WO2023210908A1 - Procédé de prédiction de l'efficacité d'un effet de traitement d'un glioblastome au moyen de lymphocytes t - Google Patents

Procédé de prédiction de l'efficacité d'un effet de traitement d'un glioblastome au moyen de lymphocytes t Download PDF

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WO2023210908A1
WO2023210908A1 PCT/KR2022/020248 KR2022020248W WO2023210908A1 WO 2023210908 A1 WO2023210908 A1 WO 2023210908A1 KR 2022020248 W KR2022020248 W KR 2022020248W WO 2023210908 A1 WO2023210908 A1 WO 2023210908A1
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cells
glioblastoma
expression level
pvr
protein
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Korean (ko)
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안스데반
최혜연
김태규
박순아
이윤경
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가톨릭대학교 산학협력단
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention provides a method for predicting the effectiveness of glioblastoma treatment effect by T cells; Composition for predicting the effectiveness of glioblastoma treatment effect by T cells; and a kit for predicting the effectiveness of glioblastoma treatment effect comprising the same.
  • Glioblastoma is a type of glioma and is known to be the most common and severe type of tumor that occurs in the brain (Journal of Neuro-Oncology, 108(1):11 ⁇ 27). Symptoms of glioblastoma include headaches, personality changes, or nausea, and as symptoms worsen, unconsciousness may occur. Glioblastoma is a representative tumor that is difficult to overcome. The median age of the disease is about 12 to 15 months, and only about 3 to 5% of patients survive for more than 5 years.
  • glioblastoma The cause of glioblastoma is not well known, but rare causes include neurofibromatosis or Li-Fraumeni syndrome, and exposure to radiation is also mentioned as a cause.
  • Glioblastoma accounts for 15% of all brain tumors and is diagnosed through imaging tests such as CT and MRI and surgical biopsy.
  • Treatment of glioblastoma consists of surgery, standard chemotherapy and radiation treatment (temozolomide-radiation combination treatment), and standard chemotherapy. Depending on the patient's condition and detailed diagnosis, additional surgery, chemotherapy, or radiation treatment may be considered.
  • Patent Document 1 Korean Patent No. 10-1692044
  • the purpose of the present invention is to provide a method for predicting the effectiveness of glioblastoma treatment by T cells, which includes measuring the expression level of the PVR (poliovirus receptor) protein or the gene encoding it from a biological sample.
  • PVR poliovirus receptor
  • Another object of the present invention is to provide a composition for predicting the effectiveness of glioblastoma treatment by T cells, which includes an agent that measures the expression level of the PVR (poliovirus receptor) protein or the gene encoding it.
  • PVR poliovirus receptor
  • Another object of the present invention is to provide a kit for predicting the effectiveness of the glioblastoma treatment effect by T cells, including the composition for predicting the effectiveness of the effect of the treatment of glioblastoma by T cells.
  • the present invention provides a method for predicting the effectiveness of glioblastoma treatment by T cells, which includes measuring the expression level of the PVR (poliovirus receptor) protein or the gene encoding it from a biological sample.
  • PVR poliovirus receptor
  • the present invention provides a composition for predicting the effectiveness of glioblastoma treatment by T cells, which includes an agent for measuring the expression level of the PVR (poliovirus receptor) protein or the gene encoding it.
  • PVR poliovirus receptor
  • the present invention provides a kit for predicting the effectiveness of the glioblastoma treatment effect by T cells, including the composition for predicting the effectiveness of the effect of the treatment of glioblastoma by T cells.
  • the present invention provides a method for predicting and treating the effectiveness of glioblastoma treatment using the above method.
  • the present invention provides a method of measuring the expression level of the PVR (poliovirus receptor) protein or the gene encoding it from a biological sample isolated from a glioblastoma patient and administering T cells if the expression level is higher than that of the normal control group. Provides a method of treating blastoma.
  • PVR poliovirus receptor
  • the method according to the present invention measures the expression level of the PVR (poliovirus receptor) protein or the gene encoding it, and when the expression level of the protein or the gene encoding it is higher than the control group, treatment with T cells It can be predicted that the effect may be effective, so it can be useful in personalized treatment of glioblastoma.
  • PVR poliovirus receptor
  • Figure 1 shows the results of measuring the expression levels (expression %) of MICA, ULBP, PVR, and Nectin in patient-derived glioblastoma cell lines using flow cytometry.
  • Figure 2 shows the results of measuring the degree of lysis (specific lysis %) after treating patient-derived glioblastoma cell lines with ⁇ T cells at a ratio of 20:1 and co-culturing them for 4 hours.
  • Figure 3 shows the results of analyzing the correlation between the degree of lysis (specific lysis %) and the expression level (expression %) of MICA, ULBP, PVR, and Nectin in patient-derived glioblastoma cell lines.
  • Figure 4 shows the results of ROC curve analysis.
  • the present invention provides a method for predicting the effectiveness of glioblastoma treatment by T cells, which includes measuring the expression level of the PVR (poliovirus receptor) protein or the gene encoding it from a biological sample.
  • PVR poliovirus receptor
  • PVR poliovirus receptor protein
  • CD155 cluster of differentiation 155
  • DNAM DNAM It is known to be one of the major ligands for the -1 receptor.
  • the biological sample may be selected from the group consisting of tissue, cells, blood, plasma, and serum, the tissue may be a tumor tissue, and the tumor tissue may be resected from a glioblastoma patient.
  • the biological sample may be a tumor tissue resected from a glioblastoma patient, and more preferably, it may be a glioblastoma cell line isolated from a tumor tissue resected from a glioblastoma patient.
  • the measurement is RT-PCR (reverse transcription polymerase chain reaction), competitive RT-PCR, real-time quantitative RT-PCR, RNase protection method, northern blot, and DNA chip. It may be performed by a method selected from the group consisting of DNA chip technology assay (DNA chip technology assay), ELISA (Enzyme-linked immunosorbent assay), western blot, immunoblot, and immunohistochemistry. It is not limited to this.
  • the step of comparing the measured expression level with a control group may be further included.
  • the control group at this time may mean normal people without glioblastoma.
  • the method may further include predicting or determining that the effect of treating glioblastoma by T cells will be effective.
  • the T cells may be ⁇ T cells (gamma delta T cells), but are not limited thereto.
  • the present invention provides a composition for predicting the effectiveness of glioblastoma treatment by T cells, which includes an agent for measuring the expression level of the PVR (poliovirus receptor) protein or the gene encoding it.
  • PVR poliovirus receptor
  • the agent may be selected from the group consisting of primers, probes, aptamers, and antibodies specific for PVR (poliovirus receptor), but is not limited thereto.
  • primer refers to a base sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and serving as a starting point for copying the template strand. It refers to a short base sequence that acts. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.
  • probe in the present invention refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a gene, and is labeled so that the presence or absence and expression level of a specific gene can be confirmed. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art.
  • the primer or probe of the present invention can be chemically synthesized using phosphoramidite solid support synthesis or other well-known methods. These base sequences can also be modified through various methods known in the art. Examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) ) or variations to charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • variations to charged linkages e.g. phosphorothioate, phosphorodithioate, etc.
  • aptamer in the present invention refers to an oligonucleotide or peptide molecule that specifically binds to a target molecule.
  • an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment) is used. By using evolutionary methods, oligomers that bind to specific chemical or biological molecules with high affinity and selectivity can be separated and obtained.
  • antibody in the present invention refers to a substance that reacts when an antigen, a foreign substance, invades while circulating in the blood or lymph in the body's immune system. It is a globulin-based protein formed in lymphoid tissue and is also called immunoglobulin. Antibodies are produced by B cells and bind specifically to antigens. One antibody molecule has two heavy chains and two light chains, and each heavy chain and light chain each contain a variable region at the N-terminus. Each variable region is composed of three complementarity determining regions (CDRs) and four framework regions (FRs). The complementarity determining regions determine the antigen-binding specificity of the antibody and determine the structure of the variable region. It exists as a relatively short peptide sequence held in forming sites.
  • CDRs complementarity determining regions
  • FRs framework regions
  • the expression level of the PVR (poliovirus receptor) protein or the gene encoding it may be measured in a glioblastoma cell line isolated from tumor tissue resected from a glioblastoma patient.
  • the present invention provides a kit for predicting the effectiveness of the glioblastoma treatment effect by T cells, including a composition for predicting the effectiveness of the glioblastoma treatment effect by T cells.
  • the kit may be selected from the group consisting of microarray, gene amplification kit, immunoassay kit, protein microarray kit, and ELISA kit.
  • the present invention includes the steps of (a) measuring the expression level of a PVR (poliovirus receptor) protein or a gene encoding it from a biological sample isolated from a patient; (b) comparing the measured expression level with a normal control group;
  • biological samples, tissues, measurement methods, and T cells can be applied in the same manner as described above, so their description is omitted.
  • the present invention provides a method of measuring the expression level of the PVR (poliovirus receptor) protein or the gene encoding it from a biological sample isolated from a glioblastoma patient and administering T cells if the expression level is higher than that of the normal control group. Provides a method of treating blastoma.
  • PVR poliovirus receptor
  • glioblastoma treatment method biological samples, tissues, measurement methods, and T cells can be applied in the same manner as described above, so their description is omitted.
  • Example 1 Preparation and culture of patient-derived glioblastoma cell lines
  • glioblastoma cell lines were isolated from the tumor tissues. Briefly, glioblastoma tissue isolated from the patient was washed with cold phosphate buffer saline (PBS), and after washing, 1% Glutamax (Gibco), 1% Penicillin/streptomycin (Gibco), and 2.5 ⁇ g/ml of amphotericin B (Gibco) were added. The tissue was cut into small pieces of 0.5 to 1 mm in size with surgical scissors while applying cold Hibernate-A medium containing this.
  • PBS cold phosphate buffer saline
  • the cut tumor tissue pieces were collected in a conical tube, washed three times with room temperature PBS, and then applied with RBC (red blood cell) lysis buffer for 5-10 minutes. Afterwards, only the patient-derived glioblastoma cell line was isolated by centrifugation.
  • RBC red blood cell
  • ⁇ T cells were obtained from peripheral blood from normal individuals or glioblastoma patients. Briefly, peripheral blood was added onto Ficoll-Hypaque at a ratio of 1:1 and then centrifuged at 2500 rpm for 20 minutes. Afterwards, peripheral blood mononuclear cells (PBMC) in the buffy coat layer were collected and washed with RPMI medium. Washing was performed four times, at 1800 rpm for 15 minutes, 1600 rpm for 10 minutes, 1400 rpm for 5 minutes, and 1400 rpm for 5 minutes, respectively. Afterwards, the separated PBMCs were suspended in freezing media, placed in a freezing bottle containing isopropyl alcohol, stored in a -80°C freezer for 16 hours, and then stored in a liquid nitrogen tank.
  • PBMC peripheral blood mononuclear cells
  • the stored PBMCs were quickly dissolved in a 37°C bath, suspended in RPMI containing 10% FBS, and washed. Washed PBMCs were treated with 5 ⁇ M zoledronic acid and 1000 U/mL IL-2, and then dispensed into 24 well-plates. On the 3rd day, 1000 U/mL of IL-2 was additionally treated, and on the 7th day, the cells were harvested and washed. Afterwards, the cells were treated with 1000 U/mL of IL-2 and cultured, or K562 cells expressing CD32, CD80, CD83, 4-1BBL, CD40L, and CD70 as artificial antigen-presenting cells were irradiated (100 Gy) and co-cultured. . The cultured ⁇ T cells were harvested on day 14 or on day 21 by repeating the same process once more, suspended in freezing media, frozen using a CRF (controlled-rate-freezer), and stored in liquid nitrogen until the experiment.
  • CRF controlled-rate-freezer
  • the present inventors performed an experiment to evaluate the effectiveness of glioblastoma treatment effect by T cells.
  • ligands such as MICA (MHC class I polypeptide-related sequence A), ULBP (UL16 binding protein 1), PVR (Poliovirus receptor, CD155), and Nectin in patient-derived glioblastoma cell lines was measured using flow cytometry.
  • the patient-derived glioblastoma cell line was labeled with Calcein-AM, and the patient-derived glioblastoma cell line labeled with Calcein-AM was treated with ⁇ T cells at an E(effector):T(target) ratio of 20:1. Co-cultured for 4 hours.
  • the degree of degradation by T cells was low in cell lines 5 and 8, the degree of degradation by T cells was low in cell lines 7 and 9, and the degree of degradation was about 20-30% in cell lines 7 and 9, and in the remaining cell lines, the degree of degradation by T cells was low.
  • the degree of degradation by T cells was low.
  • the correlation between the expression level (expression %) of MICA, ULBP, PVR, and Nectin and the degree of lysis by T cells (specific lysis %) was analyzed.
  • the correlation between PVR expression and the degree of degradation by T cells was found to be significant.
  • the degree of degradation by T cells is high, and it was confirmed that the effectiveness of treatment by T cells can be predicted or evaluated by measuring the PVR expression level.
  • the expression of the remaining MICA, ULBP, and Nectin was not related to the degree of degradation by T cells.
  • the present inventors performed ROC curve analysis to evaluate the accuracy of predicting the effectiveness of glioblastoma treatment effect by T cells from the presence or absence of PVR expression. As a result, sensitivity of 71.4% and specificity of 100% were confirmed in more than 89.2% of patient-derived glioblastoma cell lines expressing PVR ( Figure 4).
  • the present invention can predict or determine whether the therapeutic effect by T cells is effective in the treatment of glioblastoma by measuring the expression level of the PVR protein or the gene encoding it in the patient-derived glioblastoma cell line. was confirmed.

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Abstract

La présente invention concerne un procédé de prédiction de l'efficacité d'un effet de traitement d'un glioblastome au moyen de lymphocytes T, le procédé comprenant une étape de mesure, à partir d'un échantillon biologique, du niveau d'expression d'une protéine de récepteur de poliovirus (PVR) ou d'un gène codant pour celle-ci. En mesurant le niveau d'expression d'une protéine de PVR ou d'un gène codant pour celle-ci dans un traitement de glioblastome, l'efficacité d'un effet de traitement au moyen de lymphocytes T peut être prédite, et ainsi la présente invention peut être utilisée efficacement pour un traitement de glioblastome personnalisé.
PCT/KR2022/020248 2022-04-28 2022-12-13 Procédé de prédiction de l'efficacité d'un effet de traitement d'un glioblastome au moyen de lymphocytes t WO2023210908A1 (fr)

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Citations (3)

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WO2018067446A1 (fr) * 2016-10-06 2018-04-12 Duke University Détection de cd -155, le récepteur de poliovirus
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WO2018067446A1 (fr) * 2016-10-06 2018-04-12 Duke University Détection de cd -155, le récepteur de poliovirus
WO2019102456A1 (fr) * 2017-11-27 2019-05-31 University Of Rijeka Faculty Of Medicine Immunotoxines pour le traitement du cancer
WO2021070181A1 (fr) * 2019-10-08 2021-04-15 Nectin Therapeutics Ltd. Anticorps dirigés contre le récepteur du poliovirus (pvr) et leurs utilisations

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