blood 105 (8): 3127-32 (2005). Other small molecule inhibitors of ALK are currently under development at Novartis, Inc. and Cephalon Inc.

Inhibitors targeting small molecules specifically and often irreversibly bind to the catalytic site of the enzyme and / or inhibit the constellation-derived enzyme required for its activity, ATP within the enzyme. -A class of molecules that bind to a binding gap or other binding site and primarily inhibit the activity of the enzyme it targets. Small molecule inhibitors can be reasonably designed using X-ray crystallography or computer modeling of the three-dimensional structure of ALK kinase, or can be found by high-speed, high-volume screening of compound libraries for inhibition of ALK. Such methods are known in the art and have already been described. The specificity of ALK inhibition is, for example, by testing the ability of such compounds to inhibit ALK activity but not other kinase activities in the kinase group, and / or as described above, lung tumor cells. It can be confirmed by testing the inhibition of ALK activity in the contained biological sample. Such screening methods are further described below.

Antibody Inhibitors The therapeutic agents that inhibit ALK kinase, which are useful in the methods of the invention, specifically bind to the critical catalytic sites or domains required for ALK activity to allow secondary proteins to approach ligands, substrates or ALK. Antibodies can also be targeted that inhibit the kinase by interfering and / or by inhibiting the enzyme from the coordination required for its activity. The production, screening and therapeutic use of antibodies specific for humanized targets is well documented. See Merluzzi et al., Adv. Clin. Path. 4 (2): 77-85 (2000). Commercially available technologies such as Morphosys, Inc.'s Human Combinatorial Antibody Library (HuCAL®) for the production and screening of antibodies that specifically inhibit humanized targets at high speed and high volume. The system is available.

The production of various anti-receptor kinase target antibodies and their use for inhibiting the activity of the target receptor has been described. For example, US Patent Application Publication No. 20040202655 published on October 14, 2004, "Antibodies to IGF-I Receptor for the Treatment of Cancers", Morton et al .; US Patent Application Publication No. published on April 15, 2004. 2004086503, "Human anti-Epidermal Growth Factor Receptor Single-Chain Antibodies", Raisch et al .; US Patent Application Publication No. 20040033543, published February 19, 2004, "Treatment of Renal Carcinoma Using Antibodies Against the EGFr", See Schwab et al. Standardized methods for the production and use of antibodies that inhibit receptor tyrosine kinase activity are known in the art. See, for example, European Pat. No. 14,234,428, registered June 2, 2004, "Antibodies that Block Receptor Tyrosine KInase Activation, Methods of Screening for and Uses Thereof", Borges et al.

Phage presentation methods can also be utilized to generate ALK-specific antibody inhibitors, a well-known reference for protocol and recombinant antibody selection for bacteriophage library constructs, CURRENT PROTOCOLS IN. It is described in IMMUNOLOGY, Colligan et al. (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1. US Pat. No. 6,319,690, registered November 20, 2001, Little et al .; US Pat. No. 6,300,064, registered October 9, 2001, Knappik et al .; November 1998. See also U.S. Pat. No. 5,840,479, registered 24th, Little et al .; U.S. Patent Application Publication No. 2003219839, published November 27, 2003, Bowdish et al.

A library of antibody fragments displayed on the surface of bacteriophage can be produced (see, eg, US Pat. No. 6,300,064, registered October 9, 2001, Knappik et al.). Binding of the receptor protein tyrosine kinase (such as ALK) to soluble dimeric forms can be screened. Antibody fragments that bind to the soluble dimeric form of RTK used in screening are identified as candidate molecules that interfere with the structural activation of target RTK in cells. See European Pat. No. 14,234,428, Borges et al., Supra.

Antibodies targeting ALK binding identified by screening antibody libraries as described above are for their ability to inhibit ALK activity both in vitro kinase assays and in vivo cell lines and / or tumors. Further screening is possible. Inhibition of ALK includes, for example, the ability to inhibit the ALK kinase activity of such antibody therapeutics, but not the activity of other kinases in the kinase group, and / or, as described above, cancer cells. Inhibition of ALK activity in living biological samples can be tested and confirmed. Methods for screening such compounds for ALK kinase inhibition are described above.

Indirect Inhibitors ALK-inhibiting compounds useful in carrying out the disclosed methods are compounds that indirectly inhibit ALK activity by inhibiting the activity of other proteins or molecules other than ALK kinase itself. May be good. Such inhibitory therapeutics target inhibitors that regulate the activity of major regulatory kinases that phosphorylate or dephosphorylate (and activate or deactivate) ALK itself, or interfere with ligand binding. Can be. Like other receptor tyrosine kinases, ALK regulates downstream signaling via a network of adapter proteins and downstream kinases, including STAT5 and AKT. As a result, the induction of cell growth and survival by ALK activity can be inhibited by targeting these interactions or downstream proteins. Among the agents currently under development for use in this manner include Wartmanin.

ALK kinase activity can also be indirectly inhibited by using a compound that inhibits the binding of the activating molecule required to make ALK its active conformation. Similarly, for example, the production and use of anti-PDGF antibodies that down-regulate the PDGF receptor tyrosine kinase has been described. See U.S. Patent Application Publication No. 2003219839, "Anti-PDF Antibodies and Methods for Producing Engineered Antibodies", Bowdish et al.

Indirect inhibitors of ALK activity can be reasonably designed using X-ray crystallography or computer modeling of the three-dimensional structure of ALK, or lead to inhibition of ALK kinase, major upstream regulatory enzymes and / or It can be found by screening fast mass processing of compound libraries for inhibition of the required binding molecule. Such means are known in the art and have already been described. Inhibition of ALK by such therapeutic agents, for example, tests the ability of compounds that inhibit ALK activity but not other kinase activities in the kinase group and / or as described above, cancer cells such as NSCLC. Inhibition of ALK activity in biological samples containing cells can be tested and confirmed. Methods for identifying cancer-inhibiting compounds characterized by EML4-ALK or TFG-ALK fusion polynucleotides and / or fusion polypeptides are further described below.

Therapeutic agents that inhibit the antisense and / or the transcription inhibitor ALK enhance ALK kinase activity by inhibiting the transcription of the ALK and / or EML4-ALK or TFG-ALK fusion gene or the gene encoding the truncated ALK gene. It may contain antisense and / or transcriptional inhibitory compounds that inhibit. For example, inhibition of a wide variety of receptor kinases, including VEGFER, EGFR, and IGFR, and FGFR, by antisense therapies for the treatment of cancer has already been described. For example, U.S. Pat. Nos. 6,734,017, 6,710,174, 6,617,162, 6,340,674, 5,783,683, and 5,783,683. See Nos. 5,610,288.

Antisense oligonucleotides can be designed, constructed and used as therapeutic agents for target genes according to known techniques. For example, Cohen J., Trends in Pharmacol. Sci. 10 (11): 435-437 (1989); Marcus-Sekura, Anal. Biochem. 172: 289-295 (1988); Weintraub, H., Sci. AM. pp.40-46 (1990); Van Der Krol et al., Bio Techniques 6 (10): 958-976 (1988); Skorski et al., Proc. Natl. Acad. Sci. USA (1994) 91: 4504 See -4508. It has recently been described that EGER's antisense RNA inhibitors are used to inhibit the growth of human carcinoma in vivo. See US Patent Application Publication No. 20040047847, published March 11, 2004, "Inhibition of Human Squamous Cell Carcinoma Growth in vivo by Epidermal Growth Factor Receptor Antisense RNA Transcribed from Pol III Promoter", He et al. Similarly, the mammalian ALK gene (see FIG. 4 (SEQ ID NO: 6)) or EML4-ALK or TFG-ALK fusion polynucleotide or cleaved ALK polynucleotide (FIGS. 2AC (SEQ ID NOs: 2, 19 and 21). An ALK-inhibiting therapeutic agent containing at least one of the antisense oligonucleotides against) can be prepared according to the method described above. A pharmaceutical composition containing an antisense compound that inhibits ALK can be prepared. , Can be administered as further described below.

Small interfering RNA
Small interfering RNA molecules (siRNAs) that inhibit translation through RNA interference processes and thereby inhibit the activity of ALK can also preferably be used in the methods of the invention. Selective silencing of the target gene by RNA interference and the introduction of exogenous low molecular weight double-stranded RNA containing a sequence complementary to the mRNA encoding the target protein is well documented. For example, US Patent Application Publication No. 20040038921 published February 26, 2004, "Composition and Method for Inhibiting Expression of a Target Gene", Kreutzer et al .; US Patent Application Publication No. 200886356 published June 12, 2003. See, "RNA Sequence-Specific Mediators of RNA Interference", Tuschl et al .; US Patent Application Publication No. 20040229266, published November 18, 2004, "RNA Interference Mediating Small RNA Molecules", Tuschl et al. I want to.

For example, as shown herein (see Example 2), siRNA-mediated silencing of EML4-ALK fusion protein expression in human NSCLC cell lines expressing the fusion protein is diseased in these cells. Although it selectively inhibited the progression of the disease, it did not inhibit the progression of the disease in control cells that did not express the mutant ALK protein.

Double-stranded RNA molecules (dsRNA) have been shown to inhibit gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). That is, RNAse III Dicer processes the dsRNA into a small interfering RNA (siRNA) of approximately 22 nucleotides, which serves as a guide sequence to induce target-specific mRNA cleavage by the RNA-induced silencing complex RISC. It works (see Hammond et al., Nature (2000) 404: 293-296). RNAi is involved in a catalytic reaction in which new siRNAs are produced by continuous cleavage of longer dsRNAs. Therefore, unlike antisense, RNAi degrades target RNA in a non-stoichiometric way. It has been shown that when administered to cells or organs, exogenous dsRNA promotes sequence-specific degradation of exogenous messenger RNA (mRNA) via RNAi.

A wide variety of target-specific siRNA products, including vectors and systems for their expression and use in mammalian cells, are currently available and can be used in mammalian cells. See, for example, Promega, Inc. (promega.com); Dharmacon, Inc. (dharmacon.com.). Detailed technical manuals for the design, construction and use of dsRNA for RNAi are available. See, for example, Dharmacon's "RNAi Thechnical Reference & Application Guide"; Promega's "RNAi: A Guide to Gene Silencing." SiRNA products that inhibit ALK are also commercially available and are suitable for use in the methods of the invention. For example, Dharmacon, Inc., Lafayette, CO (Cat Nos. M-003162-03, MU-003162-03, D-003162-07 thru -10; siGENOME® SMART selection and SMARTpool® siRNAs) Please refer.

The dsRNA optimally contains at least one sequence having at least one 1-4 nucleotide overhang at its end, which is substantially homologous to a portion of the target mRNA sequence, 49 in length. It has recently been demonstrated that small dsRNAs, shorter than nucleotides, preferably 19-25 nucleotides, are most effective in mediating RNAi in mammals. See U.S. Patent Application Publication No. 20040038921, Kreutzer et al., Supra; U.S. Patent Application Publication No. 2004022926, Tuschl et al., Supra. The constructs of such dsRNAs, and their use in pharmaceutical formulations for eliminating expression of target proteins in vivo, are described in detail in the above literature.

If the sequences of the genes of interest in mammals are known, 21-23 ntRNAs produce them, for example, those that mediate RNAi in mammalian cells, such as human or other primate cells. Can be tested for ability. Those 21-23 ntRNA molecules that have been shown to mediate RNAi can optionally be tested in a suitable animal model to further assess their in vivo effects. Known target sites, such as sites that have been determined to be effective target sites based on studies with other nucleic acid molecules, such as ribozymes or antisense, or sites that contain mutations or deletions. These targets, such as those known to be associated with the disease or disease, can also be used to design siRNAs that target these sites.

Alternatively, a valid dsRNA sequence can be reasonably designed / predicted screened for the target mRNA of interest for the target site, eg, using a computer-incorporated algorithm. Target sequences are analyzed in a computer using a custom Perl script or a commercially available sequence analysis program such as Oligo, MacVector, or GCG Wisconsin Package and subsequences of the entire fragment or of a particular length, such as 23 nucleotides. It can be a list of fragments.

A variety of parameters can be used to determine which site in the target RNA sequence is the most suitable target site. These parameters include, but are not limited to, the secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between the various regions of the target sequence, or the relative position of the target sequence in the RNA copy. To do. Based on these measurements, any number of target sites in the RNA copy can be selected to screen for the efficacy of siRNA molecules, eg, using in vitro RNA cleavage assays, cell cultures, or animal models. it can. See, for example, US Patent Application Publication No. 20130178091, published September 11, 2003, McSwiggen J. Algorithms for identifying and selecting RNAi target sites have also recently been described. See U.S. Patent Application Publication No. 20040236517, "Selection of Target Sites for Antisense Attack of RNA," published November 25, 2004, Drlica et al.

Commonly used gene transfer techniques include calcium phosphate, DEAE dextran, electroporation and microinjection and viral methods (Graham et al., (1973) Virol. 52: 456; McCutchan et al., (1968), J. . Natl. Cancer Inst. 41: 351; Chu et al. (1987), Nucl. Acids Res. 15:1311; Fraley et al. (1980), J. Biol. Chem. 255: 10431; Capecchi (1980), cell 22:479). DNA can also be introduced into cells using cationic liposomes (Feigner et al. (1987), Proc. Natl. Acad. Sci. USA 84: 7413). Commercially available cationic lipid formulations include Tfx50 (Promega) or Lipofectamin 200 (Life Technologies). Viral vectors can also be used to deliver dsRNA to cells and intervene RNAi. See U.S. Patent Application Publication No. 20040023390, published February 4, 2004, "siRNA-mediated Gene Silencing with Viral Vectors", Davidson et al.

RNAi transfection and vector / expression systems in mammalian cells are commercially available and well described. See, for example, Dharmacon, Inc., DharmaFECT® system; Promega, Inc., siSTRIKE® system. Gou et al. (2003) FEBS. 548, 113-118; Sui et al. A DNA vector-based RNAi technology to supress gene expression in mammalian cells (2002) Proc. Natl. Acad. Sci. 99, 5515-5520; Yu et al. (2002) Proc. Natl. Acad. Sci. 99, 6047-6052; Paul, C. et al. (2002) Nature Biotechnology 19,505-508; McManus et al. (2002) RNA 8, 842-850 See also.

SiRNA interference in mammals using the prepared dsRNA molecules is achieved by administering to the mammal a pharmaceutical formulation containing the dsRNA. The pharmaceutical composition is administered at a dose sufficient to inhibit the expression of the target gene. The dsRNA is usually administered at a dose of less than 5 mg per kilogram of body weight per day, which dose is sufficient to inhibit or completely stop the expression of the target gene. In general, a suitable dose of dsRNA is in the range of 0.01-2.5 milligrams per kilogram of body weight per day, preferably in the range of 0.1-200 micrograms per kilogram of body weight per day, and more. Preferably within the range of 0.1 to 100 micrograms per kilogram of body weight per day, even more preferably within the range of 1.0 to 50 micrograms per kilogram of body weight per day, and most preferably per kilogram of body weight per day. It will be in the range of 1.0 to 25 micrograms. Pharmaceutical compositions containing dsRNA are administered once or several times daily at low doses, eg, using sustained release formulations known in the art. Preparation and administration of such pharmaceutical compositions can be carried out according to standard techniques, as further described below.

Such dsRNAs are then prepared for therapeutically effective amounts of such dsRNAs as described above to prepare EML4-ALK or TFG-ALK fusion proteins or cleaved active ALK kinases. The formulation can be used to inhibit the expression and activity of ALK in cancer, for example by direct injection into the tumor, to human subjects with developing cancer. Such inhibition of other receptor tyrosine kinases such as VEGFR and EGFR has recently been described using siRNA inhibitors. U.S. Patent Application Publication No. 20040209832 published October 21, 2004, McSwiggen et al .; U.S. Patent Application Publication No. 20130178091 published September 11, 2003, McSwiggen; U.S. Patent Application published September 9, 2004 See Publication No. 20040175703, Kreutzer et al.

Therapeutic Compositions, Administration Therapeutic compositions that inhibit ALK kinase useful in the practice of the methods of the invention are not limited to the oral or peritoneal route, but are intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, intratracheal. It can be administered to mammals by any means known in the art, including (aerosol), rectal, vaginal and topical (including intraoral and sublingual) administration.

For oral administration, therapeutic agents that inhibit ALK will generally be provided as powders or granules in the form of tablets or capsules, or aqueous solutions or suspensions. Tablets for oral use were mixed with pharmaceutically acceptable excipients such as Inactive diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, colorants, and preservatives. It may contain an active ingredient. Suitable Inactive Diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, and lubricants, if added, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a substance such as glyceryl monostearate or glyceryl distearate for delayed absorption in the gastrointestinal tract.

Oral capsules include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil. are doing.
For intramuscular, intraperitoneal, subcutaneous and intravenous applications, the pharmaceutical compositions of the present invention are generally provided in sterile aqueous solutions or suspensions buffered at appropriate pH and isotonicity. Let's go. Suitable aqueous excipients include Ringer's solution and saline. The carrier alone can constitute an aqueous buffer (“only” means the absence of an adjunct or encapsulant that may affect or intervene in the absorption of therapeutic agents that inhibit ALK. Means). Such substances include micellar structures, such as liposomes or capsids, as described below. The aqueous suspension may contain a cellulose derivative, a suspending agent such as sodium alginate, polyvinylpyrrolidone and tragacant rubber, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl p-hydroxybenzoate and n-propyl.

Therapeutic compositions that inhibit ALK kinase also include encapsulating formulations that protect therapeutic agents (eg, dsRNA compounds) from premature excretion from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. can do. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods of making such formulations will be apparent to those skilled in the art. This material is also commercially available from Alza Corporation and Nova Pharmaceuticals Inc. Liposomal suspensions, which contain liposomes that target cells infected with monoclonal antibodies to viral antigens, can also be used as pharmaceutically acceptable carriers. These are manufactured according to methods known to those of skill in the art, such as those described in US Pat. No. 4,522,811, PCT International Publication WO 91/06309, and European Patent Publication EP-A-43075. be able to. Capsule formulations can contain viral coat proteins. The viral coat protein may be obtained from or associated with the virus, such as polyomavirus, or may be partially or completely artificial. For example, the coating protein may be viral protein 1 and / or viral protein 2 of polyomavirus, or derivatives thereof.

Compositions that inhibit ALK can also contain delivery excipients, carriers, and diluents and salts thereof, including liposomes, for administration to the subject, and / or in pharmaceutically acceptable formulations. Can be present in. For example, methods for delivering nucleic acid molecules include Akhtar et al., 1992, Trends Cell Bio., 2, 139; DELIVERY STRATEGIES FOR ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS, ed. Akbtar, 1995; Maurer et al., 1999, Mol. Membr. Described in Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192. ing. US Pat. No. 6,395,713 of Beigelman et al. And PCT Publication WO 94/02595 of Sullivan et al. Further describe general methods for delivering nucleic acid molecules. These protocols can be used to deliver virtually any nucleic acid molecule.

Therapeutic agents that inhibit ALK are, but are not limited to, encapsulated in liposomes, by iontophoresis, or in other excipients such as hydrogels, cyclodextrins, biodegradable nanocapsules and bioadhesive granules. It can be administered to mammalian cancer by many methods known to those of skill in the art, including by incorporation into or by a proteinaceous vector (O'Hare amd Normand PCT WO 00/53722). Gazette). Also, the therapeutic / excipient mixture is delivered topically by direct injection or the use of an infusion pump. Direct injection of the composition, whether subcutaneously, intramuscularly, or intradermally, using standard needle and syringe techniques, or Conry et al., 1999, Clin. Cancer Res., 5, 2330- This can be done by needleless techniques as described in 2337 and Barry et al. PCT WO 99/31262.

Pharmaceutically acceptable formulations of therapeutic agents that inhibit ALK kinase include salts of the above compounds, such as acid addition salts, such as hydrochloric acid, hydrobromic acid, acetic acid and benzenesulfonic acid salts. A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, eg, systemic administration, to a patient, including cells or, for example, a human. Appropriate forms depend in part on the application or route of administration, eg, by oral, transdermal or injectable. Such morphology should not prevent the composition or formulation from reaching the target cells. For example, the pharmacological composition injected into the bloodstream must be soluble. Other factors are known in the art and include considerations such as toxicity and forms in which the composition or formulation does not affect its effectiveness.

Routes of administration that result in systemic absorption (ie, systemic absorption or accumulation of the drug into the bloodstream, and then systemic distribution) are preferred, but not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and. Intramuscular. Each of these routes of administration exposes a therapeutic agent that inhibits ALK to reachable lesion tissue or tumor. The rate of influx of the drug into the blood circulation has been shown to be a function of molecular weight or size. The use of liposomes or other drug carriers containing the compounds of the invention potentially localizes the drug to certain tissue types, such as, for example, the tissue of the reticuloendotheliate (RES). Make it possible. Liposomal formulations, such as lymphocytes and macrophages, that can promote the binding of cell surfaces to drugs are also useful. This technique takes advantage of the immune recognition specificity of macrophages and lymphocytes of abnormal cells, such as cancer cells, to enable enhanced delivery of the drug to target cells.

"Pharmaceutically acceptable formulation" means a composition or formulation capable of effectively distributing the nucleic acid molecule of the invention to a body part most suitable for its desired activity. A non-limiting example of a drug suitable for a formulation containing the nucleic acid molecule of the invention is a P-glycoprotein inhibitor (such as Pluronic P85) (Jolliet-) that can enhance the influx of the drug into the CNS. Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); Poly (DL-lactide-coglycolide) microspheres for slow broadcasts after transplantation into the cerebrum (Emerich et al., 1999, Cell Transplant , 8, 47-58; biodegradable polymers such as Alkermes, Inc. Cambridge, Mass.); Poly, which can deliver drugs across the blood-cerebral barrier and alter the mechanism of uptake into neurons. Includes loaded nanoparticles (Prog Neuro-Psychopharmacol Biol Psychiatry, 23, 941-949, 1999), such as those made of butyl cyanoacrylate. Other examples of non-limiting delivery strategies for compounds that inhibit ALK useful in the methods of the invention are Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA, 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev. 15, 73-107; Aldrian-Herrada et al. , 1998, Nucleic Acid Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

Therapeutic compositions containing poly (ethylene glycol) lipids containing surface-modified liposomes (PEG-modified or long-term blood-retaining liposomes or stealth liposomes) can also be appropriately used in the methods of the invention. These formulations provide a way to increase drug accumulation in the target tissue. This type of drug carrier resists opsonization and excretion by the mononuclear phagocyte lineage (MPS or RES), allowing enhanced long-term blood circulation and tissue exposure of the encapsulated drug (Lasic et. al., Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 10005-1011). Such liposomes have been shown to selectively accumulate in tumors, presumably by extraductal migration and capture in neoangiogenic target tissues (Lasic et al., Science 1995, 267, 1275). -1276; Oku et al., 1995, Biochem. Biophys. Acta, 1238, 86-90). Long-term circulating liposomes enhance the pharmacokinetics and efficacy of DNA and RNA, especially when compared to conventional cationic liposomes, which are known to accumulate in MPS tissues (Liu et al., J. Biol. Chem. 1995, 42,24864-24870; Choi et al., PCT International Publication WO 96/10391; Ansell et al., PCT International Publication WO 96/10390; Holland et al., International Patent Application Published WO 96/10392). Long-term circulating liposomes may also protect the drug from nuclease degradation more strongly than cationic liposomes due to their ability to inhibit accumulation in metabolically active MPS tissues such as the liver and spleen.

Therapeutic compositions may contain a pharmaceutically effective amount of the desired compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents used for treatment are known in the pharmaceutical art and are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co. (A.R. Gennaro, Ed. 1985). For example, preservatives, stabilizers, dyes and flavoring agents can be presented. These include esters of sodium benzoate, sorbic acid and P-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is the dose required to prevent, prevent, or treat the condition (to some extent, preferably all of the symptoms). Pharmaceutically effective doses include the type of disease, composition used, route of administration, type of mammal to be treated, physical characteristics of the particular mammal under consideration, concomitant medications, and others recognized by those skilled in the art Depends on the factors of. Generally, the active ingredient is administered from 0.1 mg / kg to 100 mg / kg body weight / day, depending on the potency of the negatively charged polymer.

Dose levels of about 0.1 mg to about 140 mg / kg body weight per day are useful in treating the above conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be mixed with the carrier material to produce a single dose form will vary depending on the subject being treated and the particular method of administration. The dose unit form generally contains from about 1 mg to about 500 mg of active ingredient. Specific dose levels for any particular patient are the activity, age, weight, overall health, sex, diet, frequency of administration, route of administration, and excretion rate of the specific compound used, the combination of drugs and the specific disease to be treated It will be understood that it depends on a number of factors, including the severity of the disease.

The composition can also be added to animal food or drinking water for administration to non-human animals. Animal feed or drinking water compositions can be conveniently formulated so that the animal can ingest the appropriate amount of the composition with the meal. It is also convenient to keep the composition as a mixed feed for addition to feed or drinking water.

ALK-inhibiting therapeutic agents useful in the practice of the present invention are single compounds, or combinations of multiple compounds, as described above, of the same type of inhibitor (ie, antibody inhibitor) or of different types (ie, antibody inhibitors). That is, it may be contained in either an antibody inhibitor or a small molecule inhibitor). Such a combination of compounds can enhance the overall therapeutic effect in inhibiting the progression of cancer expressing the fusion protein. For example, a therapeutic composition may be a combination of small molecule inhibitors such as WHI-131 and / or WHI-154, or other inhibitors and / or other small molecule inhibitors that target ALK activity. You can. The therapeutic composition may contain a non-specific chemotherapeutic agent in addition to the one or more target inhibitors. Such a combination has recently been shown to have a synergistic tumorigenic effect in many cancers. The combined effect of such ALK activity and inhibition of tumor growth in vivo can be assessed as described below.

Identification of Compounds that Inhibit Mutant ALK Kinases The present invention, in one aspect, confirms whether a compound inhibits the activity of an EML4-ALK or TFG-ALK fusion fusion polypeptide or ALK kinase polypeptide in cancer. Provides a method of ascertaining whether a compound can inhibit the progression of cancer characterized by EML4-ALK or TFG-ALK fusion fusion polynucleotides and / or fusion polypeptides. In some preferred embodiments, inhibition of ALK activity is confirmed by testing biological samples containing cells from bone marrow, blood, pleural effusion, or tumor. In another preferred embodiment, inhibition of ALK activity is confirmed using reagents specific for the mutant ALK polynucleotides or polypeptides of the invention.

The test compound may be of any type of therapeutic agent or composition described above. Methods for assessing the effectiveness of compounds, both in vitro and in vivo, are well established and known in the art. For example, the composition can be tested for its ability to inhibit ALK in vitro using cells or cell extracts in which ALK is activated. The group of compounds can be used to test the specificity of a compound for ALK (in contrast to other targets, such as EGFR or PDGFR).

Other techniques for screening for available agents provide high-speed, high-dose screening with appropriate binding affinity for the protein of interest, as described in PCT WO 84/03564. To do. In this method, a number of different small molecule test compounds are synthesized on solid substrates, such as plastic pins or some other surface, for application to mutant ALK polypeptides. The test compound is washed by reacting it with a mutant ALK polypeptide or fragment thereof. The bound mutant polypeptide (eg, EML4-ALK fusion polypeptide) is then detected by methods known in the art. The purified mutant ALK polypeptide can also be applied directly onto the plate for use in the drug screening techniques described above. Unneutralized antibodies can also be used to capture the peptide and anchor it to a solid support.

Compounds found to be effective inhibitors of ALK activity in vitro are subsequently used in vivo using xenografts of mammals carrying human tumors, such as NSCLC. EML4-ALK or TFG-ALK fusion polypeptides and / or cleaved ALK kinase polypeptides can be tested for their ability to inhibit the progression of cancer. This method allows the effect of the drug to be observed in a biological environment that most closely resembles the patient. The ability of agents to alter signal transduction in cancer cells or surrounding stromal cells can be confirmed by analysis with phosphorylation-specific antibodies. The effectiveness of agents that induce inhibition of cell death or cell proliferation can also be analyzed and observed with apoptosis-specific markers such as cleaved caspase 3 and cleaved PARP. Similarly, mammalian bone marrow transplants (eg, mice) carrying human leukemia promoted by the mutant ALK protein can be used. In this procedure, mice are transplanted with bone marrow cells that are known to be promoted by mutant ALK kinase. The growth of carcinoma cells may be observed. Mice can then be treated with drugs and the effect of drug treatment on cancer phenotype or progression can be observed externally. The mice are then killed and the transplanted bone marrow is removed and analyzed by IHC and Western blot.

Determining the toxicity and therapeutic effect of such compounds, such as LD50 (lethal dose to 50% of the population) and ED50 (effective therapeutic dose in 50% of the population), is a standard pharmaceutical procedure in cell cultures or laboratory animals. Can be confirmed by. The dose ratio of toxicity to therapeutic effect is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds that exhibit a high therapeutic index are preferred.

The teachings of all the references cited above and below are incorporated herein by reference. The following examples are provided solely to further illustrate the invention and are not intended to limit the scope of the invention, except as claimed herein. .. The present invention includes modifications and modifications that will be apparent to those skilled in the art of the methods taught herein.

Verification of ALK Kinase Activity in Solid Tumors by Comprehensive Phosphopeptide Profiling A. Profiling Human NSCLC Cell Lines Comprehensive phosphorylation profiles of kinase activity in 22 human NSCLC cell lines, including H2228, have recently been described influential to characterize by isolation and mass spectrometry of modified peptides from complex mixtures. Tests were performed using different techniques (see "IAP" technique, Rush et al., Supra). IAP technology was performed using a phosphotyrosine-specific antibody (CELL SIGNALING TECHNOLOGY, INC., Beverly, MA, 2003/04 Cat. # 9411) to obtain a peptide containing phosphotyrosine derived from an extract of an NSCLC cell line. Separated and then characterized.

Specifically, the IAP approach was used to facilitate the identification of tyrosine kinases involved in protein phosphorylation in each NSCLC cell line. In particular, special or abnormal kinase activity was considered.

Cell Culture All cell culture reagents were obtained from Invitrogen, Inc. A total of 41 human NSCLC cell lines were tested. 2 mM L -In RPMI1640 medium containing 10% FBS adjusted to contain glutamine, 1.5 g / L sodium bicarbonate, 4.5 g / L glucose, 10 mM HEPES, 10 mM sodium pyruvate, penicillin / streptomycin. Was cultured in. Six additional human NSCLC cell lines, HCC78, Cal-12T, HCC366, HCC44 and LOU-NH91, were obtained from DSMZ and cultured in RPMI1640 containing 10% FBS and penicillin / streptomycin. Cells were kept in a 5% CO ₂ incubator at 37 ° C.

For immunoaffinity precipitation and immunoblotting tests, cells were grown to 80% confluence and then starved overnight in RPMI medium without FBS prior to harvesting.

Immunoprecipitation with phosphopeptides 1 million cells in urea lysis buffer (20 mM Hepes (pH 8.0), 9 M urea, 1 mM sodium vanadium, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate) ) Dissolved in. The lysate was sonicated and centrifuged to make it transparent. The clarified lysate was reduced with DTT and alkylated with iodoacetamide as described above (see Rush et al., Nat. Biotechnol. 23 (1): 94-101 (2005)). I want to be). The sample was then diluted 4-fold with 20 mM Hepes to reduce the urea concentration to 2M and digested with trypsin overnight with gentle stirring at room temperature.

Digested peptides were crudely purified on a Sep-Pak C18 column as described above (see Rush et al., Supra). The eluate was lyophilized and the dried peptide was dissolved in 1.4 ml MOPS IP buffer (50 mM MOPS / NaOH, pH 7.2, 10 mM Na ₂ PO ₄ , 50 mM NaCl) to remove the insoluble material. It was removed by centrifugation. Immunoprecipitation was performed overnight at 4 ° C. using 160 μg of phosphotyrosine 100 antibody (Cell Signaling Technology) bound to protein G agarose beads (Roch). In a cool place, the beads were washed 3 times with 1 ml of MOPS IP buffer and 2 times with 1 ml of HPLC grade dH20. The phosphopeptide was eluted from the beads with 60 μl of 0.1% TFA followed by a second elution with 40 μl of 0.1% TFA to collect this fraction. The eluted peptides were concentrated using a ZipTip column (Millipore) and analyzed by LC-MS / MS. Mass spectra were collected on an LTQ ion trap mass spectrometer (ThermoFinnigan).

Analysis by LC-MS / MS Mass Spectrometer Concentrate peptides in IP eluate (100 μl) to Stop and Go Extraction Chips (Stage Tips) (Rappsilber et al., Anal. Chem., 75 (3): 663): 663 -70 (2003)) was used to separate from the eluted antibody. Peptides were eluted from the microcolumn with 1 μl 60% MeCN, 0.1% TFA in 7.6 μl 0.4% acetic acid / 0.005% heptafluorobutyric acid (HFBA).

Each phosphopeptide sample was analyzed twice by LC-MS. A fused silica microcapital column (125 μm × 18 cm) was packed with C18 reverse phase resin (Magic C18AQ, 5 μm particles, pore diameter 200 Å, Microchrom Bioresources, Auburn, CA). Samples (4 μL) were mounted on this column using an automatic sampler (LC Packings Famos, San Francisico, CA) with a 55-minute linear concentration gradient of 7-30% acetonitrile in 0.1% formic acid. It was eluted in a mass spectrometer by the method. This gradient method was performed at approximately abc nn / min using a dual HPLC pump (Agilent 1100, Palo Alto, CA) equipped with an in-line flow splitter. The ions of the eluted peptide were mass spectrometrically analyzed with a hybrid linear ion trap-7 Tesla ion cyclotron responce Fourier transform instrument (LTQ-FT, Thermo Finnigan, San Jose, CA).

Simultaneously with the operation of the linear ion trap and Fourier transform device, collect MS / MS scans with 7 data in the linear ion trap based on the measurements made during the MS survey scan previously performed on the ICR cells. The top-seven method was used. MS scans were performed at 375-1800 m / z with an 8 × 106 automatic gain control (AGC) target and a mass resolution of 105. AGC for MS / MS was 8 × 106, moving body ejection rate was 25 s, and slightly charged ions were eliminated by charge state screening.

Database analysis and assigned peptide sequences were assigned to MS / MS spectra using TurboSequest software (v.27, rev.12) (ThermoFinnigan) and a composite forward / reverse IPI human protein database. The search parameters are: trypsin as a protease; mass tolerance of the precursor of 1.08 Da; static modification on cysteine (+57.02146, carboxamide methylation); and serine, threonine and tyrosine (+79.96633 Da, phosphorylation), Dynamic modification on lysine (+8.01420, ¹³ C6 ¹⁵ N2), arginine (+6.02013, ¹³ C6) and methionine (+15.94941, oxidation). The target / decoy database method was used to establish appropriate score selection criteria with an approximate false positive allocation ratio of <1%. The allocation is to exceed the charge-dependent XCorr threshold (z = 1, XCorr ≥ 1.5, z = 2, XCorr ≥ 2.2, z = 3, XCorr ≥ 3.3). In addition, it contains phosphotyrosine, has a mass accuracy of -5 to +25 ppm, and contains lysine / arginine residues in either all-light or all-heavy form. Needed to do.

A custom quantification program, Vista (Bakalarsky et al., Manuscript in preparatin), to calculate the peak area and finally the relative abundance of heavy and light forms of each peptide for assignments that pass these criteria Was further evaluated using. Peptides identified with a signal vs. noise of less than 15 on MS scans were not considered for quantification. For these peptides found in only one state, the signal-to-noise ratio was used instead.

A search was performed on the NCBI human database published on August 24, 2004, which contained 27,175 proteins capable of methionine oxide (M + 16) and phosphorylation (Y + 80) as dynamic modifications. All spectra supporting the final list of assigned sequences (not shown herein) were reviewed by at least three scientists to establish reliability.

The above IAP analysis identified over 2000 novel, non-overlapping phosphotyrosine-containing peptides, over 1,500 phosphotyrosine sites, and over 1,000 tyrosine phosphorylated proteins from the cell lines tested. (Data not shown). Receptor tyrosine kinases known to be involved in NSCLC signaling have been observed as phosphorylated tyrosine in many cell lines, such as EGFR, Her2, Her3, EphA2 and Met. High levels of EGFR phosphopeptides have been observed in several cell lines, including two cell lines known to express the genetically activated form of amplified levels of EGFR, HCC827 and H3255. It was confirmed that this method identifies the receptor tyrosine kinase that is known to be activated in NSCLC cell lines.

Three cell lines expressed receptor tyrosine kinases not observed in other NSCLC cell lines. Large amounts of tyrosine phosphorylated peptides from Ros, ALK, and PDGFRα were observed in the HCC78, H2228, and H1703 cell lines, respectively. The NSCLC cell line H2228, which highly expresses ALK, was selected for further experimentation.

B. Profiling Human NSCLC Tumor Samples As described in detail in column A above, IAP technology was subsequently applied to test the overall phosphorus profile of 154 human tumor sample groups from NSCLC patients. The organization was obtained from Second Xiangya Hospital, China.

Frozen tissue samples are cut into small pieces and supplemented with lysis buffer (20 mM HEPES (pH 8.0), 9 M urea, 1 mN sodium vanadium, 2.5 mM sodium pyrophosphate, 1 mM β-glycerin phosphate , 1 ml of lysis buffer per 100 mg of frozen tissue), homogenized twice with Polytron for 20 seconds each time. The homogenate was then sonicated for a short time. The clarified lysate was reduced with DTT and alkylated with iodoacetamide as described above (see Rush et al., Nat. Biotechnol. 23 (1): 94-101 (2005)). .. The sample was then diluted 4 times with 20 mM Hepes to reduce the urea concentration to 2M and digested with trypsin overnight at room temperature with gentle stirring.

The digested peptide was crudely purified on a Sep-Pak C18 column as described above (see Rush et al., Supra.). The eluate was lyophilized and the dried peptide was dissolved in 1.4 ml MOPS IP buffer (50 mM MOPS / NaOH, pH 7.2, 10 mM Na ₂ PO ₄ , 50 mM NaCl) to remove the insoluble material. It was removed by centrifugation. Immunoprecipitation was performed overnight at 4 ° C. using 160 μg of phosphotyrosine 100 antibody (Cell Signaling Technology) bound to protein G agarose beads (Roch). In a cool place, the beads were washed 3 times with 1 ml of MOPS IP buffer and 2 times with 1 ml of HPLC grade dH20. The phosphopeptide was eluted from the beads with 60 μl of 0.1% TFA followed by a second elution with 40 μl of 0.1% TFA to collect this fraction. The eluted peptides were concentrated using a ZipTip column (Millipore) and analyzed by LC-MS / MS. Mass spectra were collected on an LTQ ion trap mass spectrometer (ThermoFinnigan).

Immunoprecipitation of the phosphopeptide was followed by LC-MS / MS spectroscopy as described in column A above. Database searches and sequence assignments were performed as detailed in column A above, using the NCBI human database published on August 24, 2004, containing 27,970 proteins.

The IAP analysis identified more than 2000 non-overlapping phosphotyrosine-containing peptides from the human tumor samples tested, more than 1,500 phosphotyrosine sites, and more than 1,000 tyrosine phosphorylated proteins (data shown). It has not been). Receptor tyrosine kinases known to be involved in NSCLC signaling have been re-observed as phosphorylated tyrosine in many tumors, such as EGFR, Her2, Her3, EphA2 and Met. High levels of EGFR phosphate polypeptide were re-observed in some tumor samples, confirming that this method identifies receptor tyrosine kinases known to be active in NSCLC cell lines. ..

Five patient samples expressed receptor tyrosine kinases not observed in other NSCLC cell lines and tumors. Large amounts of ALK-derived tyrosine-phosphorylated peptides were observed in patients CS010 / 11, CS045, and CS110. These three tumors, which highly express ALK, were selected for further experimentation.

Isolation and Sequencing of Three ALK Fusion Genes
A. Sequencing in human NSCLC cell line ALK kinase to determine the presence of chimeric ALK transcripts, given the detection of high phosphorylation levels of ALK kinase in NSCLC cell line H2228. 5'Rapid amplification of the cDNA ends of the domain-encoding sequence was performed.

Rapid amplification of complementary DNA ends
The RNeasy Mini Kit (Quiagen) was used to extract RNA from the H2228 cell line. DNA was extracted using the DNeasy Tissue Kit (Quiagen). Rapid amplification of the cDNA ends was performed using the 5'RACE system (Invitrogen) with primers ALK-GSP1 for cDNA synthesis and ALK-GSP2 and ALK-GSP3 for nested PCR reactions.

5'RACE
FIG. 5A shows the detection of the EML4-ALK fusion gene (short mutant) by 5'RACE and the detection of the PCR amplification product after the second round. PCR products were purified with a PCR purification kit (Qiagen) and sequenced using ALK-GSP3 and ABI3130 capillary automated DNA sequencers (Applied Biosystems). Sequence analysis of the resulting product revealed that the kinase domain of ALK and the C-terminus were fused to the N-terminus of the EML-4 gene (Fig. 1AC, see figure below). The EML4-ALK fusion gene (short variant) is frame-by-frame and fused the first 233 amino acids of EML-4 with the last 562 amino acids of ALK (Fig. 1AC, see figure below). ). Since both the EML-4 and ALK genes are located on chromosome 2, the fusion gene was created by a gene deletion between the two loci.

The following primers were used:
ALK-GSP1: 5'-GCAGTAGTTGGGGGTTGTAGTC (SEQ ID NO: 9)
ALK-GSP2: 5'-GCGGAGCTTGCTCAGCTTGT (SEQ ID NO: 10)
ALK-GSP3: 5'-TGCAGCTCTCTGGTGCTTCC (SEQ ID NO: 11).

PCR Assay RT-PCR analysis was performed to confirm that the N-terminus of EML-4 was intact in the fusion protein (see Figure 6). The first strand of cDNA was synthesized from 2.5 mg of total RNA using the SuperScript® III First Strand Synthesis System (Invitrogen) at Oligo (dT) ₂₀ . The EML4-ALK fusion gene was then amplified using a primer pair of EML-Atg and ALK-GSP3. Mutual fusion was detected using primer pairs of EML-4-43 and ALK-GSP3, EML-4-94 and ALK-GSP3, and EML-4-202 and ALK-GSP3. For genomic PCR, fusion gene amplification was performed with a primer pair of EML-4atg and ALK-tga using Platinum Taq DNA polymerase Invitrogen.

The following primers were used:
ALK-GSP3: 5'-TGCAGCTCCTGGTGCTTCC (SEQ ID NO: 12)
EML4-Atg: 5'-CGCAAGATGGACGGGTTTGGC (SEQ ID NO: 13)
EML4-43: 5'-TGTTCAAGATCGCCCTGTCAGCCTCT (SEQ ID NO: 14)
EML4-94: 5'-TGAAATCACTGTGCTAAAGGCGGC (SEQ ID NO: 15)
EML4-202: 5'-AAGCCCTCGAGCAGTATTCCCAT (SEQ ID NO: 16)
ALK-Tga: 5'-GAATTCCGCCGAGCTCAGGCCCAG (SEQ ID NO: 17).

Notably, in the EML4-ALK fusion (short variant), the ALK moiety is EML at exactly the same position as the ALK observed in other ALK fusions, such as the NPM-ALK fusion that occurs in ALCL. -4 It is to fuse with the part. The kinase domain of ALK in the H2228 cell line was further sequenced from genomic DNA and found to be wild-type. Therefore, the deletion mutations found in H2228 do not affect the ALK kinase domain. In addition, wild-type EML-4 has an N-terminal coiled coil domain in which tyrosine is phosphorylated only at sites that are not present in the EML4-ALK fusion protein (short mutant) and is conserved in the fusion protein (Fig. (See 1A) suggests that it may play a role in dimerizing and activating ALK, as well as promoting interaction with wild-type ALK.

B. Similar to sequencing in human NSCLC cell lines , chimeric ALK transcripts were found in view of the detection of high phosphorylated levels of ALK kinase in human NSCLC tumor samples from patients CS010 / 11, CS045, and CS110. 5'Rapid amplification of the cDNA ends of the sequence encoding the kinase domain of ALK was performed to determine if it was present in these tumors.

Rapid amplification of complementary DNA ends and 5'RACE was performed using primers ALK-GSP1 for cDNA synthesis and ALK-GSP2 and ALK-GSP3 for nested PCR reactions, as detailed in column A above. did.

FIG. 5C shows the detection of the EML4-ALK fusion gene (both short and long mutants) by 5'RACE in two patient samples, and the detection of the TFG-ALK fusion gene in one patient, and a second. It shows the detection of PCR amplification products after the round. The PCR product was purified and sequenced as detailed in column A above. Sequence analysis of the resulting product revealed that the kinase domain of ALK and the C-terminus were fused to the N-terminus of the EML-4 gene of two different mutants (Fig. 1A-1B, figure below). Please refer to). The EML4-ALK fusion gene is on a frame-by-frame basis, and the first 233 amino acids (short variants) or the first 495 amino acids (long variants) of EML-4 are fused with the 562 amino acids at the end of ALK (Figure). 1A-1B, see figure below). Since both the EML-4 and ALK genes are located on chromosome 2, the fusion gene was created by a gene deletion between the two loci. Observation of the fusion gene (short mutant) in patient CS045 confirmed the discovery of this mutant gene in human cell line H2228.

The TFG-ALK fusion gene was also on a frame-by-frame basis, with the first 138 amino acids of TFG fused to the last 562 amino acids of ALK (Fig. 1C, see figure below). Since the TFG and ALK genes are located on different chromosomes (chromosomes 6 and 2 respectively), the fusion gene was created by a gene translocation between the two loci. Interestingly, fusion of TFG with ALK occurs at exactly the same location of ALK observed for fusion of two EML4-ALK variants, with frequent cleavage of ALK at this location in solid tumors. It suggests that it may be.

The same primers as described in column A above were used. RT-PCR analysis was performed as detailed in column A above to confirm that the N-terminus of EML-4 and TFG was intact in the fusion protein (see Figure 6). ). The primer pairs for EML-4 and ALK are as described in column A above, and the following primer pairs were used for TFG.
TFG-F1: 5'-TTTGTTTAATGGCCAGCCAAAGACCC-3 (SEQ ID NO: 28).

Of note, in both EML4-ALK fusion mutants, the ALK moiety is EML-at exactly the same position as the ALK observed in other ALK fusions, such as the NPM-ALK fusion that occurs in ALCL. It means that it fuses with the four parts. In addition, wild-type EML-4 has an N-terminal coiled coil domain (see FIG. 1A-1B) in which tyrosine is phosphorylated only at sites not present in the EML4-ALK fusion protein and is conserved in the fusion protein. It suggests that it may play a role in dimerizing and activating ALK, as well as promoting interaction with wild-type ALK. Also noteworthy is that the fusion of the TG portion with ALK also occurs at the exact same position of ALK, and certainly the fusion of TFG with ALK at this position has been described in human lymphoma (Hernandez). Et al. (2002), Supra.), But have not previously been described in human solid tumors such as NSCLC.

Growth Inhibition of ALK Fusion Expressing Mammalian Solid Tumors Using SiRNA Cell Growth and Survival in NSCLC Tumor Samples Derived from NSCLC Cell Line H2228, Patients CS010 / 11, CS045, and CA110 with ALK cleavage / fusion form To confirm that it promotes, the ability of siRNA (against ALK) to inhibit the growth of these cells and tumors can be tested.

ALK's SMART pool siRNA double strand (preferred target sequence-data not shown) is available, for example, from Dharmacon Research, Inc. (Lafayette, CO). A non-specific SMART pool siRNA is used as a control. Cells are transfected with siRNA by electroporation. That is, 2 × 10 ⁷ cells (H2228) are given a single pulse (20 ms; 275 V, K562 20 ms; 285 V) using a square wave electropolator (BTX Genetronics San Diego, CA) and cultured at room temperature for 30 minutes. Then, transfer to a T150 flask using 30 ml RPMI-1640 / 10% FBS.

The number of surviving cells is measured with the CellTiter 96 AQ _ueuous One solution Cell Proliferation Assay (Promega). Calculate the IC ₅₀ using OriginPro 6.1 software (OriginLab). The percentage of apoptotic cells at 48 hours can be measured by flow cytometric analysis of Cleaved-Caspase-3 (Cell Signaling Technolgy).

Immunoblotting analysis will reveal that ALK expression is specifically and significantly reduced 72 hours after transfection of siRNA into H2228 cells or tumor cells from patients CS010 / 11, CS045, and CS110. .. It is expected that reduced expression of ALK will result in a strong inhibition of cell growth. Treatment with ALK siRNA is also expected to result in increased apoptosis of these solid tumor cells. These results further indicate that mutant / fused ALK kinases in the H2228 cell line and patient tumors promote the growth and growth of these NSCLC cells, and such growth and growth is ALK using siRNA. It would suggest that it can be blocked by inhibiting the expression and activity of the kinase.

Growth inhibition of solid mammalian tumors expressing ALK fusion using WI-131 and / or WI-154 Growth of mutant ALK fusion proteins in NSCLC cell line H2228 and patients CS010 / 11, CS045, and CS110-derived NSCLC tumor lines And to further confirm that it promotes survival, cells may be treated with targeted inhibitors of ALK kinase such as WI-131 and / or WI-154. WI-131 and WI-154 are small molecule inhibitors of quinazoline-type ALK kinase and their activity against the NPM-ALK fusion protein in T-cell lymphoma has been described. See Marzec et al., Supra.

That is, NSCLC cells are cultured and a cell growth inhibition assay is performed in the CellTiter 96 AQueous One solution cell proliferation assay (Promega) according to the manufacturer's suggestion. That is, 1000-5000 cells are sown on a flat-bottomed 96-well plate and grown in complete medium containing 10% FBS. After 24 hours, the cell medium is replaced with 100 μl complete growth medium containing 10% FBS containing various concentrations of drug and the cells are cultured for an additional 72 hours. Each drug concentration is applied to the wells of three cells. At the end of culturing, _add 20 μl of CellTiter 96 AQ _euous One solution to each well and incubate the plates for 1-4 hours. Read the absorbance at 490 nm using a Titan Multiskan Ascent microplate reader (Titertek Instrument). Growth inhibition can be expressed as the mean ± SD value of the percentage of absorbance read from treated cells vs. untreated cells.

In such analysis, ALK fusion proteins (EML4-ALK (short and long mutants), TFG-ALK) promote the growth and survival of a subset of human NSCLC tumors expressing these mutant proteins. It is expected that it can be confirmed that such cells can be inhibited by inhibiting the activity of the fusion ALK kinase with a target inhibitor such as WI-131 and / or WI-154.

ALK fusion proteins promote the growth and survival of transformed mammalian cell lines 3T # cells, to see if expression of one or more ALK fusion proteins can convert normal cells to a cancer phenotype. It may be transformed with the cDNA construct of the above (Example 2), which expresses EML4-ALK (short mutant and long mutant) or TFG-ALK fusion protein, respectively.

That is, cells are retained in RPMI-1640 medium (Invitogen) containing 10% fetal bovine serum (FBS) (Sigma) and 1.0 ng / ml IL-3 (R & D Systems). Production of retrovirus supernatant and transduction are carried out as described above. See Schwaller et al., Embo J. 17 (18): 5321-33 (1988). 3T3 cells were transduced with a retrovirus supernatant containing the MSCV-Neo / EML4-ALK (or TFG-ALK) vector and selected for G418 (1 mg / ml). The viability of the transformed cells on soft agar was then evaluated by washing the cells 3 times in PBS and then sowing the transduced cells on a plate. If desired, for dose-response curves, _ALKs are treated with siRNA as described above (see Example 3) to determine the number of surviving cells in the CellTiter 96 AQ _ueuous One solution. Measured by cell proliferation assay (Promega). IC _50s can be calculated using OriginPro 6.1 software (OriginLab). The percentage of apoptotic cells at 48 hours can be measured by flow cytometric analysis of Cleaved-Caspase-3 (Cell Signaling Technology) using antibodies specific for this target. By such analysis, expression of the EML4-ALK fusion protein (short or long variant) or TFG-ALK fusion protein transforms 3T3 cells and these cells are promoted by this ALK fusion protein. In some cases, it will be shown that survival and growth on soft agar can be confirmed, and that inhibition of ALK expression in transformed cells reduces viability and increases apoptosis.

Detection of EML4-ALK Fusion Protein Expression in Human Solid Tumors Using FISH Assay The presence of the EML4-ALK fusion protein (short mutant) in human NSCLC tumor samples is demonstrated by fluorescence in situ hybridization as described above. Detected using a FISH) assay. See, for example, Verma et al. HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York, NY (1988). Over 200 paraffin-embedded human NSCLC tumor samples were tested.

ALK's dual color, break-apart rearrangement probe was obtained from Vysis (Vysis, Dowers Grove, IL, USA) and used with the following modifications according to the manufacturer's instructions. .. That is, the paraffin-embedded tissue section was rehydrated, and the antigen was recovered by ultrasonic waves in 0.01 M citrate buffer (pH 6.0) for 11 minutes. Sections were digested with protease (4 mg / ml pepsin, 2000-3000 U / mg) at 37 ° C. for 25 minutes, dehydrated and hybridized at 37 ° C. for 18 hours using the FISH probe set. After washing, 4', 6-diamidino-2-phenyllindol (DAPI; mg / ml) in Vectashield-encapsulated medium was applied for nuclear counterstaining.

The ALK rearrangement probe contains two different labeled probes contralateral to the cut point (at nucleotide 3171) of the ALK gene in the wild-type sequence (SEQ ID NO: 6). When hybridized, the original ALK region is shown as an orange / green fusion signal, while rearrangement at this locus results in a separated orange and green signal (if it occurs in an EML4-ALK deletion mutant). There will be. See FIG.

This FISH analysis revealed a relatively low incidence of this short form of EML4-ALK mutation in the sample group examined (one of 229 samples). However, given the high incidence of NSCLC worldwide (more than 151,000 new cases annually in the United States alone), it is expected that there will be a significant number of patients carrying this mutant ALK. This patient can benefit from a treatment plan that inhibits ALK.

Detection of ALK Fusion Protein Expression in Human Solid Tumors Using PCR Assays The presence of one or more ALK fusion proteins in human solid tumor samples is, as previously described, genomic or reverse transcriptase (RT) polymerase linkage. It can also be detected using any of the reactions (PCR). See, for example, Cools et al., N. Engl. J. Med. 348: 1201-12014 (2003). Thus, for example, solid tumor samples may be obtained from patients with, for example, NSCLC, using standard techniques. Construct a PCR probe for the cleaved ALK kinase or EML4-ALK fusion protein (short or long mutant) or TFG-ALK fusion protein. The RNeasy Mini Kit (Qiagen) may be used to extract RNA from tumor samples. DNA may be extracted using the DNeasy Tissue Kit (Quiagen). For RT-PCR, the first strand is synthesized, for example, from 2.5 μg of total RNA using, for example, SuperScript® III First Strand Synthesis System (Invitrogen) at oligo (dT) ₂₀ .

Primer pairs such as EML4-202 and ALK-GSP3 (see Example 2 above) are then used to amplify the ALK fusion gene. For genomic PCR, fusion gene amplification can be performed with a primer pair, eg EML4-202 and ALK-GSP3 (see Example 2 above), using Platinum Taq DNA polymerase Invitrogen. .. Such analysis will identify patients with solid tumors characterized by expression of cleaved ALK kinase (and / or EML4-ALK fusion protein (s) or TFG-ALK fusion protein). .. This patient is a candidate for treatment with a therapeutic agent that inhibits ALK, such as WHI-131 and / or WHI-154.

Claims (4)

A method for detecting the presence of mutated anaplastic lymphoma kinase (ALK) polynucleotides in biological samples obtained from human non-small cell lung cancer (NSCLC) patients.
It is detected that an ALK fusion polynucleotide encoding an ALK fusion polypeptide containing the amino acid residue at position 1116-1383 and having ALK kinase activity in the amino acid sequence represented by SEQ ID NO: 5 is present in the sample. A method comprising performing a polymerase chain reaction (PCR) with a pair of oligonucleotides whose detection hybridizes to the ALK portion of the ALK fusion polynucleotide. The method of claim 1, wherein the ALK fusion polynucleotide is mRNA or DNA. The method of claim 1, wherein the detection comprises sequencing the nucleic acid. The method according to any one of claims 1 to 3, wherein the biological sample is a biopsy tissue sample or a pleural effusion sample.

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