WO2017008682A1 - Application de ny-eso-2 dans le diagnostic et le traitement du cancer colorectal à instabilité microsatellitaires - Google Patents

Application de ny-eso-2 dans le diagnostic et le traitement du cancer colorectal à instabilité microsatellitaires Download PDF

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WO2017008682A1
WO2017008682A1 PCT/CN2016/089135 CN2016089135W WO2017008682A1 WO 2017008682 A1 WO2017008682 A1 WO 2017008682A1 CN 2016089135 W CN2016089135 W CN 2016089135W WO 2017008682 A1 WO2017008682 A1 WO 2017008682A1
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eso
cells
intestinal cancer
cancer
protein
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万涛
虞淦军
曹雪涛
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中国人民解放军第二军医大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/52—Intestine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

Definitions

  • the present invention relates to the fields of biology and medicine, and more particularly to the use of NY-ESO-2 in immunotherapy of microsatellite unstable colorectal cancer.
  • Colorectal cancer is one of the most common malignant tumors in the world. Its incidence rate is the third and second in malignant solid tumors in men and women, ranking fourth among cancer-related deaths. In recent years, the incidence of colorectal cancer has been rising, and China's rate of increase is twice that of the international average growth rate (2%), especially in developed cities such as Shanghai, where the incidence rate reaches the second place in solid tumors (30). 100,000), seriously threatening human health and quality of life.
  • a NY-ESO-2 gene for the preparation of (a) diagnostic microsatellite unstable intestinal cancer; and/or (b) determination of prognosis of a colon cancer patient Kit.
  • the NY-ESO-2 gene is derived from a mammal, preferably from a mouse, rat or human.
  • the kit comprises: a detection reagent for quantitative detection of NY-ESO-2 protein or mRNA, and a corresponding label or instructions.
  • the kit further comprises a detection reagent for quantitative detection of NY-ESO-1 protein or mRNA.
  • the detection reagent comprises a NY-ESO-2 specific primer, a specific antibody, a probe or a chip.
  • the detection reagent is a NY-ESO-2 specific primer.
  • the reagent described above includes a detection chip including a nucleic acid chip and a protein chip.
  • the nucleic acid chip comprises a substrate and a specific oligonucleotide probe of a microsatellite unstable intestinal cancer related gene spotted on the substrate, the specificity of the cancer associated gene Oligonucleotide probes include probes that specifically bind to the NY-ESO-2 gene or mRNA.
  • the protein chip comprises a substrate and a specific antibody specific for a cancer-associated protein spotted on the substrate, and the specific antibody of the microsatellite unstable intestinal cancer-associated protein comprises anti-NY- Specific antibodies to the ESO-2 protein.
  • the prognosis includes predicting the survival of a patient with a confirmed bowel cancer.
  • the label or the description states the following:
  • the ratio of the mRNA expression level of the NY-ESO-2 relative reference gene to the mRNA expression level of the NY-ESO-2 relative reference gene of the adjacent tissue is ⁇ 1, it indicates that the test subject suffers from microsatellite instability.
  • the risk of cancer is higher than the general population, and / or
  • the intestinal cancer comprises colon cancer or rectal cancer.
  • the kit is for detecting a human intestinal cancer tissue sample or a blood sample.
  • the invention also provides a kit for detecting microsatellite unstable intestinal cancer, wherein the kit contains a NY-ESO-2 gene, a protein or a detection reagent thereof, and a corresponding label or instruction.
  • the kit further contains a NY-ESO-2 gene and a protein as a positive control.
  • a NY-ESO-2 gene, protein or agonist thereof for the preparation of a pharmaceutical composition for the treatment of intestinal cancer.
  • the pharmaceutical composition is a vaccine composition.
  • the agonist comprises 5-aza-2'deoxycytidine (DAC), an anti-PD-1 antibody, an anti-CTLA-4 antibody, and the like.
  • DAC 5-aza-2'deoxycytidine
  • the intestinal cancer is microsatellite unstable intestinal cancer.
  • the pharmaceutical composition can be prepared as follows:
  • step (B1) further comprises: further incubating the NY-ESO-2 sensitized antigen presenting cells with T cells to obtain anti-tumor immune activity activated by NY-ESO-2 T cells.
  • the antigen presenting cell is a dendritic cell.
  • the preparation of the pharmaceutical composition can also be as follows:
  • A2 providing a vector that co-expresses an anti-NY-ESO-2 antibody gene and a T cell activating gene
  • (B2) transfecting T cells with the vector of (A2) to obtain chimeric antigen receptor T cells (CAR-T);
  • a method for screening a candidate compound for treating intestinal cancer comprising the steps of:
  • test compounds were added to the culture system of the cells, and the expression amount and/or activity of NY-ESO-2 in the intestinal cancer cells of the test group was observed; in the control group, in the same cells No test compound was added to the culture system, and the expression amount and/or activity of NY-ESO-2 in the cells of the control group was observed;
  • test compound is a therapeutic agent for the treatment of intestinal cancer which promotes the expression and/or activity of NY-ESO-2.
  • the method may further include the steps of:
  • a method for inhibiting microsatellite unstable intestinal cancer cells in vitro comprising the steps of: transfecting NY-ESO-2-activated T cells having antitumor immunological activity and/or T cells expressing an anti-NY-ESO-2 antibody and/or T cells expressing a TCR having a NY-ESO-2 epitope recognized are co-incubated with intestinal cancer cells, thereby inhibiting microsatellite unstable intestinal cancer cells.
  • the invention also provides a method for treating microsatellite unstable intestinal cancer, comprising the steps of: administering NY-ESO-2 activated T cells with anti-tumor immunoreactivity and/or expressing anti-NY-ESO to a subject in need thereof T cells of the -2 antibody and/or T cells expressing a TCR recognizing the NY-ESO-2 epitope, thereby treating microsatellite unstable intestinal cancer.
  • the desired subject is a subject having microsatellite unstable intestinal cancer.
  • the desired subject is a mammal, including a mouse, a rat or a human.
  • Figure 1A shows the preferred NY-ESO-2 cDNA sequence (SEQ ID NO.: 1 provides the original cDNA sequence, the optimized sequence can indicate the specific position and the specifically optimized codon);
  • Figure 1B shows NY-ESO Amino acid sequence of -2 (SEQ ID NO.: 2).
  • Figure 2A shows that NY-ESO-2 significantly up-regulated NY-ESO-2 expression in LS180-MSI-H/S model MSI-H cells in gene chip analysis;
  • Figure 2B shows qPCR analysis results in NY-ESO -2 significantly up-regulated NY-ESO-2 expression in LS180-MSI-H/S model MSI-H cells;
  • Figure 2C shows expression of NY-ESO-1, NY-ESO-2 in MSI-H colorectal cancer cell lines, The average level of MAGEA4 was higher than that of the 3 MSS colorectal cell lines.
  • Figure 3 shows the expression of NY-ESO-2 in cancer tissues of patients.
  • Figure 4 shows the effect of differences in NY-ESO-2 expression on three-year survival in MSI-H patients ( Figure 4A) and MSS patients ( Figure 4B) in a retrospective analysis.
  • Figure 5A shows tumor growth curves after LS180-MSI-H tumor-bearing nude mice and subjected to peptide-sensitized DC reinfusion treatment;
  • Figure 5B shows survival curves of tumor-bearing nude mice.
  • NY-ESO-2 is highly expressed in microsatellite unstable intestinal cancer, and uses NY-ESO-2 as a molecular marker of intestinal cancer, which contributes to the intestinal tract.
  • the cancer is classified to guide the treatment of different types of intestinal cancer; in addition, the inventors have also found that the prognosis of patients with intestinal cancer has a strong correlation with the expression of NY-ESO-2, microsatellite unstable colon cancer Patients with high expression of NY-ESO-2 can have a better prognosis.
  • the present inventors have also found through experiments that DC cells sensitized by NY-ESO-2 can more effectively activate the tumor immunological activity of T cells in vivo, thereby achieving the purpose of treating microsatellite unstable intestinal cancer. On the basis of this, the present invention has been completed.
  • Colorectal cancer is one of the most common malignant tumors in the world. Its incidence rate is the third and second in malignant solid tumors in men and women, ranking fourth among cancer-related deaths. In recent years, the incidence of colorectal cancer has been rising, and China's rate of increase is twice that of the international average growth rate (2%), especially in developed cities such as Shanghai, where the incidence rate reaches the second place in solid tumors (30). 100,000), seriously threatening human health and quality of life.
  • Microsatellite refers to a simple repeat sequence of ⁇ 10 nucleotides in the genome.
  • the repeat sequence consisting of two nucleotides is the most abundant, and the number of repetitions is 10 to 50 times, which is mainly included in the non-coding region of the gene, and the sequence is short. Most ⁇ 200 bp, the change in the number of repeat units can cause quite high polymorphism. Simple repeat Adding or losing is called MSI.
  • MSI microsatellite instability
  • MSS microsatellite stability
  • NY-ESO-2 is used as a molecular marker to assist in the diagnosis of microsatellite unstable intestinal cancer, and NY-ESO-2 has a significant correlation with the survival of intestinal cancer patients, using NY- ESO-2 can contribute to the prognosis of patients with colorectal cancer, that is, patients with high expression of NY-ESO-2 have better prognosis than patients with low expression of NY-ESO-2.
  • NY-ESO-2 is an important member of the cancer-testis antigen gene family.
  • the encoded protein has a relative molecular weight of about 18kD, 180 amino acids, and a glycine-rich region at the N-terminus. Contains a hydrophobic amino acid tail. It has strong immunogenicity in tumor antigens, but its expression in normal tissues is limited to testis and embryonic tissues.
  • NY-ESO-2 is known to be expressed in malignant melanoma, hepatocellular carcinoma, ovarian cancer and the like. However, in colorectal cancer, the expression of NY-ESO-2 is uncertain, and the frequency of expression is quite different.
  • the present inventors have found that the expression of NY-ESO-2 has a good correlation with the microsatellite stability typing of intestinal cancer by retrospective study and experimental verification of a large number of different intestinal cancer cell lines. That is, NY-ESO-2 is more expressed in microsatellite unstable intestinal cancer. Thus, the expression of NY-ESO-2 can be used to classify intestinal cancer, which is helpful for the guidance of the treatment plan.
  • the present inventors have found that using NY-ESO-2 as an antigen to stimulate antigen presenting cells, thereby stimulating T cells, or T cells expressing anti-NY-ESO-2 antibodies or expressing TCRs recognizing NY-ESO-2 T cells have good microsatellite instability intestinal cancer inhibitory activity. This tumor suppressing activity is weaker for microsatellite stable intestinal cancer.
  • the anti-NY-ESO-2 antibody which can be used in the present invention is not particularly limited and may be a monoclonal antibody which specifically binds to NY-ESO-2.
  • the monoclonal may be obtained by a conventional method, for example, using hybridoma cells.
  • T cells producing tumor immunological activity or chimeric antigen receptor T cells expressing anti-NY-ESO-2 antibody or T cells expressing TCR recognizing NY-ESO-2 may produce strong resistance to microsatellite unstable intestinal cancer Tumor inhibition.
  • the NY-ESO-2 antigen or nucleic acid encoding the same used in the present invention is an isolated protein or a nucleic acid encoding the same.
  • the terms "NY-ESO-2 protein”, “NY-ESO-2 antigen”, “NY-ESO-2 polypeptide”, “molecular marker of the invention” are used interchangeably and refer to both having NY.
  • - ESO-2 amino acid sequence of protein or more Peptide the sequence is SEQ ID NO.: 2.
  • a preferred polynucleotide sequence encoding the NY-ESO-2 amino acid is SEQ ID NO.: 1.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified, as separated from other substances present in the natural state.
  • isolated NY-ESO-2 protein or polypeptide means that the NY-ESO-2 protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify the NY-ESO-2 protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • the NY-ESO-2 protein includes a fusion protein and a non-fusion protein.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the invention may also or may not include an initial methionine residue.
  • the protein of the present invention can also be co-expressed with T cell activating molecules and transfected into T cells to obtain chimeric antigen receptor T cells, and the killing of tumor cells is carried out with NY-ESO-2 as a target.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • Polynucleotides encoding mature polypeptides of NY-ESO-2 include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and Non-coding sequence.
  • the term "polynucleotide encoding a polypeptide" can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.
  • nucleic acid fragments that hybridize to the sequences described above, including sense and antisense nucleic acid fragments.
  • a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, More preferably, it is at least 50 nucleotides, preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding the NY-ESO-2 protein.
  • the human NY-ESO-2 nucleotide full length sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art.
  • the template is amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vectors of the invention or the NY-ESO-2 protein coding sequences, and methods of producing the polypeptides of the invention by recombinant techniques.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant NY-ESO-2 protein by conventional recombinant DNA techniques. Generally there are the following steps:
  • Methods well known to those skilled in the art can be used to construct expression vectors containing human NY-ESO-2 encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, or 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • An antigen presenting cell refers to a type of cell that has an ingestion, treatment of an antigen and presentation of antigen information to T lymphocytes, and is also called a helper cell.
  • APC antigen presenting cell
  • the antigen-presenting cells which can be used in the present invention are not particularly limited, and are any antigen-presenting cells which function as antigen cross-presentation (or cross-sensitization) in the immune system and stimulate T cells to generate an immune response (especially a tumor immune response).
  • the antigen presenting cell is a dendritic cell which is the most functional APC in the immune system, preferably an antigen presenting cell sensitized with the NY-ESO-2 antigen of the present invention.
  • substances which interact with the NY-ESO-2 protein can be screened by various conventional screening methods.
  • An agonist of the NY-ESO-2 protein of the present invention when administered therapeutically (administered), can promote the expression and/or activity of NY-ESO-2 protein, using a highly expressed NY-ESO-2 antigen, In turn, T cells are stimulated to produce tumor immunological activity, thereby inhibiting the growth or proliferation of microsatellite unstable intestinal cancer.
  • compositions of the invention can be prepared by a variety of methods:
  • an antigen-presenting cell sensitized with NY-ESO-2 and a pharmaceutically acceptable carrier are contained. Applying the pharmaceutical composition to a patient suffering from microsatellite unstable intestinal cancer,
  • these agonists can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the substance being formulated and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe and effective amount of a NY-ESO-2 protein of the invention or an agonist thereof, and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be formulated into an injection form, for example, with physiological saline or containing glucose and An aqueous solution of other adjuvants is prepared by a conventional method.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram to 10 milligrams per kilogram of body weight per day.
  • the invention also relates to diagnostic assays for quantifying and localizing the detection of human NY-ESO-2 protein levels or mRNA levels. These tests are well known in the art.
  • the human NY-ESO-2 protein level detected in the test can be used to diagnose intestinal cancer and liver cancer.
  • a method for detecting the presence or absence of the NY-ESO-2 protein in a sample is to detect the specific antibody of the NY-ESO-2 protein, which comprises: contacting the sample with a NY-ESO-2 protein-specific antibody; observing whether or not it is formed
  • the antibody complex, which forms an antibody complex indicates the presence of the NY-ESO-2 protein in the sample.
  • the NY-ESO-2 protein or polynucleotide thereof can be used for the diagnosis and treatment of NY-ESO-2 protein related diseases.
  • a part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • the antibody against NY-ESO-2 can be immobilized on a protein chip for detection of NY-ESO-2 protein in the sample.
  • the present invention also provides a kit for detecting liver cancer, which comprises a primer pair specifically for amplifying NY-ESO-2 and/or a NY-ESO-2 specific antibody.
  • the invention also provides a method for drug screening based on NY-ESO-2.
  • One method is to first screen for compounds that affect (promote) NY-ESO-2 expression or activity, and then further test the selected compounds against cancer cells.
  • One screening method can be based on the expression level of mRNA for NY-ESO-2.
  • representative cancer cells include, but are not limited to, microsatellite unstable intestinal cancer cells.
  • NY-ESO-2 has a significant correlation with the prognosis of patients with intestinal cancer, that is, the survival time of NY-ESO-2 positive expression is longer.
  • DC cells sensitized by NY-ESO-2 can be used to stimulate T cells to produce anti-tumor activity, This anti-tumor activity is more effective for microsatellite unstable intestinal cancer.
  • Example 1 Clinical retrospective analysis of the difference in expression of NY-ESO-2 in different microsatellite states on patient survival time
  • RNA from three MSI-H/S cell models of HCT116, LS180, and LS174t was extracted by RNA extraction kit.
  • Target preparation firstly prepare Poly-A RNA Controls dilution, synthesize First-Strand and Second-Strand cDNA, and synthesize biotin-labeled cRNA from in vitro transcription, further purify and fragment the cRNA, and then carry out the next chip hybridization. jobs
  • Chip hybridization the chip balance chip was taken out to room temperature, and then the hybridization solution was placed on a heating block at 99 ° C for 5 min, during which a corresponding volume of pre-hybrid solution was injected into the chip, placed in a hybridization furnace, 45 ° C, 60 rpm. /min, 10 min, then the hybridization solution which had been incubated at 99 ° C for 5 min was transferred to another heating fast, 45 ° C for 5 min. After the incubation, the maximum speed of the microcentrifuge was centrifuged for 5 min to remove insoluble matter from the hybridization solution. Remove the chip from the hybridization furnace and suck it out Pre-Hybridization Mix, then inject the corresponding hybridization solution. Finally, the chip was placed in a hybridization furnace at 45 ° C, 60 rpm / min, and hybridized for 16 hr.
  • Protein expression profiling (1) Adjust the cell concentration to 1 ⁇ 10 7 /ml, take 500ul in a 10cm diameter cell culture dish, make up the cell culture medium to 10ml, and after the cells are attached, wash the saline twice, change The tumor cell-free medium was cultured, and after 48 hours of culture, the cell supernatant was collected, and the protein concentration of the cells in the plate was collected. (2) Add 100 ⁇ l of the sample dilution to the chip and incubate for 30 min on a room shaker to block the quantitative antibody chip. The buffer in each well was aspirated, 100 ⁇ l of standard solution and sample were added to the wells and incubated overnight at 4 ° C on a shaker.
  • RNA extraction kit Real-time quantitative PCR analysis: adherent tumor cells were trypsinized, collected by centrifugation, washed twice with PBS, 1 ⁇ 10 6 cells, RNA extraction kit, and extracted total RNA. Finally, the concentration and purity were determined and stored in -80. °C. 2 ⁇ g of total RNA was reverse-transcribed into cDNA according to the reverse transcription kit instructions, and the mixture was incubated at 42 ° C for 30 minutes, inactivated at 99 ° C for 5 minutes, and then diluted with 16 ⁇ l of double distilled water to prepare a template for qPCR reaction.
  • Quantitative PCR primers for molecules such as NY-ESO-2 and Actin F: 5'-CAGACCACCGCCAACTGCA-3' (SEQ ID NO.: 3); R: 5'-TGAGGGAGGCTGAGCCAAA-3' (SEQ ID NO.: 4)
  • Quantitative PCR amplification was performed using Green Realtime PCR Master Mix. As shown in Figure 2B.
  • Flow cytometry analysis the adherent tumor was digested with trypsin to prepare a single cell suspension, adjust the cell concentration to 1 ⁇ 10 6 /ml, and take 100 ul in a 1.5 ml centrifuge tube at 2000 r/min, 4 ° C. After centrifugation for 5 min, the cells were washed twice with cold PBS, and finally resuspended in 200-400 ul of PBS, labeled with antibodies according to the antibody instructions, and finally resuspended in 200 ul PBS to prepare for the machine.
  • Flow cytometry was used to detect the average level of NY-ESO-2 expression in three MSI-H colorectal cancer cell lines. The results showed that NY-ESO-1, NY was expressed in MSI-H colorectal cancer cell lines. -ESO-2, the average level of MAGEA4 is higher than 3 MSS colorectal cell lines. As shown in Figure 2C.
  • the double-blind measurement was performed using the intestinal cancer specimen of the known microsatellite typing in Example 1, thereby typing the intestinal cancer specimen according to the positive expression of NY-ESO-2.
  • the results showed that the sensitivity of microsatellite instability in colorectal cancer based on NY-ESO-2 expression was 90% and the specificity was 78%. Therefore, microsatellite stability typing of intestinal cancer can be performed according to the expression of NY-ESO-2.
  • the bacterial suspension was resuspended in the buffer, and the Escherichia coli was disrupted by ultrasonication at 60-70 Hz for 10-15 minutes, centrifuged at 15,000 rpm for 30 minutes, and the precipitate was collected and denatured and renatured.
  • the protein solution obtained by denaturation and renaturation treatment was subjected to preliminary purification by HisTrap affinity column, and the target protein was eluted by 400-600 mM/L imidazole.
  • the above step eluate was further purified by ion exchange column Sepharose QFF, and the flow-through and gradient eluate were collected, concentrated, and stored frozen at -20 ° C until use.
  • the transfected PVDF membrane was stained with Ponceau red, and the strip to be tested was cut and washed with TBST for 3 times, 5 min/pass. 5% skim milk powder was sealed for 2 h, and TBST was washed 3 times for 5 min/pass.
  • the primary antibody was added in the appropriate ratio, incubated overnight at 4 ° C, and washed 3 times with TBST for 5 min/pass.
  • Horseradish peroxidase-conjugated secondary antibody was added in the appropriate ratio and incubated for 2 h at room temperature. The TBST was washed 3 times, 5 min/pass, and the color developing solution was added, and the reaction was stopped in the dark water until the band appeared.
  • HLA-A2.1/Kb mice were sacrificed by cervical dislocation. The femur was taken aseptically. The bone marrow cells were washed out in serum-free RPMI1640 medium. After centrifugation at 1000g ⁇ 5 minutes, carefully discard the medium supernatant, 3-5ml of Tris-NH4Cl. The solution was dissolved in red blood cells, and anti-Ia, B220, CD4, CD8 monoclonal antibody (final concentration was 10 ⁇ g/ml) and complement (10:1 dilution) were added, and T, B and Ia+ cells were removed in a 37 C water bath for 45 minutes.
  • Mouse DCs cultured to the sixth day were collected, and the cell concentration was adjusted to 2 ⁇ 10 6 cells/ml using mouse DC medium (RPMI1640 complete medium, 10 ng/ml mGM-CSF, 1 ng/ml mIL-4). Divided into 24-well plates, 1 ml per well, the number of wells was the same as the number of mice to be immunized, and 10 ⁇ g/ml NY-ESO-2 polypeptide, NY-ESO-2 polypeptide, MAGE-A4 were added according to the experimental group. The polypeptide, OVA polypeptide, and the same number of wells without any stimulation of DC were used as a blank control group.
  • the cells were collected, the medium supernatant was discarded, and the cells were washed twice with PBS to remove the stimuli present in the original medium. Finally, the cell concentration was adjusted to a concentration of 1 ⁇ 10 6 cells/100 ⁇ l with PBS for a total of 200 ⁇ l (100 ⁇ l of which was used to fill the dead volume of the syringe in the future to ensure the number of cells immunized), and then used to immunize the mice.
  • mice Seven days after the last immunization, each group of mice was sacrificed. The spleens of the mice were aseptically removed. The residual blood was rinsed with sterile PBS, soaked in RPMI1640 medium, and gently ground on a 400 mesh steel wire with a sterile syringe needle. A single cell suspension was obtained and large tissue blocks were removed by stencil filtration. After centrifugation of the filtered single cell suspension at 1000 x g for 5 minutes, the medium supernatant was discarded and suspended in hypotonic NH 4 Cl solution (0.15 M NH 4 Cl, 1 M KHCO 3 , 0.1 mM Na 2 EDTA, pH 7.2).
  • hypotonic NH 4 Cl solution (0.15 M NH 4 Cl, 1 M KHCO 3 , 0.1 mM Na 2 EDTA, pH 7.2).
  • erythrocyte-depleted mouse spleen cells were suspended in RPMI1640 medium containing 10% fetal calf serum and counted.
  • mice spleen cells obtained by the above method were suspended in RPMI1640 medium containing 10% fetal bovine serum, and LS180-MSI-H, LS180-MSS was used, and the final concentration of mitomycin in both cells was 1 mg/ml, 37 ° C, Incubate with 5% CO 2 for 1 h, and then use as a stimulating cell after proliferation.
  • the final concentration of 10 ug/ml of OVA polypeptide was used as a control stimulation group, and the concentration of the stimulated cells was adjusted to 2 ⁇ 10 5 /ml, and then the cell suspension was separately added to the reaction cells.
  • T cell colonies secreting IFN- ⁇ were detected by the method described in the IFN- ⁇ ELISPOT assay kit.
  • Each of the above tests has three duplicate holes. It can be seen that NY-ESO-2/2 sensitized DC cells have stronger stimulating effect on T cells secreting IFN- ⁇ in MSI-H intestinal cancer, but worse in MSS intestinal cancer.
  • LS180-MSI-H, LS180-MSS, T2 cells were used as target cells, and the target cell concentration was adjusted to 4 ⁇ 10 7 /ml, 500 ul with physiological saline, CFSE was added, the final concentration was 2.5 uM, 37 ° C, 10 min, and then added to 1640 culture.
  • the reaction was stopped at 5 ml, the cells were washed three times with PBS, the target cell concentration was adjusted to 2 ⁇ 10 6 /ml, and the 96-well round bottom plate was added to 100 ⁇ l/well.
  • the mouse lymphocytes stimulated by the antigen were used as effector cells. Add lymphocytes at 5:1, 10:1, and 20:1.
  • the proportion of double positive cells was determined by the ratio of CFSE-PI double positive cells in the target cells without stimulating cells, and the killing rate of CTL was calculated.
  • the calculation formula is as follows. (There are three sub-holes for each gradient detection above)
  • Killing rate % (experimental group CFSE-PI double positive cells % - blank control %) / (1 - blank control %).
  • the dose was 2 x 10 6 cells/mouse.
  • the above nude mice were inoculated with tumor cells for 5 days.
  • the experimental groups were grouped according to the above-described immunized HLA-A2.1/Kb transgenic mice.
  • the spleen cells of each group of immunized mice were collected as described above, the concentration of PBS was adjusted, and the tail vein was reinfused into the tumor-bearing nude mice, and the number was 1 ⁇ 10 8 cells/200 ⁇ l ⁇ rat, and the transfusion was performed 3 times, each time interval. one week. Tumor growth was observed and tumor growth curves and mouse survival curves were plotted. As shown in Figure 5.

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Abstract

La présente invention concerne un gène et une protéine NY-ESO-2, ou l'utilisation d'un réactif de détection de ceux-ci, qui sont utilisés pour préparer un kit pour (a) diagnostiquer le cancer colorectal à instabilité microsatellitaire, et/ou (b) déterminer le pronostic d'un patient atteint de cancer colorectal, et appliqués à (c) une méthode thérapeutique destinée à traiter le cancer colorectal à instabilité microsatellitaire, NY-ESO-2 étant une cible.
PCT/CN2016/089135 2015-07-13 2016-07-07 Application de ny-eso-2 dans le diagnostic et le traitement du cancer colorectal à instabilité microsatellitaires WO2017008682A1 (fr)

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HUANG, XIAOHUI: "Identification of the Differentially Expressed Molecules in MSI-H/S Colorectal Cancer and Their Effects on Immunotherapy", MEDICINE & PUBLIC HEALTH, CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, NO. 04, 15 April 2014 (2014-04-15), pages E72 - 41 *

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