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WO2022169249A1 - Method for predicting treatment response to cancer immunotherapy in lung cancer - Google Patents

Method for predicting treatment response to cancer immunotherapy in lung cancer Download PDF

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Publication number
WO2022169249A1
WO2022169249A1 PCT/KR2022/001649 KR2022001649W WO2022169249A1 WO 2022169249 A1 WO2022169249 A1 WO 2022169249A1 KR 2022001649 W KR2022001649 W KR 2022001649W WO 2022169249 A1 WO2022169249 A1 WO 2022169249A1
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cells
therapeutic response
lung cancer
predicting
gzmb
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French (fr)
Korean (ko)
Inventor
김혜련
하상준
이인석
조재원
김가민
박세연
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Industry Academic Cooperation Foundation of Yonsei University
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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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • G01N33/57484—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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • 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 relates to a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer.
  • Lung cancer is one of the most common cancers in both men and women.
  • non small lung cancer is a kind of epithelial cancer (carcinoma) and refers to all epithelial lung cancers, not small lung cancer.
  • non-small cell lung cancer occupies a high proportion in the overall incidence of lung cancer.
  • non-small cell lung cancer is divided into several subtypes according to the size, shape, and chemical composition of cancer cells, and typical examples include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.
  • Adenocarcinoma is found in the outer region of the lung and tends to progress more slowly than other lung cancers.
  • Squamous cell carcinoma starts in the early version of the cells that make up the airway, and shows a high incidence mainly in smokers.
  • large cell cancer can develop in any part of the lung, and its treatment is still a challenge because the progression rate is fast enough to be similar to that of small cell lung cancer.
  • Non-small cell lung cancer may include persistent cough, chest pain, weight loss, nail damage, joint pain, and shortness of breath.
  • non-small cell lung cancer progresses more slowly than other cancers, it rarely shows symptoms in the early stages. Therefore, early detection and treatment of non-small cell lung cancer is difficult, and it is highly likely to be detected after metastasis to the bones, liver, small intestine, and brain. Accordingly, at the time of diagnosis of non-small cell lung cancer, more than half of the patients are advanced enough to not be able to perform surgery, so early treatment is difficult in reality.
  • radical resection is performed, but only about 30% of cases in which radical resection can be performed. Furthermore, the majority of all patients who underwent radical resection appear to recur and die from a more aggressive disease after surgical resection.
  • TKIs tyrosine kinase inhibitors
  • tumor PD-L1 expression by immunohistochemistry can be currently used as the best predictive biomarker for PD-1 blockade.
  • IHC immunohistochemistry
  • the accuracy of predicting the therapeutic response of PD-L1 dependent on tumor PD-L1 expression is not high enough to confirm drug efficacy. More specifically, PD-L1-expressing negative patients may respond to PD-1 blockade, and PD-L1-expressing positive patients may not respond to PD-1 blockade. Furthermore, some responders without PD-L1 may have a similar response duration if they were PD-L1 positive in the Checkmate 057 trial. Moreover, PD-L1 expression is dynamic and can change temporally and spatially. This change in PD-L1 expression may be adaptive immune resistance exerted by the tumor.
  • biomarkers for predicting therapeutic response of PD-L1 blockade CD8 T cell infiltration, inflammatory tumor markers, T cell receptor clonality and somatic mutational burden can be used.
  • the above biomarkers have difficulties in obtaining tumor tissue, heterogeneity of tumors and the possibility of inducing some biomarkers, so it may be difficult to use them as single markers in predicting therapeutic response to PD-1 blockade. have.
  • the inventors of the present invention have recognized that the reactivity to immuno-cancer therapy is reduced in patients with EGFR mutations, and by identifying differences in individuals with reduced drug reactivity using EGFR mutations as an example, their drug reactivity to overcome the limitations of Accordingly, the inventors of the present invention have discovered that the composition of specific immune cells they have is differentiated from patients having responsiveness to immuno-cancer therapy, and accordingly, it can be found that the responsiveness to immuno-cancer therapy can be reduced.
  • the inventors of the present invention have found that specific immune cells that are differentially distributed have different mechanisms from cells by conventional sorting. That is, the inventors of the present invention have discovered a gene signature capable of characterizing a specific immune cell that can predict the responsiveness to immunotherapy.
  • the inventors of the present invention have developed a novel therapeutic response prediction method using GZMB+CD103+CD8+T cells as a biomarker for predicting the therapeutic response to immune anticancer therapy, that is, an immune checkpoint inhibitor. .
  • the problem to be solved by the present invention is to measure the level of GZMB+CD103+CD8+T cells, and to measure the level of GZMB+CD103+CD8+T cells so as to maximize the therapeutic effect by predicting the responsiveness of the immunotherapy for the subject at an early stage.
  • An object of the present invention is to provide a method for predicting a therapeutic response, which can be provided by determining the therapeutic response of an immuno-cancer therapy for an individual based on it.
  • Another problem to be solved by the present invention is to provide a kit capable of predicting a therapeutic response to an immuno-cancer therapy, based on the above-described method for predicting a therapeutic response.
  • the present invention provides, for a biological sample isolated from an individual, ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 measuring the level of mRNA or protein thereof for at least one marker gene of at least one of To provide a method for predicting a therapeutic response to immunotherapy in lung cancer.
  • the at least one genetic marker is ITGAE and/or GZMA
  • the mRNA is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerization Enzyme reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting), measured by any one method selected from the group consisting of a DNA chip
  • the protein is, Western blot, enzyme linked immunosorbent assay, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry (immunohistochemistry, IHC), Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and protein chip.
  • RT-PCR reverse transcription polymerase reaction
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • the present invention provides the steps of measuring the level of GZMB + CD103 + CD8 + T cells, and the measured GZMB + CD103 + CD8 + T cells in a biological sample isolated from a subject.
  • a method of predicting a therapeutic response to an immuno-cancer therapy in lung cancer comprising determining a therapeutic response of the immuno-cancer therapy for an individual based on the level.
  • GZMB+CD103+CD8+T cells are ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT , HAVCR2, LAG3, ICOS, TOX and TCF7 mRNA or a protein thereof for at least one of the marker genes may be expressed, but is not limited thereto, and may further include various marker genes that can be expressed by CD8 + T cells.
  • the log FC (fold change) of GNLY is 1.5 or more
  • the log FC (fold change) of GZMB is 1.2 or more
  • the log FC of ITGAE may be 0.4 or more, and they each contain a positive log FC.
  • GZMB + CD103 + CD8 + T cells may have the above-described expression of GNLY, GZMB and ITGAE increased (up-regulation).
  • the predictive power for GZMB+ITGAE(CD103)+CD8+T cells may be AUC 0.854.
  • the measuring step includes measuring GZMB + CD103 + CD8 + T cells with respect to a sample of a normal control group, and determining the therapeutic response of the immune anticancer therapy is the measured If the level of GZMB+CD103+CD8+T cells for the subject exhibits a reduced level than the level of GZMB+ITGAE(CD103)+CD8+T cells for a normal control sample, the subject is negatively responsive to immune anticancer therapy. It may include the step of determining that
  • the measuring step further comprises measuring CXCL13 + CD4 + T cells for a sample of a normal control, and the determining step is, CXCL13 + CD4 + T for the measured individual determining that the subject is negative for responsiveness to the immuno-cancer therapy if the level of cells indicates a reduced level than the level of CXCL13 + CD4 + T cells relative to a normal control.
  • CXCL13 + CD4 + T cells CD2, CD6, CD82, TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN, and can express mRNA or a protein thereof for at least one marker gene of GATA3, but is not limited thereto, and further, CXCL13 + CD4 + T cells, PDCD1, MAF and SH2D1A mRNA for at least one marker gene or a protein thereof may be further expressed.
  • the measuring step further comprises measuring the level of CXCR5 + CD20 + B cells with respect to a sample of a normal control, and the determining includes: CXCR5 + CD20 for the measured individual if the level of +B cells indicates a reduced level than the level of CXCR5 + CD20 + B cells relative to the normal control sample, determining by the subject that the therapeutic response to the immuno-cancer therapy is negative.
  • the CXCR5+CD20+B cells may express at least one antigen presentation molecule of HLA-DPB1, HLA-DRB5, HLA-DQA2, and HLA-C, and may express the TAP1 marker gene.
  • the antigen marker and the above-mentioned marker gene may show a reduced level than the antigen marker and the above-mentioned marker gene expression level of CXCR5 + CD20 + B cells for a normal control.
  • the measuring step further comprises measuring the number of GZMB+CD103+CD8+T cells, and the determining comprises: If the number of +CD103+CD8+ T cells is 1 cells/mm 2 or less, or the ratio of GZMB+CD103+CD8+ T cells to total CD103+CD8+ T cells is less than 30%, the subject is treated with immunotherapy determining that the reaction is negative.
  • the measuring step further comprises measuring the area of tertiary lymphoid structures (TLS) for the sample of the subject and the normal control, and the determining comprises: If the area of TLS for the isolated biological sample exhibits a level that is at least 70% reduced compared to the level of the TLS area for the normal control sample, or the area of TLS for the subject is less than 5 mm 2 , the subject is on immunotherapy determining that the therapeutic response to the patient is negative.
  • TLS tertiary lymphoid structures
  • the subject may be a suspected lung cancer subject, but is not limited thereto, and CXCR5 + CD20 + B cells, CD4 + T cells according to an embodiment of the present invention through tumors and various diseases.
  • CXCL13+CD4+T cells, CD8+T cells, CD103+CD8+T cells, and GZMB+CD103+CD8+T cells can all be included.
  • the biological sample may include at least one selected from the group consisting of tissue, peripheral blood, serum and plasma, but is not limited thereto, and may include all samples in which immune cells can be measured.
  • the present invention provides an immuno-cancer therapy for lung cancer individuals, comprising an agent configured to measure the level of GZMB + CD103 + CD8 + T cells in a biological sample isolated from the individual.
  • a kit for predicting treatment response may be provided.
  • the present invention has the effect of providing a new biomarker that can predict the therapeutic response to PD-1 blockade and various immune checkpoint inhibitors.
  • the present invention has the effect of predicting the therapeutic response to PD-1 blockade based on the ratio to the biomarker in various samples within the subject. Accordingly, the present invention predicts an early treatment response to PD-1 blockade for an individual using a biomarker, so it is possible to provide information to quickly determine whether to proceed with anti-PD-1 treatment, and the treatment responsiveness By early selection of patients who can be effective and non-responsive patients, side effects and unnecessary cost consumption can be reduced, and the therapeutic effect in the clinic can be maximized.
  • the effect according to the present invention is not limited by the contents exemplified above, and more various effects are included in the present specification.
  • FIG. 1 exemplarily shows a procedure for a method for predicting a therapeutic response to an immune anticancer therapy in lung cancer according to an embodiment of the present invention.
  • FIG. 2 exemplarily shows the mechanism of action of immune cells used in immune anticancer therapy in lung cancer containing an EGFR mutation according to an embodiment of the present invention.
  • 3A to 3I show results for CD8 cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.
  • 4A to 4B show results of predicting an anti-PD-1 treatment response through a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention.
  • 5A to 5D show the results of an overall analysis of immune cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.
  • 6A to 7 show results for NKT and B cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.
  • FIGS. 8A to 8E show results for CD4 and Treg cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.
  • 9a to 10d show the results of verifying the activation of T cells through homeostasis and TLS for immune cells used in immune anticancer therapy of an individual containing an EGFR mutation according to an embodiment of the present invention.
  • subtype or subpopulation refers to a cell population constituting a part of the entire cell population, and can be pathologically distinguished and classified according to the biomarkers contained in the cells. have.
  • non-small cell lung cancer refers to any epithelial lung cancer that is not small lung cancer as a type of epithelial cancer.
  • anti-PD-1 treatment may be used as an immunotherapy for non-small cell lung cancer.
  • anti-PD-1 treatment may be a therapy configured to block a mechanism by which T cells fail to attack cancer cells. More specifically, anti-PD-1 treatment may be based on blocking the binding of PD-L1 and PD-L2, which are surface proteins of cancer cells, to PD-1, a protein on the surface of T cells. For example, when an immune anticancer agent binds to the PD-1 receptor of T cells, it can inhibit the evasion function of T cells against cancer cells. Therefore, in the present specification, “anti-PD-1 treatment” may be used in the same sense as "PD-1 blocking".
  • the term “therapeutic response negative” immunotherapy that is, the binding response of receptors and ligands on the surface of T cells by immune checkpoint inhibitors does not block, or secretion of cytotoxic substances such as cytokines according to blocking It may mean that the power is not improved. However, it is not limited thereto, and may include the occurrence of any response associated with alleviation of lung cancer symptoms or good prognosis by immuno-cancer therapy.
  • the term “positive or +” may mean that a marker capable of characterizing a cell is expressed.
  • cytotoxic T cells CD8 is expressed on the cell surface, and thus, cytotoxic T cells may be denoted as CD8 + T cells, but are not limited thereto. Except for ', it can also be expressed as CD8 T cells.
  • the term “about” refers to a common error range for each value readily known to one of ordinary skill in the art. References herein to “about” a value or parameter include embodiments relating to that value or parameter per se.
  • the term "patient or subject” is used interchangeably and includes any single animal in need of treatment, more preferably a mammal (such as a non-human animal such as a cat, a dog, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates).
  • the patient herein is a human.
  • a patient may be a “cancer patient,” ie, a person suffering from, at risk of, or suffering from cancer, one or more symptoms of cancer.
  • level of expression or “expression level” are used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which information (eg, gene-coding and/or epigenetic information) is converted into a functioning construct and present in the cell.
  • expression refers to transcription into a polynucleotide, translation into a polypeptide, or further polynucleotide and/or polypeptide modifications (eg, post-translational modification of a polypeptide).
  • Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotides and/or polypeptide modifications also include transcripts or cleavages that are produced by alternative splicing (alternative splicing) It should be considered expressed, whether derived from a modified transcript or from a post-translational process of the polypeptide, eg, by proteolysis.
  • an “expressed gene” includes those transcribed into a polynucleotide into mRNA and then translated into a polypeptide and also those transcribed into RNA but not translated into a polypeptide (eg, mobile and ribosomal RNAs).
  • Expression levels for more than one gene of interest may be determined by aggregation methods known to those skilled in the art and disclosed herein, including, for example, calculating the median or average of all expression levels of the gene of interest. can be decided. Prior to aggregation, the expression level of each gene of interest is, for example, normalized to the expression level of one or more housekeeping genes, or normalized to the total library size, or to a median or average expression level value across all genes measured.
  • Normalization can be normalized using statistical methods known to those of skill in the art and also disclosed herein.
  • the normalized expression level of each gene of interest is determined in the art, including, for example, by calculating a Z-score of the normalized expression level of each gene of interest. It can be standardized by using statistical methods known to those skilled in the art and also disclosed herein.
  • the method for predicting treatment response to immuno-cancer therapy in lung cancer includes measuring the level of GZMB+CD103+CD8+T cells in a biological sample isolated from an individual ( S110) and determining the therapeutic response of the immunotherapy for the subject based on the measured level of GZMB+CD103+CD8+T cells (S120).
  • the step of measuring the level of GZMB+CD103+CD8+T cells includes not only the aforementioned GZMB+CD103+CD8+T cells, but also CXCR5+CD20+B cells, CD4+T cells, and CXCL13+CD4+T cells.
  • Various immune cells such as CD8 + T cells and CD103 + CD8 + T cells can be further measured.
  • CXCR5 + CD20 + B cells, CD4 + T cells, CXCL13 + CD4 + T cells, CD8 + T cells, CD103+CD8+T cells and GZMB+CD103+CD8+T cells are immune cells that can have an immune response to a tumor, that is, an anticancer effect through a chain reaction between each cell.
  • the T FH cells secrete CXCL13 to induce B cells, that is, to infiltrate into the follicles of the cells.
  • CXCL13 secreted by T FH cells can activate B cells by binding to CXCR5 expressed in B cells. Then, it can be expressed on the surface of the cell. Then, the antigen expressed by the B cells may bind to the TCR of the T RM cells and subtypes thereof having a cytotoxic effect, thereby activating the T RM cells and the subtypes thereof.
  • the antigens expressed by B cells not only activate T RM cells, but also T FH capable of secreting CXCL13.
  • T FH binds to cellular TCR
  • T FH By activating the cells, the secretion of CXCL13 can be further promoted. Accordingly, immune homeostasis and toxic effects may be further improved.
  • activated T RM cells and subtypes thereof contain CD103, a tumor-specific marker that can target tumors, and thus do not respond to cells other than tumors, and secrete GZMB, a strong cytotoxic molecule, to kill tumors. You can attack directly.
  • TLS tertiary lymphoid structures
  • EGFR mutations can inhibit the formation of such TLS and the expression of immune cells, which are components thereof. Accordingly, in the case of an individual containing an EGFR mutation, the effect on immunotherapy may be reduced.
  • the effect on immunotherapy may be reduced.
  • the immune cells that can most effectively predict the therapeutic response to immuno-cancer therapy are, preferably, GZMB+CD103+CD8+T It may be a cell (T RM -like cells).
  • GZMB+CD103+CD8+T cells are ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS , TOX and TCF7 may express mRNA for at least one marker gene or a protein thereof, and a specific marker among them may be differentially expressed.
  • GZMB+CD103+CD8+T cells may have differentially increased expression levels of GZMB, ITGAE (CD103) and GNLY.
  • CD4 + T cells and CXCL13 + CD4 + T cells were CD2, CD6, and CD82. Marker of at least one of TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN and GATA3 mRNA for the gene or a protein thereof may be expressed, and the CXCL13 + CD4 + T cells may further express mRNA or a protein thereof for at least one marker gene of PDCD1, MAF, and SH2D1A.
  • CXCR5+CD20+B cells can express at least one antigen presentation molecule among HLA-DPB1, HLA-DRB5, HLA-DQA2 and HLA-C and TAP1, and are capable of expressing the TAP1 marker gene.
  • the immune cells may further include NK cells (Natural killer cells) or NKT cells (Natural Killer T cells).
  • the subject is a subject suspected of lung cancer, and may contain an EGFR mutation, but is not limited thereto.
  • the biological sample may include at least one selected from the group consisting of tissue, peripheral blood, serum and plasma, and the immuno-cancer therapy may be anti-PD-1 treatment, but is not limited thereto, and tumor microscopic It may include all immune checkpoint inhibitors for a variety of ligands that may be inhibited from the environment.
  • GZMB + CD103 + CD8 + T cells can be measured for the sample of the normal control group, and further, GZMB + CD103 + CD8 + T cells as well as the normal control sample.
  • Various immune cells such as CXCR5+CD20+B cells, CD4+T cells, CXCL13+CD4+T cells, CD8+T cells and CD103+CD8+T cells can be further measured for the sample.
  • the measurement of the transcript of a molecule characteristically expressed may be used instead of the aforementioned measurement of the immune cells.
  • the CD8 + T cells used in the method for predicting therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention are not only the aforementioned GZMB and CD103 (ITGAE), but also ID2, IFNG, CD7, CXCR6, At least one marker gene of PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 may be differentially expressed.
  • the above-described marker gene may be used in the measuring step ( S110 ). That is, in the measuring step (S110), at least one marker gene of ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2, and LAG3 for individuals and normal controls The level of mRNA or a protein thereof can be measured.
  • the number of GZMB+CD103+CD8+T cells that is, the number of GZMB+CD103+CD8+T cells per unit area in a biological sample isolated from an individual, may be further measured.
  • the transition probability of GZMB+CD103+CD8+T cells to GZMB+CD103+CD8+T cells may be further measured.
  • the level of CXCR5 + CD20 + B cells when the level of CXCR5 + CD20 + B cells is measured, the level of CXCR5 + CD20 + B cells relative to the sample of the normal control may be further measured, and at this time, CXCR5 + CD20 + B
  • the cell may measure an antigen presentation molecule of at least one of HLA-DPB1, HLA-DRB5, HLA-DQA2 and HLA-C, mRNA for a TAP1 marker gene, or a protein thereof, wherein the antigen is a normal control It may be a reduced level than the expression level of the antigen marker and the above-mentioned marker gene of CXCR5 + CD20 + B cells.
  • the mRNA for at least one of PRF1, GZMB, TNFRSF18, IL2RB, CD7, CD44, TYROBP and TNFRSF1B or NKT cells expressing a protein thereof (Natural Killer T cells) and a normal control NKT cells for the sample can be further measured, and at this time, the mRNA or protein thereof for the gene expressed in NKT may be a reduced level than the mRNA or protein expression level of the marker gene of NKT cells for a normal control. have.
  • the area of tertiary lymphoid structures (TLS) for the sample of the individual and the normal control may be further measured.
  • the expression level of mRNA is, droplet digital PCR (dd-PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction ( Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, can be measured by any one method selected from the group consisting of a DNA chip,
  • the expression level of the protein was determined by western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion method, rocket immunization.
  • ELISA enzyme linked immunosorbent assay
  • radioimmunoassay radioimmunodiffusion
  • ouchterlony immunodiffusion method rocket immunization.
  • electrophoresis immunohistochemistry (IHC), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS) and protein chip (protein chip) It can be measured by the method of, but is not limited thereto, and may include all of various methods by which immune cells, proteins, and mRNAs can be measured.
  • the determining step (S120) when the level of GZMB + CD103 + CD8 + T cells for the measured individual represents a reduced level than the level of GZMB + ITGAE (CD103) + CD8 + T cells for the normal control sample, It can be determined that the subject is negative for responsiveness to the immuno-cancer therapy.
  • the level of CXCL13 + CD4 + T cells or CXCR5 + CD20 + B cells for the measured individual is the level of CXCL13 + CD4 + T cells or CXCR5 + CD20 + B cells for the normal control sample. If it exhibits a level that is lower than the level, it can be determined that the subject is negatively responsive to the immuno-cancer therapy.
  • the determining step ( S120 ) when the level of mRNA or protein thereof for the measured individual for the marker gene is lower than the level of the normal control, it can be determined that the individual has a negative therapeutic response to the immuno-cancer therapy.
  • the level of CD4+ T cells is 750 cells/mm 2 or less, the level of CXCL13+CD4+ T cells is 230 cells/mm 2 or less, or the level of CD8+ T cells is 120 cells /mm 2 or less, or the level of CD103+CD8+ T cells is 5 cells/mm 2 or less, or the ratio of CD103+CD8+ T cells to total CD8+ T cells is less than 20%, or GZMB+CD103+CD8+ If the level of T cells is 1 cells/mm 2 or less, or the ratio of GZMB+CD103+CD8+ T cells to total CD103+CD8+ T cells is less than 30%, or the When the conversion probability to GZMB+CD103+CD8+T cells is 41% or less, it can be determined that the subject has a negative therapeutic response to the immuno-cancer therapy.
  • the determining step (S120) if the level of NKT cells for the measured individual represents an increased level than the level of NKT cells for the normal control sample, or the area of TLS for the measured individual is, the normal control sample If it represents a level that is reduced by 70% or more than the level of the TLS area for , or if the area of TLS for the subject is less than 5 mm 2 , it can be determined that the subject has a negative therapeutic response to the immunotherapy.
  • the method for predicting the therapeutic response to immuno-cancer therapy in lung cancer is to measure the level of various markers to determine the therapeutic response to the immuno-cancer therapy, in particular, the anti-PD-1 treatment for the subject. It can provide information so that it can be predicted early.
  • the predictive power of GZMB+CD103+CD8+T cells (T RM -like cells) for anti-PD-1 treatment may be AUC 0.854. That is, the method for predicting the therapeutic response to immunocancer therapy in lung cancer according to an embodiment of the present invention predicts the therapeutic response to the immune anticancer therapy with high predictive power through GZMB+CD103+CD8+T cells (T RM -like cells). can be determined and provided.
  • the present invention may provide a kit for predicting the therapeutic response to the immune anticancer therapy of a lung cancer individual according to an embodiment of the present invention based on the method for predicting the therapeutic response to the immunocancer therapy in lung cancer described above.
  • the kit for predicting the therapeutic response to the immuno-cancer therapy of the lung cancer individual is a biological sample isolated from the individual, GZMB + CD103 + CD8 + T cells (T RM -like cells) agents configured to measure levels.
  • GZMB+CD103+CD8+T cells used in a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described in detail.
  • DEGs differentially expressed genes
  • C2 a subtype of CD8 T cells, expresses PRF1, GZMA, GZMB, GNLY and GZMH, which are genes involved in cytotoxicity at the highest level, and furthermore, genes involved in the differentiation of CD8 T cells.
  • ITGAE is a marker for tissue-resident memory T cells (tissue-resident memory CD8+T, T RM )
  • C2 cells expressing it have high cytotoxicity and can promote differentiation into CD8 T cells. It may be a subtype of T RM cells (T RM -like cells).
  • the CD8 T cell subtype, C2 can differentially express PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 and LAG3, as shown in Fig. 3a.
  • the expression level results for the signature gene marker of C2 a subtype of the aforementioned CD8 T cells, are shown.
  • the expression level was derived through comparison with C0, C11 and C14, which are subtypes of CD8 T cells, and the FC (fold change) value, which is the ratio of the expression level, was shown.
  • C2 a subtype of CD8 T cells, was shown to have positive FC values with GZMB of 1.2 or more, CD7 of 0.9 or more, ID2 of 0.5 or more, and ITGAE of 0.4 or more, as in 3a. It may mean that the expression is increased (up-regulation) gene. Furthermore, as the CD8 T cell subtype, C2, was shown to have a negative FC value of less than -1.4 for GZMK, it may mean that GZMK is a down-regulation gene as in 3a. .
  • C2 a subtype of CD8 T cells used in the method for predicting therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention, shows a gene expression pattern differentiated from other CD8 T cells, and thus can be distinguished have. Moreover, as the subtype of CD8 T cells, C2, exhibits different gene expression patterns from other CD8 T cells, the characteristics thereof can also be clearly distinguished.
  • C2 a subtype of CD8 T cells used in the method for predicting a therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention, is based on the differential gene expression pattern and its characteristics. may affect the reactivity of Accordingly, referring to FIG. 3c , the results of UMAP and mosaic plots for CD8 cells with or without EGFR mutation are shown.
  • the EGFR mutation is a representative exemplary factor with low reactivity of the immunotherapy, and when the differential factor is analyzed by the EGFR mutation, the cause of the low immunotherapy, that is, the drug reactivity can be inferred. Therefore, for the prediction or treatment strategy for the responsiveness of immunotherapy, by comparing the presence or absence of an exemplary EGFR mutation, their differential configuration was derived.
  • the CD8 T cell subtype showing a statistically significant value is shown to be C2, and the Pearson residual value of C2 is shown to be -6.44. That is, when the EGFR mutation is included, it appears that C2 cells, a subtype of CD8 T cells, are reduced. Moreover, as the CD8 T-cell subtype, C2 cell, has a statistically differential expression in an individual containing an EGFR mutation, it may mean that it may be the cause of lowering the reactivity of immuno-cancer therapy.
  • C2 a subtype of CD8 T cells, which is a factor in the reactivity of low immune anticancer therapy, is ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, mRNA for at least one marker gene of GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX and TCF7 or a protein thereof may be expressed.
  • C2 a subtype of CD8 T cell, is It may refer to cells with differentially increased expression of PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 and LAG3. Accordingly, C2, a subtype of CD8 T cells, can be discriminated by the above-described genetic marker, and the reactivity of immunotherapy to an individual can be predicted through the differentiated subtype of CD8 T cells, C2.
  • T RM cells are a subpopulation of activated memory T cells (effector memory T cells) that do not circulate and exist in peripheral tissues. gamma) can be expressed and produced. Accordingly, referring to FIG. 3D , the expression results for T RM cell-related markers in CD8 T cell subtypes (T RM -like cells) are shown.
  • PRF1, GZMA and GZMB which are genes related to cytotoxicity, appear to be most expressed in C2, a subtype of CD8 T cells, and ID2 and IFNG, which are genes related to effectors that can exhibit effects on immunity, It appears to be most expressed in C2, a subtype of CD8 T cells. Accordingly, C2, a subtype of CD8 T cells, may mean cytolytic lymphocytes having cytotoxicity in tumors. Furthermore, since CCR7 and IL7R, which are genes related to naive and memory, are most expressed in C14, it may mean that C14 is a subtype of naive T cells, and C0 and C11 are related to cytotoxicity. The expression of the related gene is low, and the expression of the gene related to the activation may mean that it is a subtype of activated T cells with low cytotoxicity.
  • C2 expression results for ITGAE (CD103), a surface marker specific to T RM cells, and ENTPD1, a marker related to a tumor-specific response, are shown in the subtype of CD8 T cells.
  • a subtype of CD8 T cells, ITGAE (CD103), a surface marker specific to T RM cells, and ENTPD1, a marker related to a tumor-specific response are the most expressed.
  • a subtype of CD8 T cells are T RM -like (subtype, tumor-reactive T RM -like cells) cells that exhibit tumor-specific reactivity, not bystander T cells, which are activated in an indiscriminate immune response.
  • the individual containing the EGFR mutation has a reduced cytotoxic subtype of C2 as described above, and thus has a reduced toxicity to tumor, that is, an anticancer effect, and thus the therapeutic reactivity to immune anticancer therapy. can be reduced.
  • the level of mRNA or protein thereof for a marker gene of ITGAE and/or GZMA is reduced compared to a normal control or an individual not containing an EGFR mutation, it is determined that the therapeutic response to the immuno-cancer therapy is low.
  • FIG. 3f expression results for genes involved in cytotoxicity and activation according to the presence or absence of EGFR gene mutation in the CD8 T cell subtype are shown.
  • GZMB a gene involved in cytotoxicity
  • CD7, CD27, and CXCR6, genes involved in activation all appear to be most expressed in C2
  • CD8 T cell subtype of an individual EGFR-WT
  • an individual containing an EGFR mutation can reduce the activity and distribution of C2, which is a T RM -like cell capable of exerting an anticancer effect by directly attacking the tumor, and due to the EGFR mutation, the T RM As C2, which is a T RM -like cell, is reduced, therapeutic responsiveness to immuno-cancer therapy may be reduced.
  • FIG. 3G immunohistochemistry results for CD8 + T cells, CD8 + CD103 + T cells, and CD8 + CD103 + GZMB + T cells in tumor tissues with or without EGFR mutation are shown.
  • CD8 + T cells, CD8 + CD103 + T cells, and CD8 + CD103 + GZMB + T cells occupy a larger area in the tumor tissue of an individual not containing the EGFR mutation. , appear to be distributed with a larger number.
  • the number of CD8 + T cells in the tumor tissue for EGFR-WT is about 200 no/mm 2 appears to be abnormal, and the number of CD8 + T cells in the tumor tissue for EGFR-MT is about 120 no/mm 2 appears to be below.
  • the number of CD8 + CD103 + T cells in the tumor tissue for EGFR-WT is about 37 no/mm 2 or more, and the number of CD8 + CD103 + T cells in the tumor tissue for EGFR-MT is about 5 no /mm 2 or less.
  • the number of CD8+CD103+GZMB+T cells in the tumor tissue for EGFR-WT was about 1.5 no/mm 2 It appears to be abnormal, and the number of CD8+ CD103+ GZMB+ T cells in the tumor tissue for EGFR-MT is about 1 no/mm 2 or less.
  • flow cytometry was performed for more accurate measurement. Accordingly, referring to FIG. 3h , flow cytometry analysis of CD8 + T cells, CD8 + CD103 + T cells and CD8 + CD103 + GZMB + T cells in tumor tissues according to the presence or absence of EGFR mutation (Fluorescence-activated cell sorting, FACS) Results are shown. At this time, the dimension of the data was reduced and visualized using t-SNE, and the observed value of each cell was expressed as a z-score expressing the distance from the average value.
  • FACS Fluorescence-activated cell sorting
  • CD8 CD103 and GZMB of an individual not containing the EGFR mutation (EGFR-WT) appeared to have a high expression level similar to the merged image. That is, cells expressing the above-mentioned markers involved in cytotoxicity are highly enriched in EGFR-WT. In contrast, when the EGFR mutation is included, cells expressing the above-mentioned markers are deficient. That is, due to the EGFR mutation, cells with cytotoxicity can be reduced.
  • the ratio of CD8+CD103+ T cells expressing CD103 to the total CD8+ T cells is about 57%, and the EGFR-MT case, it appears to be about 30%, and CD8 + CD103 + T cells are significantly reduced in EGFR-MT (p ⁇ 0.05).
  • the ratio of CD8 + CD103 + GZMB + T cells expressing GZMB in CD8 + CD103 + T cells for the above-mentioned 7h (a) is EGFR-WT, It appears to be about 67%, and in the case of EGFR-MT, it appears to be about 30%, and CD8+CD103+GZMB+T cells are significantly reduced in EGFR-MT (p ⁇ 0.05).
  • CD103 and GZMB which can exert cytotoxicity in CD8 T cells
  • the distribution and number of CD8 T cells expressing them in the tumor may be reduced. , and thus the therapeutic responsiveness to immuno-cancer therapy may be reduced.
  • an individual containing an EGFR mutation is tumor-specific and includes CD8 + T cells, preferably CD8 + CD103 + T cells and CD8 + CD103 + GZMB + T cells, which can exert a cytotoxic effect directly on the tumor.
  • CD8 + T cells preferably CD8 + CD103 + T cells and CD8 + CD103 + GZMB + T cells, which can exert a cytotoxic effect directly on the tumor.
  • a subtype of CD8 T cells which is a T RM -like cell, the resistance and toxicity to the tumor, that is, the anticancer effect, is reduced, and thus the therapeutic responsiveness to immunotherapy may be reduced.
  • the level of CD8 + T cells for tumor tissue and general tissue is 120 no/mm 2
  • the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) subjects).
  • the level of CD8 + CD103 + T cells for tumor tissue and normal tissue was 5 no/mm 2 In the case of the following subjects, it can be determined that the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) subjects).
  • the level of CD8 + CD103 + GZMB + T cells for tumor tissue and normal tissue is 1 no/mm 2 or less, it can be determined that the therapeutic response to immunotherapy is low (therapeutic response negative (-)). individual).
  • the method for predicting treatment response to immunotherapy in lung cancer is PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 and LAG3 Through C2, a subtype of CD8 T cells that differentially express the expression of
  • immunocancer therapy inhibits the function of immune evasion and suppression of tumors, thereby enhancing the activity of immune cells having cytotoxicity to exert anticancer effects.
  • an immunosuppressive agent capable of enhancing its effect that is, an immune anticancer therapy
  • the therapeutic response to immunotherapy may be positive.
  • a therapeutic response to immune anticancer therapy can be predicted.
  • C2 (T RM -like cells), a subtype of CD8 T cells of the present invention were treated with TCGA (The Cancer Genome Atlas) for lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC).
  • TCGA The Cancer Genome Atlas
  • LUAD lung adenocarcinoma
  • LUSC squamous cell carcinoma
  • FIG. 4b it is possible to predict the therapeutic response to immune anticancer therapy through C2 (T RM -like cells), a subtype of CD8 T cells showing cytotoxicity, which is one of immune cells with a specific frequency including EGFR mutation. do.
  • FIG. 4B (b) an AUC curve for sensitivity and specificity for the patient showing the highest score in FIG. 4B (a) is shown.
  • the AUC of the YCC cohort was 0.854, which was shown to have high sensitivity and specificity, indicating that the predictive power of response to anti-PD-1 treatment according to C2 (T RM -like cells), a subtype of tumor-infiltrating CD8 T cells, was excellent. can do.
  • C2 (T RM -like cells), a subtype of tumor-infiltrating CD8 T cells was excellent. can do.
  • the predictive power of C2 (T RM -like cells), a subtype of CD8 T cells, to anti-PD-1 treatment can be applied not only to individuals with EGFR mutations but also to various lung cancer individuals for anti-PD-1 treatment. That is, C2 (T RM -like cells), a subtype of CD8 T cells, can be used as a biomarker that can predict the therapeutic response to anti-PD-1 treatment in various lung cancer individuals.
  • FIGS. 5A to 5D various immune cells used in a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described in detail.
  • EGFR mutations which are representative causes that can lower the responsiveness to immunotherapy, were selected and compared.
  • tumor tissues of non-small cell lung cancer individuals (EGFR-WT) not containing EGFR mutations and non-small cell lung cancer individuals (EGFR-MT) containing EGFR mutations
  • EGFR-WT non-small cell lung cancer individuals
  • EGFR-MT non-small cell lung cancer individuals
  • tumor-infiltrating CD45+leukocytes were sorted on CD45, a common antigen for white blood cells, and single-cell RNA sequencing was performed using 10X Genomics technology. (Single cell RNA sequencing, scRNA-seq) was performed.
  • FIG. 5b the results of immune cells according to the presence or absence of EGFR mutations derived by the above-described process are shown.
  • the immune cell results according to the presence or absence of EGFR mutations were visualized by reducing the dimension through UMAP after preprocessing the scRNA-seq results derived by the above procedure using Seurat v.3.0 package16.
  • each cell was distinguished and classified into subtypes C0 to C17 according to biomarkers expressed in the cells.
  • CD3D, CD3E, CD3G, and CD28 in the case of CD4+ T cells, in the case of CD4, CD8+ cells, in CD8A and CD8B, in the case of Treg cells, FOXP3 and IL2RA, in the case of NK and NKT cells.
  • IL3RA, CLEC4C, and in the case of NRP1 and ILC2 cells immune cells and their subtypes were distinguished and classified through the expression of KIT and IL1RL1 biomarkers.
  • the immune cells are differentially distributed between the EGFR-MT group and the EGFR-WT group, and the distribution of the subtype composition according to the type of each immune cell is also different.
  • Treg cells are C4, NK cells are C13, B cells are C8, monocytes and macrophages are C15, dendritic cells are C16, and IL-2 cells are C17. It appears that the number of each immune cell is different depending on the presence or absence of EGFR mutation.
  • CD8 T cells it appears to contain the subtypes C0, C2, C11 and C14
  • CD4 T cells it appears to contain the subtypes of C1, C3, C5, C6 and C9, and NKT cells
  • C7, C10 and C12 it appears that the subtypes are included, and the number and ratio of subtypes for each immune cell is different depending on the presence or absence of EGFR mutation.
  • the macrophages and monocytes of C15, dendritic cells of C16 and IL-2 cells of C17 are mostly derived from individual 6 (P6). That is, the immune cells of C15, C16 and C17 were excluded as they were biased data derived from a specific patient.
  • FIG. 5D a mosaic plot result derived by excluding the aforementioned bias data is shown.
  • r ij Pearson residual
  • the proportional change of each immune cell was evaluated according to the presence or absence of EGFR mutation, r> 3.5 (augmentation) and r ⁇ -3.5 (depletion) Only the population with a threshold of values was evaluated (
  • i is the index of each immune cell (indices for each group), j is the subtype (indices for cell subsets), O is the observed cell counts (observed cell counts), and E is the expected cell counts. it means.
  • the area of the mosaic plot is proportional to the cell frequency, and the results for the observed cell number and the expected cell number deviation are displayed in blue, red, and gray, respectively, with blue for augmentation and red for decreasing. (depletion) and gray can mean no significant change.
  • the subtype of C2 appears to have a biased cell number depending on the presence or absence of EGFR mutation, and it appears that the subtype of C2 is depleted in the EGFR mutant population.
  • Treg cells (C4) appear to be depleted in the EGFR mutant population.
  • B cells (C8) appear to be depleted in the EGFR mutant population.
  • NKT and B cells used in a method for predicting a therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention will be described in detail.
  • FIG. 6a the results of UMAP and mosaic plots for NK and NKT cells according to the presence or absence of EGFR mutation are shown.
  • the subtypes of NKT cells showing statistically significant values are C7 and C10, and the Pearson residual values for each are 6.35 and 10.57. That is, when the EGFR mutation is included, it appears that the subtypes of NKT cells, C7 and C10, are increased.
  • DEGs Differently Expressed Genes
  • PFR1 and GZMB which are genes involved in cytotoxicity, are shown to have decreased expression in the C10 subtype of NKT cells, and TNFRSF18, IL2RB, CD7, CD44, Both TYROBP and TNFRSF1B were expressed in the C7 and C10 subtypes of NKT cells.
  • C7 a subtype of NKT cells, is a cell with reduced cellular activity (activation-L), and C7 is a cell with reduced cytotoxicity and cytotoxicity (cytotoxicity-L, activation-L).
  • the subject containing the EGFR mutation includes a number of subtypes with reduced cellular activity and cytotoxicity, as described above, resistance and toxicity to the tumor, that is, the anticancer effect, is reduced, and accordingly, the immune anticancer therapy treatment response may be reduced.
  • the number of NKT cells expressing mRNA for at least one marker gene of PRF1, GZMB, TNFRSF18, IL2RB, CD7, CD44, TYROBP and TNFRSF1B or a protein thereof is a normal control or an individual that does not contain an EGFR mutation (EGFR-WT) ), it can be determined that the therapeutic responsiveness to immunotherapy is low (therapeutic response negative (-) subjects).
  • HLA-DPB1, HLA-DRB5, HLA-DQA2, HLA-C and TAP1 for MHC are all reduced in individuals including the EGFR gene.
  • MHC expression when MHC expression is deficient or reduced, the individual's immune response to the foreign antigen may be weakened.
  • the MHC expression for the external antigen presentation of B cells is reduced, the activation of T cells (CD4 + T cells and CD8 + T cells) by B cells is reduced. and may reduce the therapeutic response to immune anticancer therapy.
  • cytokine secretion, ie, cytotoxic effect, of T cells according to immune anticancer therapy through an immune checkpoint inhibitor may be reduced.
  • CD20 + B cells shows a reduced level than that of a normal control or a subject not containing an EGFR mutation (EGFR-WT)
  • EGFR-WT EGFR-WT
  • CD4 and Treg cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described in detail.
  • FIG. 8a the results of UMAP and mosaic plots for CD4 and Treg cells with or without EGFR mutation are shown.
  • CD4 T cell subtypes of CD4 T cells showing statistically significant values are shown to be C1, C3 and C9, and the Pearson residual values for each are 8.48, 7.66, and -7.37, respectively, and the Pearson residual of Treg cells (C4).
  • CD52, ANXA1, ANXA2, and CXCR4 which are genes involved in immune suppression, appear to be most expressed in the C1 and C3 subtypes. That is, C1 and C3 of CD4 T cells, which are helper T cells, are cells that suppress immunity.
  • the individual containing the EGFR mutation contains a number of subtypes of C1 and C2 that suppress the immune response as described above, the resistance and toxicity to the tumor, that is, the anticancer effect is reduced, and thus the immune anticancer effect Therapeutic responsiveness to therapy may be reduced.
  • CD2, CD6, CD82 At least one of TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN and GATA3 Specifically, the number of CD4 T cells expressing mRNA for a marker gene of CD52, ANXA1, ANXA2, and CXCR4 or a protein thereof is higher than that of a normal control or an individual not containing the EGFR mutation. It can be determined that the treatment response to the therapy is low (therapeutic response negative (-) subjects).
  • CD2, CD6, CD82 which are genes involved in cell activation and immune checkpoint.
  • TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1 and CTLA4 appear to be most expressed in the C9 subtype of CD4 T cells. It also appears to be associated with CXCL13, ICOS, MAF, PDCD1, and SH2D1A genes, which are markers of Follicular helper T cells (T FH ).
  • Follicle helper T cells regulate various functions such as B cell development and immune activity, and express various antigens such as IL-4, IL-21, PD-1, CD69, OX40 and CXCL13, among which PD- 1 regulates the location of the germinal centers of T FH cells and serves to support the functions of T FH cells. Therefore, when follicle helper T cells are reduced, the effect of anti-PD-1 treatment is reduced. can be
  • T FH cells produce CXCL13 to induce homing into the follicles for B cells. That is, tertiary lymphoid structures (TLS) may be formed by CXCL13 secreted by T FH cells.
  • TLS is not a lymph node, but T cells and B cells are fibroblasts-like synovial cells (FRC)-like reticular cells, HEV (high endothelial venules) and FDC (follicular dendritic cells) together with the immune system It can mean structure.
  • FRC fibroblasts-like synovial cells
  • HEV high endothelial venules
  • FDC follicular dendritic cells
  • FIG. 8c the expression results of T FH cell-related markers in the Treg cells and the CD4 T cell subtypes and the CXCL13 expression results of the C9 subtype according to the presence or absence of EGFR mutation are shown.
  • the expression of CXCL13 for CD9, a subtype of CD4 T cells is shown to be higher in subjects not containing the EGFR mutation (EGFR-WT). That is, as it was shown that the expression of CXCL12 is reduced in an individual containing an EGFR mutation (EGFR-MT), the EGFR mutation may reduce the expression of CXCL13 for CD9, a subtype of CD4 T cells.
  • CD9 a subtype of CD4 T cells, CXCR5, a receptor for CXCL13, which is another characteristic of T FH cells, and BCL6, a lineage-defining transcription factor of the T FH cell lineage, were not detected.
  • CD9 a subtype of CD4 T cells, may mean that they are T FH -like cells rather than general T FH cells, which are located in local inflammatory sites where TLS frequently occurs. mainly found
  • T FH cells As the main role of T FH cells is the induction of B cells, reduced levels of T FH -like cells in individuals with EGFR mutations result in inefficient TLS formation, resulting in reduced TLS development, thus It can reduce the immune response of immune cells to the tumor.
  • FIG. 8D the results of immunohistochemistry for CD4 + T cells and CXCL13 + CD4 + T cells in tumor tissues with or without EGFR mutation are shown.
  • CD4 + T cells and CXCL13 + CD4 + T cells occupy a larger area in the tumor tissue of an individual (EGFR-WT) that does not contain an EGFR mutation. Accordingly, referring to FIG. 8d (b), which is the result of counting the number of cells per unit area, the number of CD4 + T cells in the tumor tissue for EGFR-WT is about 1300 no/mm 2 It appears to be abnormal, and the number of CD4 + T cells in the tumor tissue for EGFR-MT is about 750 no/mm 2 or less.
  • the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-WT is about 420 no/mm 2 or more, and the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-MT is about 230 no/mm 2 or less.
  • the individual containing the EGFR mutation induces B cells as described above, and the CD4 T cell C9 subtype that produces CXCL13 capable of forming TLS, an immune response structure, is reduced. Resistance and toxicity, that is, the anticancer effect is reduced, and thus the therapeutic responsiveness to the immune anticancer therapy may be reduced.
  • the level of CD4 + T cells for tumor tissue and general tissue is 750 no/mm 2
  • the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) subjects).
  • the level of CXCL13 + CD4 + T cells for tumor tissue and normal tissue is 230 no / mm 2 or less, it can be determined that the therapeutic response to immunotherapy is low (therapeutic response negative (-) individual) .
  • C4 is a subtype of Treg cells that maintain autoimmune suppression and immune homeostasis.
  • the individual containing the EGFR mutation suppresses the autoimmune response and the C4 subtype that can maintain immune homeostasis is reduced, so the resistance and toxicity to the tumor, that is, the anticancer effect, is reduced, and thus the immunity Therapeutic responsiveness and persistence to anti-cancer therapy may be reduced.
  • CD2, CD6, CD82 At least one of TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN and GATA3 Specifically, the number of Treg cells expressing mRNA for the marker genes of FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, and GATA3 or a protein thereof is reduced compared to normal controls or individuals not containing the EGFR mutation. For individuals presenting, it can be determined that the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) individuals).
  • Treg cells are CD27, CD28, ICOS, and TNFRSF4 encoding OX40, TNFRSF9 encoding 41BB, and TNFRSF18 involved in T cell activation other than the aforementioned FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, and GATA3 gene markers.
  • the encoding GITR marker gene and immune checkpoint molecules TIGIT and CTLA4 marker genes may also have high expression levels.
  • FIG. 8E the results of gene expression related to the Treg cell activation promoting factor in C4, a subtype of Treg cells, are shown according to the presence or absence of EGFR mutations.
  • TSC22D3 is a gene encoding GILZ and NR4A2, and in particular, GILZ mediates the immunosuppressive effect in dendritic cells and T cells. may lead to tolerance.
  • FIG. 9a for homeostasis of immune cells, the results for the conversion probability between subtypes of CD8 T cells, which are immune cells with specific frequencies containing EGFR mutations, are shown.
  • the probability of converting back to C2 from C2 is shown to be 0.57 in the case of EGFR-WT, and 0.41 in the case of EGFR-MT, and when EGFR mutation is included, the probability of conversion from C2 to C2 is lower.
  • the EGFR mutation decreases the persistence of C2, a subtype for CD8 T cells, which exhibits strong cytotoxicity, and increases conversion to C0 cells, which is a subtype for CD8 T cells with low cytotoxicity.
  • the homeostasis of T RM-like cells (C2, T RM - like cells) having a tumor-specific strong cytotoxicity is reduced, so that the effect of the anti-cancer effect on the immune anti-cancer therapy, that is, the maintenance of anti-cancer is maintained. The period may be reduced.
  • immuno-cancer therapy can be determined to be low in response to treatment (negative (-) subjects responding to treatment).
  • immunotherapy may be determined to be low in response to treatment (negative (+) subjects responding to treatment).
  • the DEGs results for the transcription factors differentially expressed in C2 T RM -like cells, T RM -like cells
  • T RM -like cells which are subtypes for CD8 T cells
  • the upregulated (upregulated) transcription factors transcription factors, TFs
  • TFs transcription factors
  • T RM -like cells Transcription factors up-regulated in C2 (T RM -like cells), a subtype for CD8 T cells, are SERTAD1, IRF1, ARID5B, ELF1, SLAZ, BHLHE40, FOSL2, RBPJ, ID2 and HOPX.
  • ID2 , RBPJ and BHLHE40 may be involved in the development and homeostasis of T RM cells.
  • RBPJ is involved in the formation of a transcriptional activator required for differentiation, maintenance and activation of T RM cells in association with Notch.
  • the expression results for the RBPJ and NOTCH1 genes involved in T RM cell differentiation, maintenance and activation are shown according to the presence or absence of EGFR mutation.
  • the expression result was expressed through Spearman's rank correlation, and as the correlation coefficient increases, it may mean that the two genes are related.
  • both RBPJ and NOTCH1 in C2 (T RM -like cells), a subtype for CD8 T cells, have a high positive (+) correlation with various genes involved in the differentiation and maintenance of T RM cells.
  • both RBPJ and NOTCH1 in C2 showed a low negative correlation with various genes involved in the differentiation and maintenance of T RM cells. appears to have
  • FIG. 9d which shows the expression result of FIG. 9c described above as scatter plots and a regression curver
  • the coefficient of correlation between genes rather than EGFR-WT As is close to zero or has a negative correlation coefficient, the relationship disappears or appears to have an inverse correlation.
  • EGFR mutations can reduce the differentiation, maintenance and homeostasis of T RM cells and T RM-like cells by RBPJ and NOTCH1, and T RM cells and T RM -like cells differentiate, maintain and homeostasis by RBPJ and NOTCH1 networks. This may mean that it is regulated.
  • the result value may mean an average expression value of a gene pair.
  • the interaction between T RM cells and B cells via the CXCL13-CXCR5 axis appears to be weak. That is, the expression of the ligand and the receptor expressed in each cell is low, and thus, the immune effect generated by the binding and interaction of the ligand and the receptor may be reduced.
  • TLS formed by CD4 + T cells, CD8 + T cells and B cells occupies a larger area. appears to be Accordingly, referring to FIG. 10b (b), which is a numerical comparison result by measuring the area, the area of TLS in the tumor tissue for EGFR-WT is about 17 mm 2 appears to be abnormal, and the area of TLS in the tumor tissue for EGFR-MT is about 5 mm 2 appears to be below. That is, the EGFR mutation can reduce TLS formation, an immune cell interaction, by about 70% compared to EGFR-WT.
  • the frequency and number of cells expressing CD4, CD20, CD8, CXCL13, CXCR5 and CD103 appear to be higher in TLS of EGFR-WT. More specifically, referring to FIG. 10d , which is the result of counting the number of cells per unit area, the number of CD4 + T cells in the tumor tissue for EGFR-WT is about 27000 no/mm 2 appears to be abnormal, and the number of CD4 + T cells in the tumor tissue for EGFR-MT is about 600 no/mm 2 appears to be below.
  • the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-WT is about 10000 no/mm 2 or more, and the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-MT is about 1500 no/mm 2 or less.
  • the number of CD8 + T cells in the tumor tissue for EGFR-WT was about 7500 no/mm 2 appears to be abnormal, and the number of CD8+ CD4+ T cells in the tumor tissue for EGFR-MT is about 1000 no/mm 2 or less.
  • the number of CD103 + CD8 + T cells in the tumor tissue for EGFR-WT is about 2900 no/mm 2 or more, and the number of CD103 + CD8 + CD4 + T cells in the tumor tissue for EGFR-MT is about It appears to be less than 500 no/mm 2 .
  • an individual containing an EGFR mutation expresses CXCL12, a ligand capable of causing interaction with B cells, CD4 + T cells and CD8 + T cells, preferably, and B cells, capable of forming TLS.
  • CD4+ T cells CXCL13+CD4+ T cells
  • CD103+CD8+ T cells and GZMB+CD103+CD8+ T cells that can exhibit strong tumor-specific cytotoxicity are decreased. Resistance and toxicity, that is, the anti-cancer effect is reduced, and thus the therapeutic responsiveness to the immune anti-cancer therapy may be reduced.
  • the area of TLS for tumor tissue and general tissue is 5 mm 2 or less, or for an individual exhibiting a level that is reduced by 70% or more than the level of TLS area for tissue of a normal control, the therapeutic response to immunotherapy is low. (negative (-) subjects responding to treatment).

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Abstract

Provided in the present specification is a method for predicting a treatment response to cancer immunotherapy in lung cancer, the method comprising the steps of: measuring the mRNA or protein level of at least one marker gene from among ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2, and LAG3 in a biological sample isolated from a subject; and determining a treatment response to cancer immunotherapy in a subject on the basis of the measured mRNA or protein level of the marker gene.

Description

폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법Methods for predicting therapeutic response to immunotherapy in lung cancer

본 발명은 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법 관한 것이다. The present invention relates to a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer.

폐암 (lung cancer) 은 남녀 모두의 성별에서 흔히 발생하는 암 중 하나이다. 폐암 중에서 비소세포성 폐암(non small lung cancer, NSLC)은 상피성 암 (carcinoma)의 일종으로 폐소성암(small lung cancer)이 아닌 모든 상피성 폐암 (epithelial lung cancer)을 일컫는다. 이러한, 비소세포성 폐암은, 전체 폐암의 발병률에 있어서 높은 비율을 차지한다.Lung cancer is one of the most common cancers in both men and women. Among lung cancers, non small lung cancer (NSLC) is a kind of epithelial cancer (carcinoma) and refers to all epithelial lung cancers, not small lung cancer. Such, non-small cell lung cancer occupies a high proportion in the overall incidence of lung cancer.

한편, 비소세포성 폐암은 암세포의 크기, 모양 및 화학적 구성에 따라 몇 가지 하위 종류로 나뉘며, 대표적으로는 선암(adenocarcinoma), 편평상피암(squamous cell carcinoma), 대세포암(large cell carcinoma) 등이 있 다. 선암은 폐의 바깥부위(outer region)에서 발견되며 다른 폐암보다 천천히 진행되는 경향이 있으나, 초기에 높은 전이 경향을 보이고 또한 높은 방사선 저항성을 보인다. 편평상피암은 기도 (airway)를 이루고 있는 세포의 초기 단계(early version)에서 시작되며, 주로 흡연자에게서 높은 발병률을 보인다. 나아가, 대세포 암은 폐의 어느 부위에서나 발병할 수 있으며, 그 진행속도가 소세포성 폐암(small cell lung cancer)과 유사 할 만큼 빠르기 때문에 그 치료는 현재까지도 난제로 떠오르고 있다.On the other hand, non-small cell lung cancer is divided into several subtypes according to the size, shape, and chemical composition of cancer cells, and typical examples include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. have. Adenocarcinoma is found in the outer region of the lung and tends to progress more slowly than other lung cancers. Squamous cell carcinoma starts in the early version of the cells that make up the airway, and shows a high incidence mainly in smokers. Furthermore, large cell cancer can develop in any part of the lung, and its treatment is still a challenge because the progression rate is fast enough to be similar to that of small cell lung cancer.

이와 같은 비소세포성 폐암의 증상으로는 지속적인 기침, 흉부 통증, 체중감소, 손톱 손상, 관절 통증, 호흡의 단기화(shortness of breath)등이 나타날 수 있다. 그러나, 비소세포성 폐암은 다른 암 보다 천천히 진행되기 때문에 초기에는 그 증상을 거의 보이지 않는다. 따라서, 비소세포성 폐암의 조기 발견 및 치료가 어려우며, 뼈, 간, 소장, 및 뇌 등 전신에 전이된 후에 발견할 가능성이 높다. 이에, 비소세포성 폐암의 진단 시 환자의 반수 이상이 수술을 할 수 없을 정도로 진행된 상태이므로 조기치료는 현실적으로 어렵다. 또한, 비소세포성 폐 암은 외과적 수술을 할 수 있을 만큼 진행되지 않은 경우라면 근치절제술과 같은 우선 수술을 시행하는데, 근치절제술을 시행할 수 있는 경우는 약 30 %에 불과한 실정이다. 나아가, 근치절제술을 시행한 전체환자들 대다수는 수술 절제 후에 보다 공격적인 질환으로 재발하여 사망하는 것으로 나타난다.Symptoms of such non-small cell lung cancer may include persistent cough, chest pain, weight loss, nail damage, joint pain, and shortness of breath. However, because non-small cell lung cancer progresses more slowly than other cancers, it rarely shows symptoms in the early stages. Therefore, early detection and treatment of non-small cell lung cancer is difficult, and it is highly likely to be detected after metastasis to the bones, liver, small intestine, and brain. Accordingly, at the time of diagnosis of non-small cell lung cancer, more than half of the patients are advanced enough to not be able to perform surgery, so early treatment is difficult in reality. In addition, if non-small cell lung cancer has not progressed enough to be surgically performed, a priority operation such as radical resection is performed, but only about 30% of cases in which radical resection can be performed. Furthermore, the majority of all patients who underwent radical resection appear to recur and die from a more aggressive disease after surgical resection.

이러한 이유로 비소세포성 폐암의 조기 치료를 위해, 새로운 치료법의 개발, 나아가 기존의 치료법에 대한, 치료 반응을 예측할 수 있는 새로운 방법에 대한 개발이 지속적으로 요구되고 있는 실정이다.For this reason, for the early treatment of non-small cell lung cancer, the development of a new treatment method, and furthermore, the development of a new method for predicting the treatment response to the existing treatment is continuously required.

발명의 배경이 되는 기술은 본 발명에 대한 이해를 보다 용이하게 하기 위해 작성되었다. 발명의 배경이 되는 기술에 기재된 사항들이 선행기술로 존재한다고 인정하는 것으로 이해되어서는 안 된다. The description underlying the invention has been prepared to facilitate understanding of the invention. It should not be construed as an admission that the matters described in the background technology of the invention exist as prior art.

한편, 아시인의 경우, 비소세포성 폐암 환자 중 약 50%에서 상피세포 성장인자 수용체(Epidermal Growth Factor Receptor, EGFR)의 돌연변이가 발견된다. EGFR은 HER 또는 erbB family라고도 불리는 티로신 키나아제 수용체 그룹의 일부로서, erbB family에는 EGFR (HER1/ErbB1), HER2 (ErbB2), HER3 (ErbB3) 그리고 HER4 (ErbB4)가 포함된다. 이러한, EGFR의 돌연변이는 EGFR의 티로신 키나아제 도메 인에 발생하여 EGFR 키나아제의 활성을 증가시킴에 따라, 세포 신호전달 체계를 지속적으로 활성화시켜 세포를 무제한으로 분화 및 성장하도록 한다.On the other hand, in the case of Asians, epidermal growth factor receptor (EGFR) mutations are found in about 50% of non-small cell lung cancer patients. EGFR is part of a group of tyrosine kinase receptors, also called HER or erbB family. The erbB family includes EGFR (HER1/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These mutations in EGFR occur in the tyrosine kinase domain of EGFR and increase the activity of EGFR kinase, thereby continuously activating the cell signaling system to allow unrestricted differentiation and growth of cells.

이에, 이를 표적으로 하는 티로신 키나아제 억제제(tyrosine kinase inhibitors, TKIs)가 1차 요법으로 주로 사용되고 있으나, EGFR 돌연변이를 포함하는 비소세포성 폐암에 있어 TKIs를 통한 치료 요법은 9 내지 14개월 이내에 후천적 내성이 발생한다.Therefore, tyrosine kinase inhibitors (TKIs) that target this are mainly used as first-line therapy, but in non-small cell lung cancer containing EGFR mutations, the treatment therapy through TKIs is acquired resistance within 9 to 14 months. Occurs.

따라서, TKIs에 대한 한계를 극복하기 위하여, 2차 요법으로 PD-1 억제제(Programmed death receptor-1 inhibitors) 또는 PD-L1 억제제(programmed cell death ligand-1)가 사용될 수 있으나, PD-1 및 PD-L1 차단 치료 또한 EGFR 돌연변이 환자에 있어 낮은 민감성 및 생존율을 갖는 것으로 나타남에 따라, 이러한 한계를 극복할 수 있는 치료 전략이 필요한 실정이다.Therefore, to overcome the limitations for TKIs, PD-1 inhibitors (Programmed death receptor-1 inhibitors) or PD-L1 inhibitors (programmed cell death ligand-1) can be used as second-line therapy, but PD-1 and PD -L1 blocking treatment also appears to have low sensitivity and survival rate in patients with EGFR mutations, so there is a need for a treatment strategy that can overcome these limitations.

한편, PD-L1 차단의 치료 반응 예측에 있어서, 면역 조직 화학법 (immunohistochemistry, IHC)에 의한 종양 PD-L1 발현이 현재 PD-1 차단에 대한 최선의 예측 바이오 마커로 사용될 수 있다. 그러나, 종양 PD-L1 발현 의존적인 PD-L1의 치료 반응 예측의 정확도는 약물 효능을 확정할 정도로 높지 않다. 보다 구체적으로, PD-L1 발현 음성 환자가 PD-1 차단에 반응할 수 있고, PD-L1 발현 양성 환자가 PD-1 차단에 반응하지 않을 수 있다. 나아가, PD-L1이 없는 일부 반응 환자는 Checkmate 057 trial에서 PD-L1 양성인 경우 비슷한 반응 지속 기간을 보일 수 있다. 더욱이, PD-L1 발현은 동적이며, 시간적 및 공간적으로 변화할 수 있다. 이러한 PD-L1 발현의 변화 현상은 종양에 의해 발휘되는 적응 면역 저항성일 수 있다.Meanwhile, in predicting the therapeutic response of PD-L1 blockade, tumor PD-L1 expression by immunohistochemistry (IHC) can be currently used as the best predictive biomarker for PD-1 blockade. However, the accuracy of predicting the therapeutic response of PD-L1 dependent on tumor PD-L1 expression is not high enough to confirm drug efficacy. More specifically, PD-L1-expressing negative patients may respond to PD-1 blockade, and PD-L1-expressing positive patients may not respond to PD-1 blockade. Furthermore, some responders without PD-L1 may have a similar response duration if they were PD-L1 positive in the Checkmate 057 trial. Moreover, PD-L1 expression is dynamic and can change temporally and spatially. This change in PD-L1 expression may be adaptive immune resistance exerted by the tumor.

PD-L1 차단의 치료 반응 예측에 대한 바이오 마커로서, CD8 T세포 침윤, 염증 종양 표지, T세포 수용체 클론성 및 체성 돌연변이 부담 (somatic mutational burden) 이 이용될 수 있다. 그러나, 이상의 바이오 마커들은, 종양 조직을 얻는데 어려움을 갖고, 종양의 이질성 및 일부 바이오 마커의 유도 가능성을 가지고 있어, PD-1차단에 대한 치료 반응 예측에 있어 단일 마커로 이용되는 것에 어려움이 있을 수 있다.As biomarkers for predicting therapeutic response of PD-L1 blockade, CD8 T cell infiltration, inflammatory tumor markers, T cell receptor clonality and somatic mutational burden can be used. However, the above biomarkers have difficulties in obtaining tumor tissue, heterogeneity of tumors and the possibility of inducing some biomarkers, so it may be difficult to use them as single markers in predicting therapeutic response to PD-1 blockade. have.

한편, 본 발명의 발명자들은 EGFR 돌연변이를 포함하는 환자가 면역 항암 요법에 대한 반응성이 저하되어 있는 것을 인지하였으며, EGFR 돌연변이를 예시로 약물 반응성이 감소되어 있는 개체에서의 차이를 확인하여 이들의 약물 반응성에 대한 한계를 극복하고자 하였다. 이에, 본 발명의 발명자들은 이들이 가지고 있는 특정 면역 세포의 구성이 면역 항암 요법에 대한 반응성을 갖는 환자와 차별화되어 있다는 것을 발견하였으며, 이에 따라, 면역 항암 요법에 대한 반응성이 감소될 수 있다는 것을 발견할 수 있었다.On the other hand, the inventors of the present invention have recognized that the reactivity to immuno-cancer therapy is reduced in patients with EGFR mutations, and by identifying differences in individuals with reduced drug reactivity using EGFR mutations as an example, their drug reactivity to overcome the limitations of Accordingly, the inventors of the present invention have discovered that the composition of specific immune cells they have is differentiated from patients having responsiveness to immuno-cancer therapy, and accordingly, it can be found that the responsiveness to immuno-cancer therapy can be reduced. could

더욱이, 본 발명의 발명자들은 차별적으로 분포되어 있는 특정 면역 세포들이 종래의 분류에 의한 세포와 메커니즘이 상이하다는것을 발견하였다. 즉, 본 발명의 발명자들은 면역 항암 요법에 대한 반응성을 예측할 수 있는 특정 면역 세포를 특성화할 수 있는 유전자 지표(gene signature)를 발견하였다.Furthermore, the inventors of the present invention have found that specific immune cells that are differentially distributed have different mechanisms from cells by conventional sorting. That is, the inventors of the present invention have discovered a gene signature capable of characterizing a specific immune cell that can predict the responsiveness to immunotherapy.

그 결과, 본 발명의 발명자들은 면역 항암 요법, 즉, 면역 관문 억제제에 대한 치료 반응을 예측하기 위한 바이오 마커로서, GZMB+CD103+CD8+T세포를 통한, 새로운 치료 반응 예측 방법을 개발하기에 이르렀다.As a result, the inventors of the present invention have developed a novel therapeutic response prediction method using GZMB+CD103+CD8+T cells as a biomarker for predicting the therapeutic response to immune anticancer therapy, that is, an immune checkpoint inhibitor. .

이에, 본 발명이 해결하고자 하는 과제는, 개체에 대한 면역 항암 요법의 반응성일 조기에 예측하여, 치료 효과를 극대화시킬 수 있도록, GZMB+CD103+CD8+T세포의 수준을 측정하고, 이의 수준을 기초로 개체에 대한 면역 항암 요법의 치료 반응을 결정하여 제공할 수 있는, 치료 반응 예측 방법을 제공하는 것이다.Accordingly, the problem to be solved by the present invention is to measure the level of GZMB+CD103+CD8+T cells, and to measure the level of GZMB+CD103+CD8+T cells so as to maximize the therapeutic effect by predicting the responsiveness of the immunotherapy for the subject at an early stage. An object of the present invention is to provide a method for predicting a therapeutic response, which can be provided by determining the therapeutic response of an immuno-cancer therapy for an individual based on it.

나아가, 본 발명이 해결하고자 하는 다른 과제는 전술한 치료 반응 예측 방법에 기초한, 면역 항암 요법에 대한 치료 반응을 예측할 수 있는 키트를 제공하는 것이다.Furthermore, another problem to be solved by the present invention is to provide a kit capable of predicting a therapeutic response to an immuno-cancer therapy, based on the above-described method for predicting a therapeutic response.

본 발명의 과제들은 이상에서 언급한 과제들로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다. The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

전술한 바와 같은 과제를 해결하기 위해, 본 발명은, 개체로부터 분리된 생물학적 시료에 대한, ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준을 측정하는 단계, 및 측정된 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준을 기초로 개체에 대한 면역 항암 요법의 치료 반응을 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응을 예측하는 방법을 제공한다.In order to solve the above problems, the present invention provides, for a biological sample isolated from an individual, ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 measuring the level of mRNA or protein thereof for at least one marker gene of at least one of To provide a method for predicting a therapeutic response to immunotherapy in lung cancer.

본 발명의 특징에 따르면, 측정하는 단계는, 정상 대조군의 시료에 대한 ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준을 측정하는 단계를 포함하고, 결정하는 단계는, 개체에 대한 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준이 정상 대조군의 수준보다 낮은 경우, 개체를 면역 항암 요법에 대한 치료 반응성이 음성인 것으로 결정하는 단계를 포함할 수 있다.According to a feature of the present invention, the measuring step includes at least one marker of ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 for a sample of a normal control group. Comprising the step of measuring the level of mRNA or protein thereof for the gene, wherein the determining step is, when the level of mRNA or protein thereof for the marker gene for the subject is lower than the level of the normal control, the subject is subjected to immuno-cancer therapy determining that the therapeutic responsiveness to the drug is negative.

본 발명의 다른 특징에 따르면, 상기 적어도 하나의 유전자 마커는 ITGAE 및/또는 GZMA이며, mRNA는, 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting), DNA 칩으로 이루어진 군에서 선택되는 어느 하나의 방법으로 측정되고, 단백질은, 웨스턴 블롯(western blot), 엘라이자(enzyme linked immunosorbent assay, ELISA), 방사선면역분석(Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(ouchterlony) 면역 확산법, 로켓 면역전기영동, 면역조직화학법(immunohistochemistry, IHC), 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS) 및 단백질 칩(protein chip) 중 적어도 하나의 방법으로 측정될 수 있으나, 이에 제한되는 것은 아니며, mRNA 및 단백질이 측정될 수 있는 다양한 방법을 모두 포함할 수 있다.According to another feature of the present invention, the at least one genetic marker is ITGAE and/or GZMA, and the mRNA is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerization Enzyme reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (Northern blotting), measured by any one method selected from the group consisting of a DNA chip, the protein is, Western blot, enzyme linked immunosorbent assay, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry (immunohistochemistry, IHC), Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and protein chip. , but is not limited thereto, and may include all of various methods by which mRNA and protein can be measured.

전술한 바와 같은 과제를 해결하기 위해, 본 발명은, 개체로부터 분리된 생물학적 시료에 대한, GZMB+CD103+CD8+T세포의 수준을 측정하는 단계, 및 측정된 GZMB+CD103+CD8+T세포의 수준을 기초로 개체에 대한 면역 항암 요법의 치료 반응을 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법을 제공한다.In order to solve the above problems, the present invention provides the steps of measuring the level of GZMB + CD103 + CD8 + T cells, and the measured GZMB + CD103 + CD8 + T cells in a biological sample isolated from a subject. Provided is a method of predicting a therapeutic response to an immuno-cancer therapy in lung cancer, comprising determining a therapeutic response of the immuno-cancer therapy for an individual based on the level.

본 발명의 특징에 따르면, GZMB+CD103+CD8+T세포는, ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX 및 TCF7 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현할 수 있으나, 이에 제한되는 것은 아니며, CD8+T세포가 발현할 수 있는 다양한 마커 유전자를 더 포함할 수 있다.According to a feature of the present invention, GZMB+CD103+CD8+T cells are ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT , HAVCR2, LAG3, ICOS, TOX and TCF7 mRNA or a protein thereof for at least one of the marker genes may be expressed, but is not limited thereto, and may further include various marker genes that can be expressed by CD8 + T cells. can

이때, GNLY의 log FC(fold change)는, 1.5 이상이고, GZMB의 log FC(fold change)는, 1.2 이상이고, ITGAE의 log FC는, 0.4 이상일 수 있으며, 이들은 각각 양의 log FC를 포함함에 따라, GZMB+CD103+CD8+T세포는 전술한 GNLY, GZMB 및 ITGAE의 발현이 증가(up-regulation)되어 있을 수 있다.At this time, the log FC (fold change) of GNLY is 1.5 or more, the log FC (fold change) of GZMB is 1.2 or more, and the log FC of ITGAE may be 0.4 or more, and they each contain a positive log FC. Accordingly, GZMB + CD103 + CD8 + T cells may have the above-described expression of GNLY, GZMB and ITGAE increased (up-regulation).

본 발명의 다른 특징에 따르면, GZMB+ITGAE(CD103)+CD8+T세포에 대한 예측력은, AUC 0.854 일 수 있다.According to another feature of the present invention, the predictive power for GZMB+ITGAE(CD103)+CD8+T cells may be AUC 0.854.

본 발명의 또 다른 특징에 따르면, 측정하는 단계는, 정상 대조군의 시료에 대한 GZMB+CD103+CD8+T세포를 측정하는 단계를 포함하고, 면역 항암 요법의 치료 반응을 결정하는 단계는, 측정된 개체에 대한 GZMB+CD103+CD8+T세포의 수준이 정상 대조군 시료에 대한 GZMB+ITGAE(CD103)+CD8+T세포의 수준보다 감소된 수준을 나타내는 경우, 개체가 면역 항암 요법에 대한 반응성이 음성인 것으로 결정하는 단계를 포함할수 있다.According to another feature of the present invention, the measuring step includes measuring GZMB + CD103 + CD8 + T cells with respect to a sample of a normal control group, and determining the therapeutic response of the immune anticancer therapy is the measured If the level of GZMB+CD103+CD8+T cells for the subject exhibits a reduced level than the level of GZMB+ITGAE(CD103)+CD8+T cells for a normal control sample, the subject is negatively responsive to immune anticancer therapy. It may include the step of determining that

본 발명의 또 다른 특징에 따르면, 측정하는 단계는, CXCR5+CD20+B세포 또는 CXCL13+CD4+T세포(Follicular helper T cell)를 측정하는 단계를 더 포함할 수 있다.According to another feature of the present invention, the measuring step may further include measuring CXCR5+CD20+B cells or CXCL13+CD4+T cells (follicular helper T cells).

본 발명의 또 다른 특징에 따르면, 측정하는 단계는, 정상 대조군의 시료에 대한 CXCL13+CD4+T세포를 측정하는 단계를 더 포함하고, 결정하는 단계는, 측정된 개체에 대한 CXCL13+CD4+T세포의 수준이 정상 대조군에 대한 CXCL13+CD4+T세포의 수준보다 감소된 수준을 나타내는 경우, 개체가 면역 항암 요법에 대한 반응성이 음성인 것으로 결정하는 단계를 포함할 수 있다. According to another feature of the present invention, the measuring step further comprises measuring CXCL13 + CD4 + T cells for a sample of a normal control, and the determining step is, CXCL13 + CD4 + T for the measured individual determining that the subject is negative for responsiveness to the immuno-cancer therapy if the level of cells indicates a reduced level than the level of CXCL13 + CD4 + T cells relative to a normal control.

이때, CXCL13+CD4+T세포는, CD2, CD6, CD82, TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN 및 GATA3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현할 수 있으나, 이에 제한되는 것은 아니며, 나아가, CXCL13+CD4+T세포는, PDCD1, MAF 및 SH2D1A 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 더 발현할 수 있다.At this time, CXCL13 + CD4 + T cells, CD2, CD6, CD82, TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN, and can express mRNA or a protein thereof for at least one marker gene of GATA3, but is not limited thereto, and further, CXCL13 + CD4 + T cells, PDCD1, MAF and SH2D1A mRNA for at least one marker gene or a protein thereof may be further expressed.

본 발명의 또 다른 특징에 따르면, 측정하는 단계는, 정상 대조군의 시료에 대한 CXCR5+CD20+B세포의 수준을 측정하는 단계를 더 포함하고, 결정하는 단계는, 측정된 개체에 대한 CXCR5+CD20+B세포의 수준이 정상 대조군 시료에 대한 CXCR5+CD20+B 세포의 수준보다 감소된 수준을 나타내는 경우, 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 포함할 수 있다.According to another feature of the present invention, the measuring step further comprises measuring the level of CXCR5 + CD20 + B cells with respect to a sample of a normal control, and the determining includes: CXCR5 + CD20 for the measured individual if the level of +B cells indicates a reduced level than the level of CXCR5 + CD20 + B cells relative to the normal control sample, determining by the subject that the therapeutic response to the immuno-cancer therapy is negative.

이때, CXCR5+CD20+B세포는, HLA-DPB1, HLA-DRB5, HLA-DQA2, 및 HLA-C중 적어도 하나의 항원 표시 인자 (antigen presentation molecule) 를 발현할 수 있으며, TAP1 마커 유전자를 발현할 수 있다. 나아가, 항원 표지 인자 및 전술한 마커 유전자는, 정상 대조군에 대한 CXCR5+CD20+B세포의 항원 표지 인자 및 전술한 마커 유전자 발현 수준보다 감소된 수준을 나타날 수 있다.In this case, the CXCR5+CD20+B cells may express at least one antigen presentation molecule of HLA-DPB1, HLA-DRB5, HLA-DQA2, and HLA-C, and may express the TAP1 marker gene. can Furthermore, the antigen marker and the above-mentioned marker gene may show a reduced level than the antigen marker and the above-mentioned marker gene expression level of CXCR5 + CD20 + B cells for a normal control.

본 발명의 또 다른 특징에 따르면, 본 발명의 또 다른 특징에 따르면, 측정하는 단계는, GZMB+CD103+CD8+T세포의 수를 측정하는 단계를 더 포함하고, 결정하는 단계는, 측정된 GZMB+CD103+CD8+T세포의 수가 1 cells/mm2 이하이거나, 전체 CD103+CD8+T세포에 대한 GZMB+CD103+CD8+T세포의 비율이 30 % 미만인 경우, 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 포함할 수 있다. According to another feature of the present invention, according to another feature of the present invention, the measuring step further comprises measuring the number of GZMB+CD103+CD8+T cells, and the determining comprises: If the number of +CD103+CD8+ T cells is 1 cells/mm 2 or less, or the ratio of GZMB+CD103+CD8+ T cells to total CD103+CD8+ T cells is less than 30%, the subject is treated with immunotherapy determining that the reaction is negative.

본 발명의 또 다른 특징에 따르면, 측정하는 단계는, GZMB+CD103+CD8+T세포의 GZMB+CD103+CD8+T세포로의 전환 확률(transition probability)을 측정하는 단계를 더 포함하고, 결정하는 단계는, 전체 CD8+T세포로의 전환 확륭에 대한 상기GZMB+CD103+CD8+T세포로의 전환 확률이 41%이하인 경우, 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 더 포함할 수 있다.According to another feature of the present invention, the measuring step further comprises measuring a transition probability of GZMB+CD103+CD8+T cells to GZMB+CD103+CD8+T cells, and determining The step is the step of determining that the subject has a negative therapeutic response to the immuno-cancer therapy when the conversion probability of the GZMB+CD103+CD8+T cells with respect to the total conversion to CD8+T cells is 41% or less. may include more.

본 발명의 또 다른 특징에 따르면, 측정하는 단계는, 개체 및 정상 대조군의 시료에 대한 3 차 림프절 구조 (tertiary lymphoid structures, TLS)의 면적를 측정하는 단계를 더 포함하고, 결정하는 단계는, 개체로부터 분리된 생물학적 시료에 대한 TLS의 면적이, 정상 대조군 시료에 대한 TLS 면적의 수준보다 70 %이상 감소된 수준을 나타내거나, 개체에 대한 TLS의 면적이 5 mm2 미만인 경우, 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 포함할 수 있다.According to another feature of the present invention, the measuring step further comprises measuring the area of tertiary lymphoid structures (TLS) for the sample of the subject and the normal control, and the determining comprises: If the area of TLS for the isolated biological sample exhibits a level that is at least 70% reduced compared to the level of the TLS area for the normal control sample, or the area of TLS for the subject is less than 5 mm 2 , the subject is on immunotherapy determining that the therapeutic response to the patient is negative.

본 발명의 또 다른 특징에 따르면, 개체는, 폐암 의심 개체일 수 있으나, 이에 제한되는 것은 아니며, 종양 및 다양한 질병을 통하여 본 발명의 일 실시예에 따른 CXCR5+CD20+B세포, CD4+T세포, CXCL13+CD4+T세포, CD8+T세포, CD103+CD8+T세포 및 GZMB+CD103+CD8+T세포가 감소되어 있을 것으로 예측되는 개체는 모두 포함할 수 있다.According to another feature of the present invention, the subject may be a suspected lung cancer subject, but is not limited thereto, and CXCR5 + CD20 + B cells, CD4 + T cells according to an embodiment of the present invention through tumors and various diseases. , CXCL13+CD4+T cells, CD8+T cells, CD103+CD8+T cells, and GZMB+CD103+CD8+T cells can all be included.

생물학적 시료는, 조직, 말초 혈액, 혈청 및 혈장으로 이루어진 그룹에서 선택된 적어도 하나를 포함할 수 있으나, 이에 제한되는 것은 아니며, 면역 세포가 측정될 수 있는 모든 시료를 다 포함할 수 있다. The biological sample may include at least one selected from the group consisting of tissue, peripheral blood, serum and plasma, but is not limited thereto, and may include all samples in which immune cells can be measured.

본 발명의 또 다른 특징에 따르면, 면역 항암 요법은, 항 PD-1 치료일 수 있으나, 이에 제한되는 것은 아니며, 종양 미세 환경을 통하여 세포 독성 효과가 억제될 수 있는 요소를 차단하는 억제제 및 물질를 통한 치료 요법을 모두 포함할 수 있다.According to another feature of the present invention, the immuno-cancer therapy may be anti-PD-1 treatment, but is not limited thereto. Through the use of inhibitors and substances that block factors that can suppress the cytotoxic effect through the tumor microenvironment. All treatment regimens may be included.

본 발명의 또 다른 특징에 따르면, 면역 세포의 수준은, 웨스턴 블롯(western blot), 엘라이자(enzyme linked immunosorbent assay, ELISA), 방사선면역분석(Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(ouchterlony) 면역 확산법, 로켓 면역전기영동, 면역조직화학법(immunohistochemistry, IHC), 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS) 및 단백질 칩(protein chip) 중 적어도 하나의 방법으로 측정되는, EGFR 돌연변이를 포함할 수 있으나, 이에 제한되는 것은 아니며, 면역 세포를 측정할 수 있는 다양한 방법이 모두 포함될 수 있다.According to another feature of the present invention, the level of immune cells, western blot (western blot), ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (Radioimmunoassay), radioimmunoassay (radioimmunodiffusion), octero ouchterlony immunodiffusion method, rocket immunoelectrophoresis, immunohistochemistry (IHC), Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS) and It may include, but is not limited to, an EGFR mutation measured by at least one method of a protein chip, and various methods capable of measuring immune cells may all be included.

전술한 바와 같은 과제를 해결하기 위해, 본 발명은, 개체로부터 분리된 생물학적 시료에 대한, GZMB+CD103+CD8+T세포의 수준을 측정하도록 구성된 제제를 포함하는, 폐암 개체의 면역 항암 요법에 대한 치료 반응 예측용 키트를 제공할 수 있다.In order to solve the above problems, the present invention provides an immuno-cancer therapy for lung cancer individuals, comprising an agent configured to measure the level of GZMB + CD103 + CD8 + T cells in a biological sample isolated from the individual. A kit for predicting treatment response may be provided.

이하, 실시예를 통하여 본 발명을 보다 상세히 설명한다. 다만, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것에 불과하므로 본 발명의 범위가 이들 실시예에 의해 한정되는 것으로 해석되어서는 아니된다. Hereinafter, the present invention will be described in more detail through examples. However, since these examples are only for illustrative purposes of the present invention, the scope of the present invention should not be construed as being limited by these examples.

본 발명은, PD-1 차단 및 다양한 면역 관문 억제제에 대한 치료 반응을 예측할 수 있는 새로운 바이오 마커를 제공할 수 있는 효과가 있다.The present invention has the effect of providing a new biomarker that can predict the therapeutic response to PD-1 blockade and various immune checkpoint inhibitors.

보다 구체적으로, 본 발명은 개체 내 다양한 시료에서 바이오 마커에 대한 비율을 기초로 PD-1 차단에 대한 치료 반응을 예측할 수 있는 효과가 있다. 이에, 본 발명은 바이오 마커를 이용하여 개체에 대한 PD-1 차단에 대한 조기 치료 반응을 예측함에 따라, 항 PD-1 치료에 대한 진행 여부를 빠르게 결정하도록 정보를 제공할 수 있으며, 치료 반응성이 효과적일 수 있는 환자와 무반응성 환자를 조기 선별하여, 이에 대한 부작용 및 불필요한 비용적 소모를 감소시키며, 임상에서의 치료 효과를 극대화시킬 수 있다.More specifically, the present invention has the effect of predicting the therapeutic response to PD-1 blockade based on the ratio to the biomarker in various samples within the subject. Accordingly, the present invention predicts an early treatment response to PD-1 blockade for an individual using a biomarker, so it is possible to provide information to quickly determine whether to proceed with anti-PD-1 treatment, and the treatment responsiveness By early selection of patients who can be effective and non-responsive patients, side effects and unnecessary cost consumption can be reduced, and the therapeutic effect in the clinic can be maximized.

본 발명에 따른 효과는 이상에서 예시된 내용에 의해 제한되지 않으며, 더욱 다양한 효과들이 본 명세서 내에 포함되어 있다. The effect according to the present invention is not limited by the contents exemplified above, and more various effects are included in the present specification.

도 1은 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 대한 절차를 예시적으로 도시한 것이다. 1 exemplarily shows a procedure for a method for predicting a therapeutic response to an immune anticancer therapy in lung cancer according to an embodiment of the present invention.

도 2는 본 발명의 일 실시예에 따른 EGFR 돌연변이를 포함하는 폐암에서 면역 항암 요법에서 이용되는 면역 세포의 작용 기전을 예시적으로 도시한 것이다.2 exemplarily shows the mechanism of action of immune cells used in immune anticancer therapy in lung cancer containing an EGFR mutation according to an embodiment of the present invention.

도 3a 내지 3i는 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 이용되는 CD8 세포에 대한 결과를 도시한 것이다.3A to 3I show results for CD8 cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.

도 4a 내지 4b는 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법을 통한 항 PD-1 치료 반응 예측 결과를 도시한 것이다.4A to 4B show results of predicting an anti-PD-1 treatment response through a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention.

도 5a 내지 5d는 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 이용되는 면역 세포에 대한 전체 분석 결과를 도시한 것이다.5A to 5D show the results of an overall analysis of immune cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.

도 6a 내지 7은 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 이용되는 NKT 및 B 세포에 대한 결과를 도시한 것이다.6A to 7 show results for NKT and B cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.

도 8a 내지 8e는 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 이용되는 CD4 및 Treg 세포에 대한 결과를 도시한 것이다.8A to 8E show results for CD4 and Treg cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention.

도 9a 내지 10d는 본 발명의 일 실시예에 따른 EGFR 돌연변이를 포함하는 개체의 면역 항암 요법에서 사용되는 면역 세포에 대하여 항상성(homeostasis) 및 TLS를 통한 T세포의 활성화를 검증한 결과를 도시한 것이다.9a to 10d show the results of verifying the activation of T cells through homeostasis and TLS for immune cells used in immune anticancer therapy of an individual containing an EGFR mutation according to an embodiment of the present invention. .

본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 첨부되는 도면과 함께 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나, 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다. Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be embodied in various different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the art to which the present invention pertains It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

본 명세서에서 사용되는 용어 "아형 또는 아집단(subtype or subpopulation)" 전체 세포 집단의 일부를 구성하는 세포 집단을 의미하며, 세포에 포함되어 있는 바이오마커(biomarker)따라 병리학적으로 구별 및 분류될 수 있다. As used herein, the term "subtype or subpopulation" refers to a cell population constituting a part of the entire cell population, and can be pathologically distinguished and classified according to the biomarkers contained in the cells. have.

본 명세서에서 사용되는 용어 "비소세포성 폐암"은 상피성 암의 일종으로 폐소성암(small lung cancer)이 아닌 모든 상피성 폐암을 의미한다. 한편, 이러한 비소세포성 폐암의 면역 항암 요법으로, 항 PD-1 치료가 이용될 수 있다.As used herein, the term “non-small cell lung cancer” refers to any epithelial lung cancer that is not small lung cancer as a type of epithelial cancer. On the other hand, anti-PD-1 treatment may be used as an immunotherapy for non-small cell lung cancer.

본 명세서에서 사용되는 용어 " 항 PD-1 치료"는, T 세포가 암세포를 공격하지 못하는 기전을 차단하도록 구성된 치료법일 수 있다. 보다 구체적으로, 항 PD-1 치료는, 암세포의 표면 단백질인 PD-L1, 및 PD-L2가 T 세포의 표면에 있는 단백질인 PD-1과 결합하는 것을 차단하는 것에 기초할 수 있다. 예를 들어, 면역 항암제가 T 세포의 PD-1 수용체에 결합하면, T 세포의 암세포에 대한 회피 기능을 억제할 수 있다. 이에, 본 명세서에서 "항 PD-1 치료"는 "PD-1 차단"과 동일한 의미로 이용될 수 있다As used herein, the term “anti-PD-1 treatment” may be a therapy configured to block a mechanism by which T cells fail to attack cancer cells. More specifically, anti-PD-1 treatment may be based on blocking the binding of PD-L1 and PD-L2, which are surface proteins of cancer cells, to PD-1, a protein on the surface of T cells. For example, when an immune anticancer agent binds to the PD-1 receptor of T cells, it can inhibit the evasion function of T cells against cancer cells. Therefore, in the present specification, "anti-PD-1 treatment" may be used in the same sense as "PD-1 blocking".

본 명세서에서 사용되는 용어 " 치료 반응 음성" 면역 항암 요법 즉, 면역 관문 억제제에 의하여 T 세포의 표면의 수용체 및 리간드의 결합 반응이 차단되지 못하거나, 차단에 따른 싸이토카인과 같은 세포 독성 물질에 대한 분비력이 향상되지 못함을 의미할 수 있다. 그러나, 이에 제한되지 않고, 면역 항암 요법에 의한 폐암 증상의 완화 또는 좋은 예후와 연관된 모든 반응의 발생을 포함할 수 있다.As used herein, the term “therapeutic response negative” immunotherapy, that is, the binding response of receptors and ligands on the surface of T cells by immune checkpoint inhibitors does not block, or secretion of cytotoxic substances such as cytokines according to blocking It may mean that the power is not improved. However, it is not limited thereto, and may include the occurrence of any response associated with alleviation of lung cancer symptoms or good prognosis by immuno-cancer therapy.

본 명세서에서 사용되는 용어 "양성 또는 +"는 세포를 특징화할 수 있는 마커가 발현되었음을 의미할 수 있다. 예를 들어, 세포 독성을 가지는 T세포의 경우, 세포 표면에 CD8이 발현되어 있으며, 이에 따라, 세포 독성을 가지는 T세포는 CD8+T세포로 표기될 수 있으나, 이에 제한되는 것은 아니며, '+'에 대한 표기를 제외하고 CD8 T세포로도 표기될 수 있다.As used herein, the term “positive or +” may mean that a marker capable of characterizing a cell is expressed. For example, in the case of cytotoxic T cells, CD8 is expressed on the cell surface, and thus, cytotoxic T cells may be denoted as CD8 + T cells, but are not limited thereto. Except for ', it can also be expressed as CD8 T cells.

본 명세서에서 사용되는 용어 "약"은, 당업자에게 용이하게 알려진 각각의 값에 대한 보통의 오차 범위를 지칭한다. 본 명세서에서 "약" 값 또는 파라미터 지칭은 그 값 또는 파라미터 자체에 관한 실시예를 포함한다.As used herein, the term “about” refers to a common error range for each value readily known to one of ordinary skill in the art. References herein to “about” a value or parameter include embodiments relating to that value or parameter per se.

본 명세서에서 사용되는 용어 "환자 또는 개체"는, 상호교환적으로 사용되고 치료가 요구되는 임의의 단일 동물, 더 바람직하게는 포유동물 (그와 같은 비-인간 동물, 예를 들어, 고양이, 개, 말, 토끼, 동물원 동물, 소, 돼지, 양, 및 비-인간 영장류 포함)을 지칭한다. 특정 실시예에서, 본원에서 환자는 인간이다. 환자는 "암 환자" 즉, 암의 하나 또는 그 이상의 증상을 앓거나, 암을 앓을 위험이 있거나, 또는 암을 앓고 있는 사람일 수 있다.As used herein, the term "patient or subject" is used interchangeably and includes any single animal in need of treatment, more preferably a mammal (such as a non-human animal such as a cat, a dog, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). In certain embodiments, the patient herein is a human. A patient may be a “cancer patient,” ie, a person suffering from, at risk of, or suffering from cancer, one or more symptoms of cancer.

본 명세서에서 사용되는 용어 "발현의 수준" 또는 "발현 수준"은 상호교환하여 사용가능하고, 일반적으로 생물학적 샘플 중 바이오마커의 양을 가리킨다. "발현"은 일반적으로 정보(예컨대, 유전자-암호화 및/또는 후성적 정보)가 상기 세포에 존재하고 작동하는 구조체로 전환되는 과정을 가리킨다. 따라서, 본 명세서에서 사용된 바와 같이, "발현"은 폴리뉴클레오티드로 전사, 폴리펩티드, 또는 더욱이 폴리뉴클레오티드 및/또는 폴리펩티드 변형으로 번역 (예를 들면, 폴리펩티드의 번역후 변형)을 지칭한다. 전사된 폴리뉴클레오티드, 번역된 폴리펩티드, 또는 폴리뉴클레오티드 및/또는 폴리펩티드 변형 (예컨대, 폴리펩티드의 변역 후 변형)의 단편 또한 이들이 대안적 스플라이싱(대안적인 스플라이싱)에 의해 생성된 전사물 또는 분해된 전사물에서 유래되든지, 또는 예컨대, 단백질 가수분해에 의한 폴리펩티드의 번역후 과정에서 유래되든, 발현된 것으로 간주되어야 한다. As used herein, the terms “level of expression” or “expression level” are used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (eg, gene-coding and/or epigenetic information) is converted into a functioning construct and present in the cell. Thus, as used herein, “expression” refers to transcription into a polynucleotide, translation into a polypeptide, or further polynucleotide and/or polypeptide modifications (eg, post-translational modification of a polypeptide). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotides and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide) also include transcripts or cleavages that are produced by alternative splicing (alternative splicing) It should be considered expressed, whether derived from a modified transcript or from a post-translational process of the polypeptide, eg, by proteolysis.

"발현된 유전자"는 mRNA로 폴리뉴클레오티드로 전사되고 그리고 그 다음 폴리펩티드로 번역된 것 및 또한 RNA로 전사되었지만 폴리펩티드로 번역되지 않은 것 (예를 들면, 이동 및 리보솜 RNA들)을 포함한다. 하나 초과의 관심 유전 자에 대한 발현 수준은 당해 분야의 숙련가에게 알려져 있고 또한 본 명세서에 개시된, 예를 들어, 관심 유전자의 모든 발현 수준의 중앙 또는 평균을 계산함에 의한 것을 포함하는, 응집 방법에 의해 결정될 수 있다. 응집 전에, 각각의 관심 유전자의 발현 수준은, 예를 들어, 하나 이상의 하우스키핑 유전자의 발현 수준으로 정규화되거나, 또는 총 라이브러리 크기로 정규화되거나, 또는 측정된 모든 유전자에 걸쳐 중앙 또는 평균 발현 수준 값으로 정규화되는 것을 포함하여, 당해 분야의 숙련가에게 알려져 있고 또한 본 명세서에 개시된 통계적인 방법을 사용하여 정규화될 수 있다. 일부 사례에서, 관심 다중 유전자에 걸친 응집 전에, 각각의 관심 유전자의 정규화된 발현 수준은, 예를 들어, 각각의 관심 유전자의 정규화된 발현 수준의 Z-점수를 계산함에 의한 것을 포함하여, 당해 분야의 숙련가에게 알려져 있고 또한 본 명세서에 개시된 통계적인 방법을 사용함에 의해 표준화될 수 있다.An “expressed gene” includes those transcribed into a polynucleotide into mRNA and then translated into a polypeptide and also those transcribed into RNA but not translated into a polypeptide (eg, mobile and ribosomal RNAs). Expression levels for more than one gene of interest may be determined by aggregation methods known to those skilled in the art and disclosed herein, including, for example, calculating the median or average of all expression levels of the gene of interest. can be decided. Prior to aggregation, the expression level of each gene of interest is, for example, normalized to the expression level of one or more housekeeping genes, or normalized to the total library size, or to a median or average expression level value across all genes measured. Normalization, including being normalized, can be normalized using statistical methods known to those of skill in the art and also disclosed herein. In some instances, prior to aggregation across multiple genes of interest, the normalized expression level of each gene of interest is determined in the art, including, for example, by calculating a Z-score of the normalized expression level of each gene of interest. It can be standardized by using statistical methods known to those skilled in the art and also disclosed herein.

본 발명의 일 work of the invention 실시예에in the example 따른 폐암에서 면역 항암 Immunocancer in lung cancer following 요법에 대한 치료 반응treatment response to therapy 예측 방법 Prediction method

이하에서는 도 1 및 2를 참조하여, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 대하여 구체적으로 설명하도록 한다.Hereinafter, with reference to FIGS. 1 and 2 , a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described in detail.

도 1을 참조하면, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법은 개체로부터 분리된 생물학적 시료에 대한, GZMB+CD103+CD8+T세포의 수준을 측정하는 단계(S110) 및 측정된 GZMB+CD103+CD8+T세포의 수준을 기초로 개체에 대한 면역 항암 요법의 치료 반응을 결정하는 단계(S120)를 포함할 수 있다. Referring to FIG. 1, the method for predicting treatment response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention includes measuring the level of GZMB+CD103+CD8+T cells in a biological sample isolated from an individual ( S110) and determining the therapeutic response of the immunotherapy for the subject based on the measured level of GZMB+CD103+CD8+T cells (S120).

먼저, GZMB+CD103+CD8+T세포의 수준을 측정하는 단계(S110)는 전술한 GZMB+CD103+CD8+T세포뿐만 아니라, CXCR5+CD20+B세포, CD4+T세포, CXCL13+CD4+T세포, CD8+T세포 및 CD103+CD8+T세포와 같은 다양한 면역세포들을 더 측정할 수 있다.First, the step of measuring the level of GZMB+CD103+CD8+T cells (S110) includes not only the aforementioned GZMB+CD103+CD8+T cells, but also CXCR5+CD20+B cells, CD4+T cells, and CXCL13+CD4+T cells. Various immune cells such as CD8 + T cells and CD103 + CD8 + T cells can be further measured.

보다 구체적으로, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에서 이용되는 CXCR5+CD20+B세포, CD4+T세포, CXCL13+CD4+T세포, CD8+T세포, CD103+CD8+T세포 및 GZMB+CD103+CD8+T세포는 각 세포간의 연쇄적인 반응을 통하여 종양에 대한 면역 반응 즉, 항암 효과를 가질 수 있는 면역 세포들이다.More specifically, CXCR5 + CD20 + B cells, CD4 + T cells, CXCL13 + CD4 + T cells, CD8 + T cells, CD103+CD8+T cells and GZMB+CD103+CD8+T cells are immune cells that can have an immune response to a tumor, that is, an anticancer effect through a chain reaction between each cell.

보다 구체적으로, 도 2를 참조하면, TFH 세포는 CXCL13를 분비하여, B세포를 유도 즉, 세포의 여포 내로 침투시킬 수 있다. TFH 세포에 의하여 분비된 CXCL13는 B세포에서 발현된 CXCR5에 결합하여 B세포를 활성화시킬 수 있으며, 이에 따라, B세포는 이에 대한 항원을 분해한 뒤, 다양한 항원(HLA 및 MHC1)와 결합시킨 후, 세포의 포면에 표출시킬 수 있다. 그 다음, 이렇게 B세포의 표출된 항원이 세포 독성 효과를 갖는 TRM 세포 및 이의 아형 세포들의 TCR과 결합하여, TRM 세포 및 이의 아형세포들을 활성화시킬 수 있다. 나아가, B세포의 표출된 항원은 TRM 세포의 활성화뿐만 아니라, CXCL13을 분비할 수 있는 TFH 세포의 TCR과도 결합하여, TFH 세포를 활성화시켜 CXCL13의 분비를 더욱 촉진시킬 수 있다. 이에, 면역 항상성 및 독성 효과가 더욱 향상될 수 있다. 또한, 활성화된 TRM 세포 및 이의 아형세포들은 종양을 표적할 수 있는 종양 특이적 마커인 CD103을 포함함에 따라, 종양 이외의 다른 세포에는 반응하지 않고, 강한 세포 독성 분자인 GZMB를 분비하여 종양을 직접적으로 공격할 수 있다. 이러한, CXCR5+CD20+B세포, CD4+T세포, CXCL13+CD4+T세포, CD8+T세포, CD103+CD8+T세포 및 GZMB+CD103+CD8+T세포는, 종양 내에 침윤되어 면역 효과 즉, 면역 세포의 지속성 및 공격성을 향상시킬 수 있는 3 차 림프절 구조(tertiary lymphoid structures, TLS)를 형성할 수 있다. 이러한, TLS의 형성은, 면역 항암 요법 즉, 종양 미세 환경에 의하여 면역 세포의 세포 독성 효과를 억제시킬 수 있는 면역 관문 억제제에 대한 반응성을 향상시킬 수 있다.More specifically, referring to FIG. 2 , the T FH cells secrete CXCL13 to induce B cells, that is, to infiltrate into the follicles of the cells. CXCL13 secreted by T FH cells can activate B cells by binding to CXCR5 expressed in B cells. Then, it can be expressed on the surface of the cell. Then, the antigen expressed by the B cells may bind to the TCR of the T RM cells and subtypes thereof having a cytotoxic effect, thereby activating the T RM cells and the subtypes thereof. Furthermore, the antigens expressed by B cells not only activate T RM cells, but also T FH capable of secreting CXCL13. Also binds to cellular TCR, T FH By activating the cells, the secretion of CXCL13 can be further promoted. Accordingly, immune homeostasis and toxic effects may be further improved. In addition, activated T RM cells and subtypes thereof contain CD103, a tumor-specific marker that can target tumors, and thus do not respond to cells other than tumors, and secrete GZMB, a strong cytotoxic molecule, to kill tumors. You can attack directly. These CXCR5 + CD20 + B cells, CD4 + T cells, CXCL13 + CD4 + T cells, CD8 + T cells, CD103 + CD8 + T cells, and GZMB + CD103 + CD8 + T cells are infiltrated into the tumor and have an immune effect, i.e. , can form tertiary lymphoid structures (TLS), which can improve the persistence and aggressiveness of immune cells. The formation of TLS can improve the reactivity to immune checkpoint inhibitors that can suppress the cytotoxic effect of immune cells by immunocancer therapy, that is, the tumor microenvironment.

그러나, EGFR 돌연변이는 이러한 TLS의 형성 및 이의 구성 요소인 면역 세포들의 발현을 저해할 수 있다. 이에, EGFR 돌연변이를 포함하는 개체의 경우, 면역 항암 요법에 대한 효과가 감소될 수 있다. However, EGFR mutations can inhibit the formation of such TLS and the expression of immune cells, which are components thereof. Accordingly, in the case of an individual containing an EGFR mutation, the effect on immunotherapy may be reduced.

나아가, EGFR 돌연변이를 포함하지 않더라도, 전술한 바와 같이, TLS의 형성 및 이의 구성 요소인 면역 세포들의 발현을 저하되어 있는 경우에도, 면역 항암 요법에 대한 효과가 감소될 수 있다.Furthermore, even if the EGFR mutation is not included, as described above, even when the formation of TLS and the expression of immune cells, which are components thereof, are reduced, the effect on immunotherapy may be reduced.

이에, TLS의 형성 및 이의 구성 요소인 면역 세포들인 CXCR5+CD20+B세포, CD4+T세포, CXCL13+CD4+T세포, CD8+T세포, CD103+CD8+T세포 및 GZMB+CD103+CD8+T세포의 수준을 측정하여, 면역 항암 요법에 대한 반응성을 예측할 수 있다. 나아가, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에서, 면역 항암 요법에 대한 치료 반응을 가장 효과적으로 예측할 수 있는 면역 세포는, 바람직하게, GZMB+CD103+CD8+T세포(TRM-like cells)일 수 있다. Accordingly, CXCR5 + CD20 + B cells, CD4 + T cells, CXCL13 + CD4 + T cells, CD8 + T cells, CD103 + CD8 + T cells and GZMB + CD103 + CD8 + By measuring the level of T cells, it is possible to predict the responsiveness to immunotherapy. Furthermore, in the method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention, the immune cells that can most effectively predict the therapeutic response to immuno-cancer therapy are, preferably, GZMB+CD103+CD8+T It may be a cell (T RM -like cells).

이때, GZMB+CD103+CD8+T세포는 ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX 및 TCF7 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현할 수 있으며, 이들 중 특정 마커를 차별적으로 발현할 수 있다. 예를 들어, GZMB+CD103+CD8+T세포는 GZMB, ITGAE(CD103) 및 GNLY의 발현량이 차별적으로 증가되어 있을 수 있다. At this time, GZMB+CD103+CD8+T cells are ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS , TOX and TCF7 may express mRNA for at least one marker gene or a protein thereof, and a specific marker among them may be differentially expressed. For example, GZMB+CD103+CD8+T cells may have differentially increased expression levels of GZMB, ITGAE (CD103) and GNLY.

또한, CD4+T세포 및 CXCL13+CD4+T세포는, CD2, CD6, CD82. TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN 및 GATA3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현할 수 있으며, CXCL13+CD4+T세포는, PDCD1, MAF 및 SH2D1A 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 더 발현할 수 있다.In addition, CD4 + T cells and CXCL13 + CD4 + T cells were CD2, CD6, and CD82. Marker of at least one of TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN and GATA3 mRNA for the gene or a protein thereof may be expressed, and the CXCL13 + CD4 + T cells may further express mRNA or a protein thereof for at least one marker gene of PDCD1, MAF, and SH2D1A.

나아가, CXCR5+CD20+B세포는 HLA-DPB1, HLA-DRB5, HLA-DQA2 및 HLA-C 및 TAP1 중 적어도 하나의 항원 표시 인자 (antigen presentation molecule) 를 발현할 수 있으며, TAP1 마커 유전자를 발현할 수 있다.Furthermore, CXCR5+CD20+B cells can express at least one antigen presentation molecule among HLA-DPB1, HLA-DRB5, HLA-DQA2 and HLA-C and TAP1, and are capable of expressing the TAP1 marker gene. can

더 나아가, 면역 세포는, NK 세포(Natural killer cells) 또는 NKT세포(Natural Killer T cells)를 더 포함할 수 있다.Furthermore, the immune cells may further include NK cells (Natural killer cells) or NKT cells (Natural Killer T cells).

더 나아가, 개체는, 폐암 의심 개체이고, EGFR 돌연변이를 포함할 수 있으나, 이에 제한되는 것은 아니다. Furthermore, the subject is a subject suspected of lung cancer, and may contain an EGFR mutation, but is not limited thereto.

더 나아가, 생물학적 시료는, 조직, 말초 혈액, 혈청 및 혈장으로 이루어진 그룹에서 선택된 적어도 하나를 포함할 수 있으며, 면역 항암 요법은, 항 PD-1 치료일 수 있으나, 이에 제한되는 것은 아니며, 종양 미세 환경으로부터 억제될 수 있는 다양한 리간드에 대한 면역 관문 억제제를 모두 포함할 수 있다.Furthermore, the biological sample may include at least one selected from the group consisting of tissue, peripheral blood, serum and plasma, and the immuno-cancer therapy may be anti-PD-1 treatment, but is not limited thereto, and tumor microscopic It may include all immune checkpoint inhibitors for a variety of ligands that may be inhibited from the environment.

다시 도 1을 참조하면, 측정하는 단계(S110)에서는, 정상 대조군의 시료에 대한 GZMB+CD103+CD8+T세포를 측정할 수 있으며, 나아가, GZMB+CD103+CD8+T세포뿐만 아니라 정상 대조군의 시료에 대한 CXCR5+CD20+B세포, CD4+T세포, CXCL13+CD4+T세포, CD8+T세포 및 CD103+CD8+T세포와 같은 다양한 면역세포들을 더 측정할 수 있다.Referring back to FIG. 1 , in the measuring step ( S110 ), GZMB + CD103 + CD8 + T cells can be measured for the sample of the normal control group, and further, GZMB + CD103 + CD8 + T cells as well as the normal control sample. Various immune cells such as CXCR5+CD20+B cells, CD4+T cells, CXCL13+CD4+T cells, CD8+T cells and CD103+CD8+T cells can be further measured for the sample.

또한, 측정하는 단계(S110)에서는 전술한 면역 세포의 측정이 아닌, 이에 특징적으로 발현되는 분자의 전사체 등의 측정이 이용될 수 있다. 보다 구체적으로, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에서 이용되는 CD8+T세포는 전술한 GZMB 및 CD103(ITGAE)뿐만 아니라, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3 중 적어도 하나의 마커 유전자가 차별적으로 발현되어 있을 수 있다.In addition, in the measuring step ( S110 ), the measurement of the transcript of a molecule characteristically expressed may be used instead of the aforementioned measurement of the immune cells. More specifically, the CD8 + T cells used in the method for predicting therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention are not only the aforementioned GZMB and CD103 (ITGAE), but also ID2, IFNG, CD7, CXCR6, At least one marker gene of PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 may be differentially expressed.

이에, 측정하는 단계(S110)에서는 전술한 마커 유전자가 이용될 수 있다. 즉, 측정하는 단계(S110)에서는 개체 및 정상 대조군에 대한 ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준을 측정할 수 있다.Accordingly, the above-described marker gene may be used in the measuring step ( S110 ). That is, in the measuring step (S110), at least one marker gene of ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2, and LAG3 for individuals and normal controls The level of mRNA or a protein thereof can be measured.

또한, 측정하는 단계(S110)에서는 GZMB+CD103+CD8+T세포 수 즉, 개체로부터 분리된 생물학적 시료에서의 단위 면적당 GZMB+CD103+CD8+T세포의 갯수를 더 측정할 수 있다.In addition, in the measuring step ( S110 ), the number of GZMB+CD103+CD8+T cells, that is, the number of GZMB+CD103+CD8+T cells per unit area in a biological sample isolated from an individual, may be further measured.

또한, 측정하는 단계(S110)에서는 GZMB+CD103+CD8+T세포의 GZMB+CD103+CD8+T세포로의 전환 확률(transition probability) 더 측정할 수 있다.In addition, in the measuring step ( S110 ), the transition probability of GZMB+CD103+CD8+T cells to GZMB+CD103+CD8+T cells may be further measured.

또한, 측정하는 단계(S110)에서는 CXCR5+CD20+B세포의 수준을 측정하는 경우, 정상 대조군의 시료에 대한 CXCR5+CD20+B세포의 수준을 더 측정할 수 있으며, 이때, CXCR5+CD20+B세포는 HLA-DPB1, HLA-DRB5, HLA-DQA2 및 HLA-C 중 적어도 하나의 항원 표시 인자 (antigen presentation molecule) 와 TAP1 마커 유전자에 대한 mRNA 또닌 이의 단백질을 측정할 수 있으며, 이러한 항원은 정상 대조군에 대한 CXCR5+CD20+B세포의 항원 표지 인자 및 전술한 마커 유전자의 발현 수준보다 감소된 수준일 수 있다.In addition, in the measuring step (S110), when the level of CXCR5 + CD20 + B cells is measured, the level of CXCR5 + CD20 + B cells relative to the sample of the normal control may be further measured, and at this time, CXCR5 + CD20 + B The cell may measure an antigen presentation molecule of at least one of HLA-DPB1, HLA-DRB5, HLA-DQA2 and HLA-C, mRNA for a TAP1 marker gene, or a protein thereof, wherein the antigen is a normal control It may be a reduced level than the expression level of the antigen marker and the above-mentioned marker gene of CXCR5 + CD20 + B cells.

또한, 측정하는 단계(S110)에서는 PRF1, GZMB, TNFRSF18, IL2RB, CD7, CD44, TYROBP 및 TNFRSF1B 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는 NKT세포(Natural Killer T cells) 및 정상 대조군의 시료에 대한 NKT세포를 더 측정할 수 있으며, 이때의 NKT에서 발현되는 유전자에 대한 mRNA 또는 이의 단백질은 정상 대조군에 대한 NKT세포의 마커 유전자에 대한 mRNA 또는 이의 단백질 발현 수준보다 감소된 수준일 수 있다.In addition, in the measuring step (S110), the mRNA for at least one of PRF1, GZMB, TNFRSF18, IL2RB, CD7, CD44, TYROBP and TNFRSF1B or NKT cells expressing a protein thereof (Natural Killer T cells) and a normal control NKT cells for the sample can be further measured, and at this time, the mRNA or protein thereof for the gene expressed in NKT may be a reduced level than the mRNA or protein expression level of the marker gene of NKT cells for a normal control. have.

또한, 측정하는 단계(S110)에서는 개체 및 정상 대조군의 시료에 대한 3 차 림프절 구조 (tertiary lymphoid structures, TLS)의 면적를 더 측정할 수 있다.In addition, in the measuring step ( S110 ), the area of tertiary lymphoid structures (TLS) for the sample of the individual and the normal control may be further measured.

또한, 측정하는 단계(S110)에서 GZMB+CD103+CD8+T세포를 포함하는 다양한 면역 세포의 수준은, 웨스턴 블롯(western blot), 엘라이자(enzyme linked immunosorbent assay, ELISA), 방사선면역분석(Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(ouchterlony) 면역 확산법, 로켓 면역전기영동, 면역조직화학법(immunohistochemistry, IHC), 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS) 및 단백질 칩(protein chip) 중 적어도 하나의 방법으로 측정될 수 있으며, In addition, the level of various immune cells including GZMB + CD103 + CD8 + T cells in the measuring step (S110) is determined by western blot, ELISA (enzyme linked immunosorbent assay, ELISA), and radioimmunoassay. ), radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry (IHC), Immunoprecipitation Assay, Complement Fixation Assay , can be measured by at least one method of flow cytometry (Fluorescence Activated Cell Sorter, FACS) and protein chip,

mRNA의 발현 수준은, 드롭렛 디지털 중합효소반응(Droplet digital PCR, dd-PCR), 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RNase protection assay, RPA), 노던 블랏팅(Northern blotting), DNA 칩으로 이루어진 군에서 선택되는 어느 하나의 방법으로 측정될 수 있으며, The expression level of mRNA is, droplet digital PCR (dd-PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction ( Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, can be measured by any one method selected from the group consisting of a DNA chip,

단백질의 발현 수준은, 웨스턴 블롯(western blot), 엘라이자(enzyme linked immunosorbent assay, ELISA), 방사선면역분석(Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(ouchterlony) 면역 확산법, 로켓 면역전기영동, 면역조직화학법(immunohistochemistry, IHC), 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS) 및 단백질 칩(protein chip) 중 적어도 하나의 방법으로 측정될 수 있으나, 이에 제한되는 것은 아니며, 면역 세포, 단백질 및 mRNA가 측정될 수 있는 다양한 방법을 모두 포함할 수 있다.The expression level of the protein was determined by western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion method, rocket immunization. At least one of electrophoresis, immunohistochemistry (IHC), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS) and protein chip (protein chip) It can be measured by the method of, but is not limited thereto, and may include all of various methods by which immune cells, proteins, and mRNAs can be measured.

결정하는 단계(S120)에서는, 측정된 개체에 대한 GZMB+CD103+CD8+T세포의 수준이 정상 대조군 시료에 대한 GZMB+ITGAE(CD103)+CD8+T세포의 수준보다 감소된 수준을 나타내는 경우, 개체가 면역 항암 요법에 대한 반응성이 음성인 것으로 결정할 수 있다.In the determining step (S120), when the level of GZMB + CD103 + CD8 + T cells for the measured individual represents a reduced level than the level of GZMB + ITGAE (CD103) + CD8 + T cells for the normal control sample, It can be determined that the subject is negative for responsiveness to the immuno-cancer therapy.

또한, 결정하는 단계(S120)에서는, 측정된 개체에 대한 CXCL13+CD4+T세포 또는 CXCR5+CD20+B세포의 수준이 정상 대조군 시료에 대한 CXCL13+CD4+T세포 또는 CXCR5+CD20+B세포의 수준보다 감소된 수준을 나타내는 경우, 개체가 면역 항암 요법에 대한 반응성이 음성인 것으로 결정할 수 있다.In addition, in the determining step (S120), the level of CXCL13 + CD4 + T cells or CXCR5 + CD20 + B cells for the measured individual is the level of CXCL13 + CD4 + T cells or CXCR5 + CD20 + B cells for the normal control sample. If it exhibits a level that is lower than the level, it can be determined that the subject is negatively responsive to the immuno-cancer therapy.

또한, 결정하는 단계(S120)에서는 측정된 개체에 대한 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준이 정상 대조군의 수준보다 낮은 경우, 개체를 면역 항암 요법에 대한 치료 반응성이 음성인 것으로 결정할 수 있다.In addition, in the determining step ( S120 ), when the level of mRNA or protein thereof for the measured individual for the marker gene is lower than the level of the normal control, it can be determined that the individual has a negative therapeutic response to the immuno-cancer therapy.

또한, 결정하는 단계(S120)에서는 CD4+T세포의 수준이 750 cells/mm2 이하이거나, CXCL13+CD4+T세포의 수준이 230 cells/mm2 이하이거나, CD8+T세포의 수준이 120 cells/mm2 이하이거나, CD103+CD8+T세포의 수준이 5 cells/mm2 이하이거나, 전체 CD8+T세포에 대한 CD103+CD8+T세포의 비율이 20 % 미만이거나, GZMB+CD103+CD8+T세포의 수준이 1 cells/mm2 이하이거나, 전체 CD103+CD8+T세포에 대한 GZMB+CD103+CD8+T세포의 비율이 30 % 미만이거나, 전체 CD8+T세포로의 전환 확륭에 대한 상기GZMB+CD103+CD8+T세포로의 전환 확률이 41%이하인 경우, 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정할 수 있다.In addition, in the determining step (S120), the level of CD4+ T cells is 750 cells/mm 2 or less, the level of CXCL13+CD4+ T cells is 230 cells/mm 2 or less, or the level of CD8+ T cells is 120 cells /mm 2 or less, or the level of CD103+CD8+ T cells is 5 cells/mm 2 or less, or the ratio of CD103+CD8+ T cells to total CD8+ T cells is less than 20%, or GZMB+CD103+CD8+ If the level of T cells is 1 cells/mm 2 or less, or the ratio of GZMB+CD103+CD8+ T cells to total CD103+CD8+ T cells is less than 30%, or the When the conversion probability to GZMB+CD103+CD8+T cells is 41% or less, it can be determined that the subject has a negative therapeutic response to the immuno-cancer therapy.

또한, 결정하는 단계(S120)에서는 측정된 개체에 대한 NKT세포의 수준이 정상 대조군 시료에 대한 NKT세포의 수준보다 증가된 수준을 나타내는 경우이거나, 측정된 개체에 대한 TLS의 면적이, 정상 대조군 시료에 대한 TLS 면적의 수준보다 70 %이상 감소된 수준을 나타내는 경우 이거나, 개체에 대한 TLS의 면적이 5 mm2 미만인 경우, 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정할 수 있다.In addition, in the determining step (S120), if the level of NKT cells for the measured individual represents an increased level than the level of NKT cells for the normal control sample, or the area of TLS for the measured individual is, the normal control sample If it represents a level that is reduced by 70% or more than the level of the TLS area for , or if the area of TLS for the subject is less than 5 mm 2 , it can be determined that the subject has a negative therapeutic response to the immunotherapy.

이상의 절차에 따라, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법은 다양한 마커의 수준을 측정함으로써 개체에 대한 면역 항암 요법 특히, 항 PD-1 치료에 대한 치료 반응을 조기에 예측할 수 있도록 정보를 제공할 수 있다. 이때, GZMB+CD103+CD8+T세포(TRM-like cells)의 항 PD-1 치료에 대한 예측력은 AUC 0.854일 수 있다. 즉, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법은 GZMB+CD103+CD8+T세포(TRM-like cells)를 통하여 높은 예측력으로 면역 항암 요법에 대한 치료 반응을 결정하여 제공할 수 있다.According to the above procedure, the method for predicting the therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention is to measure the level of various markers to determine the therapeutic response to the immuno-cancer therapy, in particular, the anti-PD-1 treatment for the subject. It can provide information so that it can be predicted early. In this case, the predictive power of GZMB+CD103+CD8+T cells (T RM -like cells) for anti-PD-1 treatment may be AUC 0.854. That is, the method for predicting the therapeutic response to immunocancer therapy in lung cancer according to an embodiment of the present invention predicts the therapeutic response to the immune anticancer therapy with high predictive power through GZMB+CD103+CD8+T cells (T RM -like cells). can be determined and provided.

한편, 본 발명은 전술한 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 기초하여, 본 발명의 일 실시예에 따른 폐암 개체의 면역 항암 요법에 대한 치료 반응 예측용 키트를 제공할 수 있다.On the other hand, the present invention may provide a kit for predicting the therapeutic response to the immune anticancer therapy of a lung cancer individual according to an embodiment of the present invention based on the method for predicting the therapeutic response to the immunocancer therapy in lung cancer described above.

이때, 본 발명의 일 실시예에 따른, 폐암 개체의 면역 항암 요법에 대한 치료 반응 예측용 키트는 개체로부터 분리된 생물학적 시료에 대한, GZMB+CD103+CD8+T세포(TRM-like cells)의 수준을 측정하도록 구성된 제제를 포함할 수 있다.At this time, according to an embodiment of the present invention, the kit for predicting the therapeutic response to the immuno-cancer therapy of the lung cancer individual is a biological sample isolated from the individual, GZMB + CD103 + CD8 + T cells (T RM -like cells) agents configured to measure levels.

본 발명의 일 work of the invention 실시예에in the example 따른 폐암에서 면역 항암 Immunocancer in lung cancer following 요법에 대한 치료 반응treatment response to therapy 예측 방법의 GZMB+CD103+CD8+T세포 차이 확인 및 이에 따른 항 PD-1 치료 반응 예측 Confirmation of GZMB+CD103+CD8+T cell difference in prediction method and prediction of anti-PD-1 treatment response accordingly

이하에서는, 도 3a 내지 4b를 참조하여, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에서 이용되는 GZMB+CD103+CD8+T세포에 대하여 구체적으로 설명한다.Hereinafter, with reference to FIGS. 3A to 4B , GZMB+CD103+CD8+T cells used in a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described in detail.

먼저, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에서 이용되는 CD8 T세포의 아형에 대한 특성을 확인하기 위하여, DEGs(Differentially Expressed Genes) 분석을 수행되었다. 이에, 도 3a를 참조하면, CD8 T세포의 아형에 대한 DEGs 분석 결과 즉, mRNA 및 이의 단백질의 발현량을 도시한 도트 플롯(Dot plot)이 도시된다.First, in order to confirm the characteristics of the CD8 T cell subtype used in the method for predicting a therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention, differentially expressed genes (DEGs) analysis was performed. Accordingly, referring to FIG. 3A , a dot plot is shown showing the results of DEGs analysis for the CD8 T cell subtype, that is, the expression level of mRNA and its protein.

CD8 T세포의 아형인 C2는 가장 높은 수준으로 세포 독성(cytotoxicity)에 관여하는 유전자인 PRF1, GZMA, GZMB, GNLY 및 GZMH를 발현하고 있으며, 나아가, CD8 T세포의 분화(differentiation)에 관여하는 유전자인 ITGAE, ID2, IFNG, CD7 및 CXCR6 또한, C2 아형에서 가장 많이 발현되어 있는 것으로 나타난다. 이때, ITGAE는 조직 상주 기억 T세포 (tissue-resident memory CD8+T, TRM)에 대한 마커임에 따라, 이를 발현하는 C2 세포는 높은 세포 독성을 가지며, CD8 T세포로의 분화를 촉진시킬 수 있는 TRM 세포의 아형(TRM -like cells)일 수 있다. C2, a subtype of CD8 T cells, expresses PRF1, GZMA, GZMB, GNLY and GZMH, which are genes involved in cytotoxicity at the highest level, and furthermore, genes involved in the differentiation of CD8 T cells. ITGAE, ID2, IFNG, CD7 and CXCR6, which are also the most expressed in the C2 subtype. At this time, as ITGAE is a marker for tissue-resident memory T cells (tissue-resident memory CD8+T, T RM ), C2 cells expressing it have high cytotoxicity and can promote differentiation into CD8 T cells. It may be a subtype of T RM cells (T RM -like cells).

결국, CD8 T세포의 아형인 C2는 3a에 도시된 바와 같이, PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 및 LAG3을 차별적으로 발현할 수 있다. 이에, 도 3b를 참조하면, 전술한 CD8 T세포의 아형인 C2의 시그니처 유전자 마커에 대한 발현양 결과가 도시된다. 이때, 발현양은 CD8 T세포의 아형인 C0, C11 및 C14와의 비교를 통하여 도출되었으며, 이에 대한 발현양의 비율인 FC(fold change) 값을 나타내었다.Consequently, the CD8 T cell subtype, C2, can differentially express PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 and LAG3, as shown in Fig. 3a. Accordingly, referring to FIG. 3B , the expression level results for the signature gene marker of C2, a subtype of the aforementioned CD8 T cells, are shown. At this time, the expression level was derived through comparison with C0, C11 and C14, which are subtypes of CD8 T cells, and the FC (fold change) value, which is the ratio of the expression level, was shown.

보다 구체적으로, CD8 T세포의 아형인 C2는 GZMB가 1.2 이상, CD7이 0.9 이상, ID2가 0.5이상 및 ITGAE가 0.4 이상인 양(+)의 FC 값을 갖는 것으로 나타남에 따라, 이들이 3a에서와 마찬가지로 발현이 증가된(up-regulation) 유전자임을 의미할 수 있다. 나아가, CD8 T세포의 아형인 C2는 GZMK가 -1.4 미만인 음(-)의 FC 값을 갖는 것으로 나타남에 따라, GZMK가 3a에서와 마찬가지로 발현이 감소된(down-regulation) 유전자임을 의미할 수 있다.More specifically, C2, a subtype of CD8 T cells, was shown to have positive FC values with GZMB of 1.2 or more, CD7 of 0.9 or more, ID2 of 0.5 or more, and ITGAE of 0.4 or more, as in 3a. It may mean that the expression is increased (up-regulation) gene. Furthermore, as the CD8 T cell subtype, C2, was shown to have a negative FC value of less than -1.4 for GZMK, it may mean that GZMK is a down-regulation gene as in 3a. .

즉, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에서 이용되는 CD8 T세포의 아형인 C2는 다른 CD8 T세포와 차별화된 유전자 발현 양상을 나타내며, 이에 따라 구별될 수 있다. 더욱이, CD8 T세포의 아형인 C2는 다른 CD8 T세포와 상이한 유전자 발현 양상을 나타남에 따라, 이에 따른 특성도 분명하게 구별될 수 있다.That is, C2, a subtype of CD8 T cells used in the method for predicting therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention, shows a gene expression pattern differentiated from other CD8 T cells, and thus can be distinguished have. Moreover, as the subtype of CD8 T cells, C2, exhibits different gene expression patterns from other CD8 T cells, the characteristics thereof can also be clearly distinguished.

결국, 이러한 차별적인 유전자 발현 양상 및 이에 따른 특성에 의하여 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에서 이용되는 CD8 T세포의 아형인 C2는 개체에 대한 면역 항암 요법의 반응성에 영향을 미칠 수 있다. 이에, 도 3c를 참조하면, EGFR 돌연변이 유무에 따른 CD8 세포에 대한 UMAP 및 모자이크 플롯 결과가 도시된다. 이때, EGFR 돌연변이는 면역 항암 요법의 반응성이 낮은 대표적인 예시적 요인으로서, EGFR 돌연변이에 의하여 차별적인 요인을 분석할 경우, 낮은 면역 항암 요법 즉, 약물 반응성에 대한 원인을 유추할 수 있다. 이에, 면역 항암 요법의 반응성에 대한 예측 또는 치료 전략을 위하여, 예시적으로 EGFR 돌연변이의 유무를 비교하여, 이들의 차별적 구성을 도출하였다.After all, C2, a subtype of CD8 T cells used in the method for predicting a therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention, is based on the differential gene expression pattern and its characteristics. may affect the reactivity of Accordingly, referring to FIG. 3c , the results of UMAP and mosaic plots for CD8 cells with or without EGFR mutation are shown. In this case, the EGFR mutation is a representative exemplary factor with low reactivity of the immunotherapy, and when the differential factor is analyzed by the EGFR mutation, the cause of the low immunotherapy, that is, the drug reactivity can be inferred. Therefore, for the prediction or treatment strategy for the responsiveness of immunotherapy, by comparing the presence or absence of an exemplary EGFR mutation, their differential configuration was derived.

보다 구체적으로, EGFR 돌연변이 유무에 따라, 통계적으로 유의한 값을 나타내는 CD8 T세포의 아형은 C2인 것으로 나타나며, C2의 피어슨 잔차의 값은 -6.44인 것으로 나타난다. 즉, EGFR 돌연변이를 포함하는 경우, CD8 T세포의 아형인 C2 세포가 감소되어 있는 것으로 나타난다. 더욱이, 이러한 CD8 T세포의 아형인 C2 세포는 EGFR 돌연변이를 포함 개체에서 통계적으로 차별적인 발현은 타남에 따라, 면역 항암 요법의 반응성을 낮출수 있는 원인임을 의미할 수 있다.More specifically, depending on the presence or absence of EGFR mutation, the CD8 T cell subtype showing a statistically significant value is shown to be C2, and the Pearson residual value of C2 is shown to be -6.44. That is, when the EGFR mutation is included, it appears that C2 cells, a subtype of CD8 T cells, are reduced. Moreover, as the CD8 T-cell subtype, C2 cell, has a statistically differential expression in an individual containing an EGFR mutation, it may mean that it may be the cause of lowering the reactivity of immuno-cancer therapy.

결국, 도 3a 내지 3c의 결과에 따라, 낮은 면역 항암 요법의 반응성의 요인인 CD8 T세포의 아형인 C2는 ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX 및 TCF7 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현할 수 있으며, 바람직하게, CD8 T세포의 아형인 C2는 PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 및 LAG3의 발현이 차별적으로 증가된 세포를 의미할 수 있다. 이에, CD8 T세포의 아형인 C2는 전술한 유전자 마커에 의하여, 분별될 수 있으며, 분별된 CD8 T세포의 아형인 C2를 통하여, 개체에 대한 면역 항암 요법의 반응성을 예측할 수 있다.In the end, according to the results of FIGS. 3a to 3c, C2, a subtype of CD8 T cells, which is a factor in the reactivity of low immune anticancer therapy, is ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, mRNA for at least one marker gene of GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX and TCF7 or a protein thereof may be expressed. Preferably, C2, a subtype of CD8 T cell, is It may refer to cells with differentially increased expression of PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 and LAG3. Accordingly, C2, a subtype of CD8 T cells, can be discriminated by the above-described genetic marker, and the reactivity of immunotherapy to an individual can be predicted through the differentiated subtype of CD8 T cells, C2.

한편. TRM 세포는 순환하지 않고 말초 조직에 존재하는 활성화된 기억 T세포(effector memory T cells)의 하위 집단으로, 강한 세포독성에 관련된 분자인 PRF1(perforin), GZMB(granzyme B) 및 IFNG(IFN-gamma)을 발현 및 생산할 수 있다. 이에, 도 3d를 참조하면, CD8 T세포의 아형(TRM -like cells)에서 TRM 세포와 관련된 마커에 대한 발현 결과가 도시된다.Meanwhile. T RM cells are a subpopulation of activated memory T cells (effector memory T cells) that do not circulate and exist in peripheral tissues. gamma) can be expressed and produced. Accordingly, referring to FIG. 3D , the expression results for T RM cell-related markers in CD8 T cell subtypes (T RM -like cells) are shown.

세포 독성에 관련된 유전자인 PRF1, GZMA 및 GZMB은, CD8 T세포의 아형인 C2에서 가장 많이 발현되어 있는 것으로 나타나며, 면역에 대한 효과를 나타낼 수 있는 활성화(effector)에 관련된 유전자인 ID2 및 IFNG 또한, CD8 T세포의 아형인 C2에서 가장 많이 발현되어 있는 것으로 나타난다. 이에, CD8 T세포의 아형인 C2는 종양에서 세포 독성을 갖는 용해성 림프구(cytolytic lymphocyte)인 것을 의미할 수 있다. 나아가, 나이브(naive) 및 기억(memory)에 관련된 유전자인 CCR7 및 IL7R은 C14에서 가장 많이 발현되어 있음에 따라, C14는 나이브 T세포의 아형인 것을 의미할 수 있으며, C0 및 C11은 세포 독성에 관련된 유전자의 발현이 적으며, 활성화에 관련된 유전자가 발현되어 있음에 따라, 낮은 세포 독성을 갖는 활성화된 T세포의 아형임을 의미할 수 있다.PRF1, GZMA and GZMB, which are genes related to cytotoxicity, appear to be most expressed in C2, a subtype of CD8 T cells, and ID2 and IFNG, which are genes related to effectors that can exhibit effects on immunity, It appears to be most expressed in C2, a subtype of CD8 T cells. Accordingly, C2, a subtype of CD8 T cells, may mean cytolytic lymphocytes having cytotoxicity in tumors. Furthermore, since CCR7 and IL7R, which are genes related to naive and memory, are most expressed in C14, it may mean that C14 is a subtype of naive T cells, and C0 and C11 are related to cytotoxicity. The expression of the related gene is low, and the expression of the gene related to the activation may mean that it is a subtype of activated T cells with low cytotoxicity.

한편, 도 3e를 참조하면, CD8 T세포의 아형에서 TRM 세포에 특이적인 표면 마커인 ITGAE(CD103) 및 종양 특이적 반응에 관련된 마커인 ENTPD1에 대한 발현 결과가 도시된다. CD8 T세포의 아형인 C2는 TRM 세포에 특이적인 표면 마커인 ITGAE(CD103)와 종양 특이적 반응에 관련된 마커인 ENTPD1이 가장 많이 발현되어 있는 것으로 나타나며, 이에 따라, CD8 T세포의 아형인 C2는 무차별적 면역 반응 활성되는 방관자 T세포(bystander T cells)가 아닌, 종양 특이적 반응성을 나타내는 TRM 유사(아형, tumor-reactive TRM -like cells) 세포이다.Meanwhile, referring to FIG. 3E , expression results for ITGAE (CD103), a surface marker specific to T RM cells, and ENTPD1, a marker related to a tumor-specific response, are shown in the subtype of CD8 T cells. In C2, a subtype of CD8 T cells, ITGAE (CD103), a surface marker specific to T RM cells, and ENTPD1, a marker related to a tumor-specific response, are the most expressed. Accordingly, C2, a subtype of CD8 T cells are T RM -like (subtype, tumor-reactive T RM -like cells) cells that exhibit tumor-specific reactivity, not bystander T cells, which are activated in an indiscriminate immune response.

이에, EGFR 돌연변이를 포함하는 개체는 전술한 바와 같이 세포 독성을 가지는 C2의 아형이 감소되어 있음에 따라, 종양에 대한 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다. Accordingly, the individual containing the EGFR mutation has a reduced cytotoxic subtype of C2 as described above, and thus has a reduced toxicity to tumor, that is, an anticancer effect, and thus the therapeutic reactivity to immune anticancer therapy. can be reduced.

즉, ITGAE, ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX 및 TCF7 중 적어도 하나, 바람직하게는, ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는 CD8 T세포의 수가 정상 대조군 또는 EGFR 돌연변이를 포함하지 않는 개체보다 감소된 수준을 나타내는 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).i.e., among ITGAE, ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX and TCF7 At least one, preferably, of a CD8 T cell expressing an mRNA for a marker gene of ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 or a protein thereof For individuals whose numbers show reduced levels than normal controls or individuals not containing the EGFR mutation, it can be determined that the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) individuals).

더욱이, ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3의 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준이 정상 대조군 또는 EGFR 돌연변이를 포함하지 않는 개체보다 감소된 수준을 나타내는 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체). 특히, ITGAE 및/또는 GZMA의 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준이 정상 대조군 또는 EGFR 돌연변이를 포함하지 않는 개체보다 감소된 수준을 나타내는 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다.Moreover, the level of mRNA or protein thereof for the marker genes of ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 is a normal control or does not contain an EGFR mutation. In the case of an individual exhibiting reduced levels than the individual, it may be determined that the therapeutic responsiveness to the immuno-cancer therapy is low (therapeutic response negative (-) individual). In particular, if the level of mRNA or protein thereof for a marker gene of ITGAE and/or GZMA is reduced compared to a normal control or an individual not containing an EGFR mutation, it is determined that the therapeutic response to the immuno-cancer therapy is low. can

나아가, 도 3f를 참조하면, CD8 T세포의 아형에서 EGFR 유전자 돌연변이 유무에 따른 세포 독성(cytotoxicity) 및 활성화(activation)에 관여하는 유전자에 대한 발현 결과가 도시된다. 세포 독성에 관여하는 유전자인 GZMB 및 활성화에 관여하는 유전자인 CD7, CD27 및 CXCR6 모두 EGFR 유전자 돌연변이를 포함하지 않는 개체(EGFR-WT)의 CD8 T세포의 아형인 C2에서 가장 많이 발현되는 것으로 나타난다. 이에, EGFR 돌연변이를 포함하는 개체는 종양을 직접적으로 공격하여 항암 효과를 발휘할 수 있는 TRM 유사 세포(TRM-like cells)인 C2의 활성 및 분포를 감소시킬 수 있으며, EGFR 돌연변이로 인하여 TRM 유사 세포(TRM-like cells)인 C2가 감소됨에 따라, 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다.Furthermore, referring to FIG. 3f , expression results for genes involved in cytotoxicity and activation according to the presence or absence of EGFR gene mutation in the CD8 T cell subtype are shown. GZMB, a gene involved in cytotoxicity, and CD7, CD27, and CXCR6, genes involved in activation, all appear to be most expressed in C2, a CD8 T cell subtype of an individual (EGFR-WT) that does not contain an EGFR gene mutation. Accordingly, an individual containing an EGFR mutation can reduce the activity and distribution of C2, which is a T RM -like cell capable of exerting an anticancer effect by directly attacking the tumor, and due to the EGFR mutation, the T RM As C2, which is a T RM -like cell, is reduced, therapeutic responsiveness to immuno-cancer therapy may be reduced.

더 나아가, 도 3g를 참조하면, EGFR 돌연변이 유무에 따른 종양 조직에서의 CD8+T세포, CD8+CD103+T세포 및 CD8+CD103+GZMB+T세포에 대한 면역조직화학법 결과가 도시된다. 먼저, 도 3g의 (a)를 참조하면, EGFR 돌연변이를 포함하지 않는 개체의 종양 조직에서 CD8+T세포, CD8+CD103+T세포 및 CD8+CD103+GZMB+T세포가 더 많은 면적을 차지하며, 더 많은 수를 가지고 분포되어 있는 것으로 나타난다. Furthermore, referring to FIG. 3G , immunohistochemistry results for CD8 + T cells, CD8 + CD103 + T cells, and CD8 + CD103 + GZMB + T cells in tumor tissues with or without EGFR mutation are shown. First, referring to (a) of FIG. 3G, CD8 + T cells, CD8 + CD103 + T cells, and CD8 + CD103 + GZMB + T cells occupy a larger area in the tumor tissue of an individual not containing the EGFR mutation. , appear to be distributed with a larger number.

이에, 단위 면적당 각 세포수를 계수한 결과인 도 3g의 (b)를 참조하면, EGFR-WT에 대한 종양 조직에서의 CD8+T세포의 개수는 약 200 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CD8+T세포의 개수는 약 120 no/mm2 이하인 것으로 나타난다.Accordingly, referring to FIG. 3g (b), which is the result of counting the number of cells per unit area, the number of CD8 + T cells in the tumor tissue for EGFR-WT is about 200 no/mm 2 appears to be abnormal, and the number of CD8 + T cells in the tumor tissue for EGFR-MT is about 120 no/mm 2 appears to be below.

또한, EGFR-WT에 대한 종양 조직에서의 CD8+CD103+T세포의 개수는 약 37 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CD8+ CD103+T세포의 개수는 약 5 no/mm2 이하인 것으로 나타난다.In addition, the number of CD8 + CD103 + T cells in the tumor tissue for EGFR-WT is about 37 no/mm 2 or more, and the number of CD8 + CD103 + T cells in the tumor tissue for EGFR-MT is about 5 no /mm 2 or less.

또한, EGFR-WT에 대한 종양 조직에서의 CD8+CD103+GZMB+T세포의 개수는 약 1.5 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CD8+ CD103+ GZMB+T세포의 개수는 약 1 no/mm2 이하인 것으로 나타난다.In addition, the number of CD8+CD103+GZMB+T cells in the tumor tissue for EGFR-WT was about 1.5 no/mm 2 It appears to be abnormal, and the number of CD8+ CD103+ GZMB+ T cells in the tumor tissue for EGFR-MT is about 1 no/mm 2 or less.

더 나아가, 보다 정확한 측정을 위하여 유세포 분석을 수행하였다. 이에, 도 3h를 참조하면, EGFR 돌연변이 유무에 따른 종양 조직에서의 CD8+T세포, CD8+CD103+T세포 및 CD8+CD103+GZMB+T세포에 대한 유세포 분석(Fluorescence-activated cell sorting, FACS) 결과가 도시된다. 이때, t-SNE를 이용하여 데이터의 차원을 줄여 시각화하였으며, 각 세포의 관측된 값은, 평균값과의 거리를 표현해주는 z-score로 표현되었다.Furthermore, flow cytometry was performed for more accurate measurement. Accordingly, referring to FIG. 3h , flow cytometry analysis of CD8 + T cells, CD8 + CD103 + T cells and CD8 + CD103 + GZMB + T cells in tumor tissues according to the presence or absence of EGFR mutation (Fluorescence-activated cell sorting, FACS) Results are shown. At this time, the dimension of the data was reduced and visualized using t-SNE, and the observed value of each cell was expressed as a z-score expressing the distance from the average value.

원안의 영역에서(area within the solid circle) EGFR 돌연변이를 포함하지 않는 개체(EGFR-WT)의 CD8, CD103 및 GZMB는 융합(merged) 이미지와 유사하게 높은 발현량을 갖는 것으로 나타난다. 즉, 전술한 세포 독성에 관여하는 마커를 발현하는 세포가 EGFR-WT에서는 고도로 농축되어 있으며, 이와 대조적으로, EGFR 돌연변이를 포함한 경우, 전술한 마커를 발현하는 세포가 결핍되어 있는 것으로 나타난다. 즉, EGFR 돌연변이로 인하여, 세포 독성을 갖는 세포가 감소될 수 있다.In the area within the solid circle, CD8, CD103 and GZMB of an individual not containing the EGFR mutation (EGFR-WT) appeared to have a high expression level similar to the merged image. That is, cells expressing the above-mentioned markers involved in cytotoxicity are highly enriched in EGFR-WT. In contrast, when the EGFR mutation is included, cells expressing the above-mentioned markers are deficient. That is, due to the EGFR mutation, cells with cytotoxicity can be reduced.

보다 구체적으로, 도 3i의 (a)를 참조하면, EGFR-WT의 경우, 전체 CD8+ T세포에 대하여 CD103을 발현하는 CD8+CD103+T세포의 비율은 약 57 %인 것으로 나타나며, EGFR-MT의 경우, 약 30 %인 것으로 나타나며, EGFR-MT에서 CD8+CD103+T세포가 유의하게 감소되어 있는 것으로 나타난다(p<0.05). 나아가, 도 7h의 (b)를 참조하면, 전술한 7h의 (a)에 대한 CD8+CD103+T세포 내에서 GZMB를 발현하는 CD8+CD103+GZMB+T세포의 비율은 EGFR-WT의 경우, 약 67 %인 것으로 나타나며, EGFR-MT의 경우, 약 30 %인 것으로 나타나며, EGFR-MT에서 CD8+CD103+ GZMB+T세포가 유의하게 감소되어 있는 것으로 나타난다(p<0.05).More specifically, referring to FIG. 3i (a), in the case of EGFR-WT, the ratio of CD8+CD103+ T cells expressing CD103 to the total CD8+ T cells is about 57%, and the EGFR-MT case, it appears to be about 30%, and CD8 + CD103 + T cells are significantly reduced in EGFR-MT (p<0.05). Furthermore, referring to (b) of Figure 7h, the ratio of CD8 + CD103 + GZMB + T cells expressing GZMB in CD8 + CD103 + T cells for the above-mentioned 7h (a) is EGFR-WT, It appears to be about 67%, and in the case of EGFR-MT, it appears to be about 30%, and CD8+CD103+GZMB+T cells are significantly reduced in EGFR-MT (p<0.05).

결국, EGFR 돌연변이와 같은 다양한 원인으로 인하여, CD8 T세포에서 세포 독성을 발휘할 수 있는 CD103 및 GZMB에 대한 발현이 억제되어 있는 경우, 이를 발현하는 CD8 T세포의 종양 내 분포 및 개수가 감소될 수 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다. After all, if the expression of CD103 and GZMB, which can exert cytotoxicity in CD8 T cells, is suppressed due to various causes such as EGFR mutation, the distribution and number of CD8 T cells expressing them in the tumor may be reduced. , and thus the therapeutic responsiveness to immuno-cancer therapy may be reduced.

따라서, EGFR 돌연변이를 포함하는 개체는 종양 특이적이며, 종양에 직접적으로 세포 독성 효과를 나타낼 수 있는 CD8+T세포 바람직하게는, CD8+CD103+T세포 및 CD8+CD103+GZMB+T세포를 포함하는, TRM 유사 세포인 CD8 T세포의 아형인 C2가 감소되어 있음에 따라, 종양에 대한 저항성 및 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다.Accordingly, an individual containing an EGFR mutation is tumor-specific and includes CD8 + T cells, preferably CD8 + CD103 + T cells and CD8 + CD103 + GZMB + T cells, which can exert a cytotoxic effect directly on the tumor. According to the decrease in C2, a subtype of CD8 T cells, which is a T RM -like cell, the resistance and toxicity to the tumor, that is, the anticancer effect, is reduced, and thus the therapeutic responsiveness to immunotherapy may be reduced. .

이에, 종양 조직 및 일반 조직에 대한 CD8+T세포의 수준이 120 no/mm2 이하인 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).Accordingly, the level of CD8 + T cells for tumor tissue and general tissue is 120 no/mm 2 In the case of the following subjects, it can be determined that the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) subjects).

또한, 종양 조직 및 일반 조직에 대한 CD8+CD103+T세포의 수준이 5 no/mm2 이하인 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).In addition, the level of CD8 + CD103 + T cells for tumor tissue and normal tissue was 5 no/mm 2 In the case of the following subjects, it can be determined that the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) subjects).

또한, 종양 조직 및 일반 조직에 대한 CD8+CD103+GZMB+T세포의 수준이 1 no/mm2 이하인 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).In addition, if the level of CD8 + CD103 + GZMB + T cells for tumor tissue and normal tissue is 1 no/mm 2 or less, it can be determined that the therapeutic response to immunotherapy is low (therapeutic response negative (-)). individual).

이상의 결과에 따라, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법은 PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 및 LAG3의 발현이 차별적으로 발현하는 CD8 T세포의 아형인 C2를 통하여, 개체에 대한 면역 항암 요법의 반응성을 정확하게 예측할 수 있다.According to the above results, the method for predicting treatment response to immunotherapy in lung cancer according to an embodiment of the present invention is PRF1, GZMA, GZMB, GNLY, GZMH, ITGAE, ID2, IFNG, CD7, CXCR6, PDCD1, HAVCR2 and LAG3 Through C2, a subtype of CD8 T cells that differentially express the expression of

이에, 이하에서는, 도 4a 및 4b를 참조하여, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법을 통한 항 PD-1 치료 반응 예측에 대하여 설명하도록 한다.Therefore, in the following, with reference to FIGS. 4A and 4B, prediction of anti-PD-1 treatment response through the method for predicting treatment response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described.

먼저, 면역 항암 요법은 종양의 면역 회피 및 억제의 기능을 저해하여, 세포 독성을 갖는 면역 세포의 활성을 향상시켜 항암 효과를 발휘함에 있다. 그러나, EGFR 돌연변이를 포함하는 개체의 경우, 이러한 세포 독성을 갖는 면역 세포의 개수가 감소되어 있음에 따라, 이의 효과를 향상시킬 수 있는 면역 억제제 즉, 면역 항암 요법의 치료 반응성이 낮을 수 있다. 그러나, EGFR 돌연변이를 포함하는 개체에서도 이러한 면역 세포가 향상되어 있을 경우, 면역 항암 요법에 대한 치료 반응이 긍정적일 수 있다.First, immunocancer therapy inhibits the function of immune evasion and suppression of tumors, thereby enhancing the activity of immune cells having cytotoxicity to exert anticancer effects. However, in the case of an individual containing an EGFR mutation, as the number of immune cells having such cytotoxicity is reduced, the therapeutic responsiveness of an immunosuppressive agent capable of enhancing its effect, that is, an immune anticancer therapy, may be low. However, if these immune cells are improved even in individuals with EGFR mutations, the therapeutic response to immunotherapy may be positive.

따라서, EGFR 돌연변이를 포함하는 개체에서 특이적인 빈도를 갖는 면역세포 즉, 본 발명의 일 실시예에 따른, 다양한 면역 세포의 아형의 수준에 따라, 면역 항암 요법에 대한 치료 반응이 예측될 수 있다.Therefore, according to the level of immune cells having a specific frequency in an individual containing an EGFR mutation, that is, various subtypes of immune cells according to an embodiment of the present invention, a therapeutic response to immune anticancer therapy can be predicted.

이에, 본 발명의 CD8 T세포의 아형인 C2(TRM-like cells)를 TCGA (The Cancer Genome Atlas)의 비소세포성폐암(lung adenocarcinoma, LUAD) 및 편평세포암(lung squamous cell carcinoma, LUSC)에서 EGFR 돌연변이 유무에 따라 검증한 결과인 도 4a를 참조하면, CD8 T세포의 아형인 C2(TRM-like cells)가 EGFR-MT에서 감소되어 있는 것으로 나타나며, 본 발명의 결과와 일치하는 것으로 나타난다. 즉, CD8 T세포의 아형인 C2(TRM-like cells)가 EGFR 돌연변이를 포함하는 폐암 및 이를 포함하지 않는 다양한 폐암에 있어, 공통적으로 모두 적용될 수 있는 적절한 평가 항목임을 의미할 수 있다. Accordingly, C2 (T RM -like cells), a subtype of CD8 T cells of the present invention, were treated with TCGA (The Cancer Genome Atlas) for lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Referring to FIG. 4a, which is the result of verification according to the presence or absence of EGFR mutations, it appears that C2 (T RM -like cells), a subtype of CD8 T cells, is reduced in EGFR-MT, which is consistent with the results of the present invention. . That is, it may mean that C2 (T RM -like cells), a subtype of CD8 T cells, is an appropriate evaluation item that can be commonly applied to lung cancer containing an EGFR mutation and various lung cancers not including the EGFR mutation.

따라서, 도 4b에서는 EGFR 돌연변이를 포함하는 특이적인 빈도를 갖는 면역세포 중 하나인 세포 독성을 나타내는 CD8 T세포의 아형인 C2(TRM-like cells)를 통하여 면역 항암 요법에 대한 치료 반응을 예측하도록 한다. Therefore, in FIG. 4b, it is possible to predict the therapeutic response to immune anticancer therapy through C2 (T RM -like cells), a subtype of CD8 T cells showing cytotoxicity, which is one of immune cells with a specific frequency including EGFR mutation. do.

도 4b를 참조하면, CD8 T세포의 아형인 C2(TRM-like cells)에 대한 면역 항암 요법 즉, 항 PD-1 반응에 대한 각각의 코호트에서의 검증 결과가 도시된다.Referring to FIG. 4B , the validation results in each cohort for immuno-cancer therapy, ie, anti-PD-1 response, are shown for C2 (T RM -like cells), a subtype of CD8 T cells.

먼저, 도 4b의 (a)를 참조하면, 종양 침윤성 CD8 T세포의 아형인 C2(TRM-like cells)과 항 PD-1 치료 반응 간의 양의 상관 관계를 갖는 것으로 나타난다. First, referring to (a) of FIG. 4B , it appears that there is a positive correlation between C2 (T RM -like cells), a subtype of tumor-infiltrating CD8 T cells, and the anti-PD-1 treatment response.

나아가, 도 4b의 (b)를 참조하면, 전술한 도 4b의 (a)에서 가장 높은 점수를 나타낸 환자에 대한 민감도 및 특이도에 대한 AUC 곡선이 도시된다. Furthermore, referring to FIG. 4B (b), an AUC curve for sensitivity and specificity for the patient showing the highest score in FIG. 4B (a) is shown.

YCC 코호트의 AUC는 0.854으로 높은 민감도 및 특이도를 가지는 것으로 나타남에 따라, 종양 침윤성 CD8 T세포의 아형인 C2(TRM-like cells)에 따른 항 PD-1 치료에 대한 반응 예측력이 우수함을 의미할 수 있다. 한편, YCC의 코호트에서는 18명의 환자 중 EGFR-MT 개체가 2명이였으며, 그 중 1명만이 반응자였다. 이에, CD8 T세포의 아형인 C2(TRM-like cells) 항 PD-1 치료에 대한 반응 예측력은 EGFR 돌연변이를 포함하는 개체뿐만 아니라 항 PD-1 치료 위한 다양한 폐암 개체에서도 적용될 수 있다. 즉, CD8 T세포의 아형인 C2(TRM-like cells)는 다양한 폐암 개체에서 항 PD-1 치료에 대한 치료 반응을 예측할 수 있는 바이오 마커로 이용될 수 있다.The AUC of the YCC cohort was 0.854, which was shown to have high sensitivity and specificity, indicating that the predictive power of response to anti-PD-1 treatment according to C2 (T RM -like cells), a subtype of tumor-infiltrating CD8 T cells, was excellent. can do. On the other hand, in the YCC cohort, there were 2 EGFR-MT subjects out of 18 patients, and only 1 of them was a responder. Accordingly, the predictive power of C2 (T RM -like cells), a subtype of CD8 T cells, to anti-PD-1 treatment can be applied not only to individuals with EGFR mutations but also to various lung cancer individuals for anti-PD-1 treatment. That is, C2 (T RM -like cells), a subtype of CD8 T cells, can be used as a biomarker that can predict the therapeutic response to anti-PD-1 treatment in various lung cancer individuals.

EGFR 돌연변이에 따른 면역 세포 분포 차이 확인Confirmation of differences in immune cell distribution according to EGFR mutation

이하에서는 도 5a 내지 5d를 참조하여, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 이용되는 다양한 면역 세포에 대하여 구체적으로 설명한다.Hereinafter, with reference to FIGS. 5A to 5D , various immune cells used in a method for predicting a therapeutic response to immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described in detail.

도 5a를 참조하면, EGFR 돌연변이 유무에 따른 면역 세포 확인 과정이 도시된다. 먼저, 면역 항암 요법에 대한 반응성을 예측하기 위하여, 면역 항암 요법에 대한 반응성을 낮출 수 있는 대표적인 원인인 EGFR 돌연변이를 예시적으로 선정하여 비교하였다. Referring to Figure 5a, the immune cell identification process according to the presence or absence of EGFR mutation is shown. First, in order to predict the responsiveness to immunotherapy, EGFR mutations, which are representative causes that can lower the responsiveness to immunotherapy, were selected and compared.

EGFR 돌연변이에 따른 특이적인 면역 세포 분포 차이 확인을 위하여, EGFR 돌연변이를 포함하지 않는 비소세포성 폐암 개체(EGFR-WT) 및 EGFR 돌연변이를 포함하는 비소세포성 폐암 개체(EGFR-MT)의 종양 조직(tumor tissue)을 외과적으로 절제하여 수집한 뒤, 백혈구 공통 항원인 CD45에 종양에 침윤된 백혈구(tumor-infiltrating CD45+leukocytes)를 선별(sorting)한 뒤, 10X Genomics 기술을 사용하여 단일 세포 RNA 시퀀싱(Single cell RNA sequencing, scRNA-seq)를 수행하였다.In order to identify specific differences in the distribution of immune cells according to EGFR mutations, tumor tissues of non-small cell lung cancer individuals (EGFR-WT) not containing EGFR mutations and non-small cell lung cancer individuals (EGFR-MT) containing EGFR mutations ( After the tumor tissue was surgically excised and collected, tumor-infiltrating CD45+leukocytes were sorted on CD45, a common antigen for white blood cells, and single-cell RNA sequencing was performed using 10X Genomics technology. (Single cell RNA sequencing, scRNA-seq) was performed.

이에, 도 5b를 참조하면, 전술한 과정에 의하여 도출된 EGFR 돌연변이 유무에 따른 면역 세포 결과가 도시된다. 이때, EGFR 돌연변이 유무에 따른 면역 세포 결과는 Seurat v.3.0 package16를 사용하여 전술한 과정에 의하 도출된 scRNA-seq 결과를 전처리한 뒤, UMAP를 통하여 차원을 축소시켜 시각화하였다. 나아가, 각각의 세포는 세포 내에 발현되어 있는 바이오마커(biomarker)따라 C0 내지 C17의 아형(subtype)으로 구별 및 분류되었다. 나아가, T세포의 경우, CD3D, CD3E, CD3G, 및 CD28 이며, CD4+T세포의 경우, CD4, CD8+세포의 경우, CD8A 및 CD8B, Treg 세포의 경우, FOXP3 및 IL2RA, NK 및 NKT세포의 경우, FCGR3A, FCGR3B, NCAM1, KLRB1, KLRC1, KLRD1, KLRF1, 및 KLRK1, 대식세포 및 단핵구의 경우, CD14, CD68, CD163, 및 C8F1R, B세포의 경우, CD19, MS4A1, CD79A, CD79B, 및 BLNK, 수지상 세포의 경우, IL3RA, CLEC4C, 및 NRP1, ILC2 세포의 경우, KIT 및 IL1RL1의 바이오 마커의 발현을 통하여, 면역 세포 및 이의 아형을 구별 및 분류하였다.Accordingly, referring to FIG. 5b , the results of immune cells according to the presence or absence of EGFR mutations derived by the above-described process are shown. At this time, the immune cell results according to the presence or absence of EGFR mutations were visualized by reducing the dimension through UMAP after preprocessing the scRNA-seq results derived by the above procedure using Seurat v.3.0 package16. Furthermore, each cell was distinguished and classified into subtypes C0 to C17 according to biomarkers expressed in the cells. Furthermore, in the case of T cells, CD3D, CD3E, CD3G, and CD28, in the case of CD4+ T cells, in the case of CD4, CD8+ cells, in CD8A and CD8B, in the case of Treg cells, FOXP3 and IL2RA, in the case of NK and NKT cells. , FCGR3A, FCGR3B, NCAM1, KLRB1, KLRC1, KLRD1, KLRF1, and KLRK1 for macrophages and monocytes, CD14, CD68, CD163, and C8F1R, for B cells, CD19, MS4A1, CD79A, CD79B, and BLNK, In the case of dendritic cells, IL3RA, CLEC4C, and in the case of NRP1 and ILC2 cells, immune cells and their subtypes were distinguished and classified through the expression of KIT and IL1RL1 biomarkers.

EGFR-MT 그룹과 EGFR-WT 그룹 간의 면역 세포가 차별적으로 분포되어 있는 것으로 나타나며, 각각의 면역 세포의 종류에 따른 아형의 구성의 분포 또한 상이한 것으로 나타난다. It appears that the immune cells are differentially distributed between the EGFR-MT group and the EGFR-WT group, and the distribution of the subtype composition according to the type of each immune cell is also different.

보다 구체적으로, Treg 세포는 C4, NK 세포는 C13, B 세포는 C8, 단핵구(monocytes) 및 대식세포(macrophage)는 C15, 수지상 세포(dendritic cells)는 C16, 및 IL-2 세포는 C17인 것으로 나타나며, 각각의 면역 세포의 개수는 EGFR 돌연변이 유무에 따라 차이가 있는 것으로 나타난다.More specifically, Treg cells are C4, NK cells are C13, B cells are C8, monocytes and macrophages are C15, dendritic cells are C16, and IL-2 cells are C17. It appears that the number of each immune cell is different depending on the presence or absence of EGFR mutation.

또한, CD8 T세포의 경우, C0, C2, C11 및 C14의 아형을 포함하고 있는 것으로 나타나며, CD4 T세포의 경우, C1, C3, C5, C6 및 C9의 아형을 포함하고 있는 것으로 나타나며, NKT세포의 경우, C7, C10 및 C12의 아형을 포함하고 있는 것으로 나타나며, 각각의 면역 세포에 대한 아형의 개수 및 비율은 EGFR 돌연변이 유무에 따라 차이가 있는 것으로 나타난다.In addition, in the case of CD8 T cells, it appears to contain the subtypes C0, C2, C11 and C14, and in the case of CD4 T cells, it appears to contain the subtypes of C1, C3, C5, C6 and C9, and NKT cells In the case of C7, C10 and C12, it appears that the subtypes are included, and the number and ratio of subtypes for each immune cell is different depending on the presence or absence of EGFR mutation.

한편, 도 5c를 참조하면, 전술한 C15의 대식세포 및 단핵구, C16의 수지상 세포 및 C17의 IL-2 세포는 대부분 6번 개체(P6)로부터 파생된 것으로 나타난다. 즉, C15, C16 및 C17의 면역 세포는 특정 환자로부터 파생된 편향 데이터임에 따라, 이를 제외하였다.Meanwhile, referring to FIG. 5C , the macrophages and monocytes of C15, dendritic cells of C16 and IL-2 cells of C17 are mostly derived from individual 6 (P6). That is, the immune cells of C15, C16 and C17 were excluded as they were biased data derived from a specific patient.

이에, 도 5d를 참조하면, 전술한 편향 데이터가 제외되어 도출된 모자이크 플롯(mosaic plot) 결과가 도시된다. 이때, 하기의 수학식 1의 피어슨 잔차(Pearson residual, rij)를 통하여, EGFR 돌연변이 유무에 따라, 각 면역 세포들의 비례 변화가 평가되었으며, r> 3.5 (augmentation) 및 r <-3.5 (depletion) 값의 임계치(threshold)를 갖는 집단에 대해서만 평가되었다 (|Pearson residual| > 2 for p-value < 0.05, |Pearson residual| > 4 for p-value < 0.01).Accordingly, referring to FIG. 5D , a mosaic plot result derived by excluding the aforementioned bias data is shown. At this time, through the Pearson residual (r ij ) of Equation 1 below, the proportional change of each immune cell was evaluated according to the presence or absence of EGFR mutation, r> 3.5 (augmentation) and r <-3.5 (depletion) Only the population with a threshold of values was evaluated (|Pearson residual| > 2 for p-value < 0.05, |Pearson residual| > 4 for p-value < 0.01).

Figure PCTKR2022001649-appb-M000001
Figure PCTKR2022001649-appb-M000001

수학식의 i는 각 면역세포의 인덱스(indices for each group), j는 아형(indices for cell subsets), O는 관찰된 세포 개수(observed cell counts) 및 E는 기대 세포 개수(expected cell counts)를 의미한다.In the equation, i is the index of each immune cell (indices for each group), j is the subtype (indices for cell subsets), O is the observed cell counts (observed cell counts), and E is the expected cell counts. it means.

또한, 모자이크 플롯의 면적은 세포의 빈도수에 비례적이며, 관찰된 세포 개수와 기대 세포 개수에 대한 편차에 대한 결과는 파랑, 빨강 및 회색으로 표시하였으며, 각각 파랑은 증가(augmentation), 빨강은 감소(고갈, depletion) 및 회색은 통계적 변화 없음(no significant change) 의미할 수 있다.In addition, the area of the mosaic plot is proportional to the cell frequency, and the results for the observed cell number and the expected cell number deviation are displayed in blue, red, and gray, respectively, with blue for augmentation and red for decreasing. (depletion) and gray can mean no significant change.

먼저, CD8 T세포의 경우, C2의 아형이 EGFR 돌연변이 유무에 따라 편향된 세포 수를 갖는 것으로 나타나며, EGFR 돌연변이 개체 집단에서 C2의 아형이 고갈되어 있는 것으로 나타난다.First, in the case of CD8 T cells, the subtype of C2 appears to have a biased cell number depending on the presence or absence of EGFR mutation, and it appears that the subtype of C2 is depleted in the EGFR mutant population.

그 다음, CD4 T세포의 경우, C1, C3 및 C9의 아형이 EGFR 돌연변이 유무에 따라 편향된 세포 수를 갖는 것으로 나타나며, EGFR 돌연변이 개체 집단에서 C1 및 C3의 아형이 증가되어 있으며, C9의 아형이 고갈되어 있는 것으로 나타난다.Next, in the case of CD4 T cells, the subtypes of C1, C3, and C9 were shown to have a biased cell number depending on the presence or absence of EGFR mutation, and the subtypes of C1 and C3 were increased in the EGFR mutant population, and the subtype of C9 was depleted. appears to have been

그 다음, Treg 세포(C4)은 EGFR 돌연변이 개체 집단에서 고갈되어 있는 것으로 나타난다.Then, Treg cells (C4) appear to be depleted in the EGFR mutant population.

그 다음, NKT세포의 경우, C7 및 C10의 아형이 EGFR 돌연변이 유무에 따라 편향된 세포 수를 갖는 것으로 나타나며, EGFR 돌연변이 개체 집단에서 C7 및 C10의 아형이 모두 증가되어 있는 것으로 나타난다.Then, in the case of NKT cells, the C7 and C10 subtypes were shown to have a biased cell number depending on the presence or absence of EGFR mutations, and both C7 and C10 subtypes were found to be increased in the EGFR mutant population.

마지막으로, B 세포(C8)은 EGFR 돌연변이 개체 집단에서 고갈되어 있는 것으로 나타난다.Finally, B cells (C8) appear to be depleted in the EGFR mutant population.

결국, EGFR 돌연변이를 포함하는 개체(EGFR-MT)는 돌연변이를 포함하지 않는 개체(EGFR-WT)와 상이한 면역 세포의 분포 및 비율을 가짐에 따라, 면역 항암 요법에 대한 반응성 및 이에 대한 효과가 상이할 수 있으며, 이를 통하여 면역 항암 요법에 대한 치료 반응을 예측할 수 있다. As a result, individuals with an EGFR mutation (EGFR-MT) have different distributions and ratios of immune cells from those without the mutation (EGFR-WT), so their responsiveness to and effects on immunotherapy are different. It is possible to predict the therapeutic response to immuno-cancer therapy through this.

나아가, EGFR-MT에서 감소되어 있거나 증가되어 있는 특정 면역 세포의 조절을 통하여, 면역 항암 요법에 대한 치료 반응을 향상시킬 수 있다.Furthermore, through the regulation of specific immune cells that are decreased or increased in EGFR-MT, it is possible to improve the therapeutic response to immune anticancer therapy.

EGFR 돌연변이에 따른 NKT 및 B세포의 아형에 대한 차이 확인Confirmation of differences in subtypes of NKT and B cells according to EGFR mutation

이하에서는 도 6a 내지 7을 참조하여, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 이용되는 NKT 및 B 세포에 대하여 구체적으로 설명한다.Hereinafter, with reference to FIGS. 6A to 7 , NKT and B cells used in a method for predicting a therapeutic response to immunotherapy in lung cancer according to an embodiment of the present invention will be described in detail.

먼저, 도 6a를 참조하면, EGFR 돌연변이 유무에 따른 NK 및 NKT세포에 대한 UMAP 및 모자이크 플롯 결과가 도시된다.First, referring to FIG. 6a , the results of UMAP and mosaic plots for NK and NKT cells according to the presence or absence of EGFR mutation are shown.

도 5d에서 전술한 바와 같이, 통계적으로 유의한 값을 나타내는 NKT세포의 아형은 C7 및 C10인 것으로 나타나며, 각각에 대한 피어슨 잔차의 값은 6.35 및 10.57인 것으로 나타난다. 즉, EGFR 돌연변이를 포함한 경우, NKT세포의 아형인 C7 및 C10 가 증가되어 있는 것으로 나타난다. As described above in FIG. 5D , the subtypes of NKT cells showing statistically significant values are C7 and C10, and the Pearson residual values for each are 6.35 and 10.57. That is, when the EGFR mutation is included, it appears that the subtypes of NKT cells, C7 and C10, are increased.

이때, NK 및 NKT세포의 아형에 대한 특성을 확인하기 위하여 DEGs(Differentially Expressed Genes) 분석을 수행하였다. 이에, 도 6b를 참조하면, EGFR 돌연변이 유무에 따른 NK 및 NKT세포의 아형에 대한 DEGs 분석 결과가 도시된다. At this time, DEGs (Differentially Expressed Genes) analysis was performed to confirm the characteristics of NK and NKT cell subtypes. Accordingly, referring to FIG. 6b , the results of DEGs analysis for NK and NKT cell subtypes according to the presence or absence of EGFR mutations are shown.

보다 구체적으로, 세포독성(cytotoxicity)에 관여하는 유전자인 PFR1 및 GZMB은 NKT세포의 C10 아형에서 발현이 감소되어 있는 것으로 나타나며, 세포활성(activation)에 관여하는 유전자인 TNFRSF18, IL2RB, CD7, CD44, TYROBP 및 TNFRSF1B은 NKT세포의 C7 및 C10 아형에서 발현이 모두 감소되어 있는 것으로 나타난다.More specifically, PFR1 and GZMB, which are genes involved in cytotoxicity, are shown to have decreased expression in the C10 subtype of NKT cells, and TNFRSF18, IL2RB, CD7, CD44, Both TYROBP and TNFRSF1B were expressed in the C7 and C10 subtypes of NKT cells.

즉, NKT세포의 아형인 C7는 세포활성이 감소(activation-L)되어 있는 세포이며, C7는 세포활성 및 세포독성이 감소(cytotoxicity-L, activation-L)되어 있는 세포이다.That is, C7, a subtype of NKT cells, is a cell with reduced cellular activity (activation-L), and C7 is a cell with reduced cytotoxicity and cytotoxicity (cytotoxicity-L, activation-L).

결국, EGFR 돌연변이를 포함하는 개체는 전술한 바와 같이 세포활성 및 세포독성이 감소되어 있는 아형을 다수 포함함에 따라, 종양에 대한 저항성 및 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응이 감소될 수 있다. After all, as the subject containing the EGFR mutation includes a number of subtypes with reduced cellular activity and cytotoxicity, as described above, resistance and toxicity to the tumor, that is, the anticancer effect, is reduced, and accordingly, the immune anticancer therapy treatment response may be reduced.

이에, PRF1, GZMB, TNFRSF18, IL2RB, CD7, CD44, TYROBP 및 TNFRSF1B 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는 NKT세포의 수가 정상 대조군 또는 EGFR 돌연변이를 포함하지 않는 개체(EGFR-WT)보다 증가된 수준을 나타내는 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).Accordingly, the number of NKT cells expressing mRNA for at least one marker gene of PRF1, GZMB, TNFRSF18, IL2RB, CD7, CD44, TYROBP and TNFRSF1B or a protein thereof is a normal control or an individual that does not contain an EGFR mutation (EGFR-WT) ), it can be determined that the therapeutic responsiveness to immunotherapy is low (therapeutic response negative (-) subjects).

그 다음, 도 7을 참조하면, EGFR 돌연변이 유무에 따른 B세포에 대한 UMAP 및 모자이크 플롯 결과 및 주조직 적합 유전자 복합체(major histocompatibility complex, MHC)에 대한 발견 결과가 도시된다. 이때, B세포는 CD20 항원의 발현을 통하여 다른 면역 세포들과 구별될 수 있다.Then, referring to FIG. 7 , the results of the UMAP and mosaic plots for B cells and the findings of the major histocompatibility complex (MHC) are shown according to the presence or absence of EGFR mutations. At this time, B cells can be distinguished from other immune cells through the expression of the CD20 antigen.

B세포(C8)의 피어슨 잔차의 값은 -6.23인 것으로 나타난다. 즉, EGFR 돌연변이를 포함한 경우, 종양 침윤 B세포가 감소되어 있는 것으로 나타난다. The value of the Pearson residual of B cells (C8) appears to be -6.23. That is, when EGFR mutations are included, it appears that tumor-infiltrating B cells are reduced.

나아가, MHC에 대한 HLA-DPB1, HLA-DRB5, HLA-DQA2, HLA-C 및 TAP1의 유전자 발현은 EGFR 유전자를 포함하는 개체에서 모두 감소되어 있는 것으로 나타난다. 이때, MHC 발현이 결핍되거나 감소된 경우, 외부 항원에 대한 개체의 면역 반응이 약화될 수 있다. 이에, EGFR 돌연변이를 포함하는 개체는 전술한 바와 같이 B세포의 외부 항원 제시에 대한 MHC 발현이 감소되어 있음에 따라, B세포에 의한 T세포(CD4+T세포 및 CD8+T세포) 활성화가 감소될 수 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응이 감소될 수 있다. Furthermore, it appears that the gene expression of HLA-DPB1, HLA-DRB5, HLA-DQA2, HLA-C and TAP1 for MHC are all reduced in individuals including the EGFR gene. In this case, when MHC expression is deficient or reduced, the individual's immune response to the foreign antigen may be weakened. Accordingly, as described above, in the subject containing the EGFR mutation, as the MHC expression for the external antigen presentation of B cells is reduced, the activation of T cells (CD4 + T cells and CD8 + T cells) by B cells is reduced. and may reduce the therapeutic response to immune anticancer therapy.

즉, EGFR 돌연변이에 의하여 B세포의 항원 제시로 활성화된 T세포 수가 감소되어 있음에, 면역 관문 억제제를 통한 면역 항암 요법에 따른 T세포의 싸이토카인 분비 즉, 세포 독성 효과가 감소될 수 있다.That is, since the number of T cells activated by antigen presentation of B cells by EGFR mutation is reduced, cytokine secretion, ie, cytotoxic effect, of T cells according to immune anticancer therapy through an immune checkpoint inhibitor may be reduced.

결국, CD20+B세포의 수가 정상 대조군 또는 EGFR 돌연변이를 포함하지 개체(EGFR-WT)보다 감소된 수준을 나타내는 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).Ultimately, if the number of CD20 + B cells shows a reduced level than that of a normal control or a subject not containing an EGFR mutation (EGFR-WT), it can be determined that the therapeutic response to the immunotherapy is low (therapeutic response negative (-)). individual).

EGFR 돌연변이에 따른 CD4 및 Treg 세포의 아형에 대한 차이 확인Confirmation of differences in subtypes of CD4 and Treg cells according to EGFR mutation

이하에서는 도 8a 내지 8e를 참조하여, 본 발명의 일 실시예에 따른 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법에 이용되는 CD4 및 Treg 세포에 대하여 구체적으로 설명한다.Hereinafter, with reference to FIGS. 8A to 8E , CD4 and Treg cells used in a method for predicting a therapeutic response to an immuno-cancer therapy in lung cancer according to an embodiment of the present invention will be described in detail.

먼저, 도 8a를 참조하면, EGFR 돌연변이 유무에 따른 CD4 및 Treg 세포에 대한 UMAP 및 모자이크 플롯 결과가 도시된다.First, referring to FIG. 8a , the results of UMAP and mosaic plots for CD4 and Treg cells with or without EGFR mutation are shown.

*통계적으로 유의한 값을 나타내는 CD4 T세포의 아형은 C1, C3 및 C9인 것으로 나타나며, 각각에 대한 피어슨 잔차의 값은 8.48, 7.66 및 -7.37인 것으로 나타나며, Treg 세포(C4)의 피어슨 잔차의 값은 -7.07인 것으로 나타난다. 즉, EGFR 돌연변이를 포함한 경우, CD4 T세포의 아형인 C1 및 C3는 증가되어 있는 것으로 나타나며, CD4 T세포의 아형인 C9 및 Treg 세포는 감소되어 있는 것으로 나타난다.*The subtypes of CD4 T cells showing statistically significant values are shown to be C1, C3 and C9, and the Pearson residual values for each are 8.48, 7.66, and -7.37, respectively, and the Pearson residual of Treg cells (C4). The value appears to be -7.07. That is, when the EGFR mutation is included, CD4 T cell subtypes C1 and C3 are increased, and CD4 T cell subtypes C9 and Treg cells are decreased.

이때, CD4 T세포의 아형 및 Treg 세포에 대한 특성을 확인하기 위하여 DEGs(Differentially Expressed Genes) 분석을 수행하였다. 이에, 도 8b를 참조하면, EGFR 돌연변이 유무에 따른 CD4 T세포의 아형 및 Treg 세포에 대한 DEGs 분석 결과 즉, mRAN 및 이의 단백질의 발현량을 도시한 도트 플롯(Dot plot)이 도시된다. At this time, in order to confirm the characteristics of the CD4 T cell subtype and Treg cells, DEGs (Differentially Expressed Genes) analysis was performed. Accordingly, referring to FIG. 8b , a dot plot showing the expression levels of mRAN and its proteins is shown as a result of DEGs analysis for CD4 T cell subtypes and Treg cells according to the presence or absence of EGFR mutation.

보다 구체적으로, 면역 억제(immune suppression)에 관여하는 유전자인 CD52, ANXA1, ANXA2, 및 CXCR4은 C1 및 C3 아형에서 가장 많이 발현되어 있는 것으로 나타난다. 즉, 조력 T세포(helper T cell)인 CD4 T세포의 C1 및 C3는 면역을 억제하는 세포이다.More specifically, CD52, ANXA1, ANXA2, and CXCR4, which are genes involved in immune suppression, appear to be most expressed in the C1 and C3 subtypes. That is, C1 and C3 of CD4 T cells, which are helper T cells, are cells that suppress immunity.

결국, EGFR 돌연변이를 포함하는 개체는 전술한 바와 같이 면역반응을 억제하는 C1 및 C2의 아형을 다수 포함하고 있음에 따라, 종양에 대한 저항성 및 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다.After all, as the individual containing the EGFR mutation contains a number of subtypes of C1 and C2 that suppress the immune response as described above, the resistance and toxicity to the tumor, that is, the anticancer effect is reduced, and thus the immune anticancer effect Therapeutic responsiveness to therapy may be reduced.

이에, CD2, CD6, CD82. TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN 및 GATA3 중 적어도 하나, 바람직하게는, CD52, ANXA1, ANXA2, 및 CXCR4의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는 CD4 T세포의 수가 정상 대조군 또는 EGFR 돌연변이를 포함하지 않는 개체보다 증가된 수준을 나타내는 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).Thus, CD2, CD6, CD82. At least one of TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN and GATA3 Specifically, the number of CD4 T cells expressing mRNA for a marker gene of CD52, ANXA1, ANXA2, and CXCR4 or a protein thereof is higher than that of a normal control or an individual not containing the EGFR mutation. It can be determined that the treatment response to the therapy is low (therapeutic response negative (-) subjects).

한편, 세포활성(activation) 및 면역관문(immune checkpoint)에 관여하는 유전자인 CD2, CD6, CD82. TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1 및 CTLA4은 CD4 T세포의 C9 아형에서 가장 많이 발현되어 있는 것으로 나타나며, 이러한 CD4 T세포의 C9 아형은 전술한 유전자 이외의 자가 항체 생성에 필수적인 여포 보조 T세포(Follicular helper T cells, TFH)의 마커인 CXCL13, ICOS, MAF, PDCD1, 및 SH2D1A 유전자와도 관련되어 있는 것으로 나타난다.On the other hand, CD2, CD6, CD82, which are genes involved in cell activation and immune checkpoint. TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1 and CTLA4 appear to be most expressed in the C9 subtype of CD4 T cells. It also appears to be associated with CXCL13, ICOS, MAF, PDCD1, and SH2D1A genes, which are markers of Follicular helper T cells (T FH ).

여포 보조 T세포는 B세포의 발달 및 면역 활성 등의 다양한 기능을 조절하며, IL-4, IL-21, PD-1, CD69, OX40 및 CXCL13 등의 다양한 항원을 발현하고 있으며, 이 중 PD-1은 TFH 세포의 배아 중심(germinal centers)의 위치를 조절하고, TFH 세포의 기능을 보조하는 역할을 함에 따라, 여포 보조 T세포가 감소되어 있는 경우, 항 PD-1 치료의 효과가 경감될 수 있다.Follicle helper T cells regulate various functions such as B cell development and immune activity, and express various antigens such as IL-4, IL-21, PD-1, CD69, OX40 and CXCL13, among which PD- 1 regulates the location of the germinal centers of T FH cells and serves to support the functions of T FH cells. Therefore, when follicle helper T cells are reduced, the effect of anti-PD-1 treatment is reduced. can be

나아가, TFH 세포는 B세포에 대한 여포(follicles) 내로 침투(homing)를 유도하기 위하여, CXCL13을 생산한다. 즉, TFH 세포에서 분비하는 CXCL13에 의하여 3 차 림프절 구조(tertiary lymphoid structures, TLS)가 형성될 수 있다. 이때, TLS는 림프 노드가 아닌 곳에 T세포 및 B세포가 섬유성 그물망 세포(fibroblasts-like synovial cell, FRC)-유사 그물망 세포, HEV (high endothelial venules) 및 FDC (follicular dendritic cells)과 함께 구성된 면역 구조를 의미할 수 있다.Furthermore, T FH cells produce CXCL13 to induce homing into the follicles for B cells. That is, tertiary lymphoid structures (TLS) may be formed by CXCL13 secreted by T FH cells. At this time, TLS is not a lymph node, but T cells and B cells are fibroblasts-like synovial cells (FRC)-like reticular cells, HEV (high endothelial venules) and FDC (follicular dendritic cells) together with the immune system It can mean structure.

이에, 도 8c를 참조하면, Treg 세포 및 CD4 T세포에서의 아형에서의 TFH 세포와 관련된 마커에 대한 발현 결과 및 EGFR 돌연변이 유무에 따른 C9 아형의 CXCL13 발현 결과가 도시된다. Accordingly, referring to FIG. 8c , the expression results of T FH cell-related markers in the Treg cells and the CD4 T cell subtypes and the CXCL13 expression results of the C9 subtype according to the presence or absence of EGFR mutation are shown.

먼저, 도 8c의 (a)를 참조하면, TFH 세포의 마커 유전자인 CXCL13, PDCD1, MAF 및 SH2D1A의 발현은, CD4 T세포의 아형인 CD9에서 증가되어 있는 것으로 나타난다. First, referring to (a) of FIG. 8C , the expression of CXCL13, PDCD1, MAF and SH2D1A, which are marker genes of T FH cells, is increased in CD9, a subtype of CD4 T cells.

나아가, 도 8c의 (b)를 참조하면, CD4 T세포의 아형인 CD9에 대한 CXCL13의 발현은 EGFR 돌연변이를 포함하지 않는 개체(EGFR-WT)에서 더 많은 것으로 나타난다. 즉, EGFR 돌연변이를 포함하는 개체(EGFR-MT)에서 CXCL12의 발현이 감소되어 있는 것으로 나타남에 따라, EGFR 돌연변이는 CD4 T세포의 아형인 CD9에 대한 CXCL13의 발현을 감소시킬 수 있다.Furthermore, referring to (b) of FIG. 8C , the expression of CXCL13 for CD9, a subtype of CD4 T cells, is shown to be higher in subjects not containing the EGFR mutation (EGFR-WT). That is, as it was shown that the expression of CXCL12 is reduced in an individual containing an EGFR mutation (EGFR-MT), the EGFR mutation may reduce the expression of CXCL13 for CD9, a subtype of CD4 T cells.

한편, CD4 T세포의 아형인 CD9에서는 TFH 세포의 또 다른 특징 중 하나에 해당하는 CXCL13에 대한 수용체인 CXCR5과 TFH 세포 계통의 전사 인자(lineage-defining transcription factor)인 BCL6가 검출되지 않았다. 즉, CD4 T세포의 아형인 CD9은 일반적인 TFH 세포가 아닌 TFH 유사 세포(TFH-like cell)인 것을 의미할 수 있으며, 이는 TLS가 자주 발생하는 국부적인 염증 부위(local inflammatory sites)에서 주로 발견된다.On the other hand, in CD9, a subtype of CD4 T cells, CXCR5, a receptor for CXCL13, which is another characteristic of T FH cells, and BCL6, a lineage-defining transcription factor of the T FH cell lineage, were not detected. In other words, CD9, a subtype of CD4 T cells, may mean that they are T FH -like cells rather than general T FH cells, which are located in local inflammatory sites where TLS frequently occurs. mainly found

결국, TFH 세포의 주요 역할이 B세포의 유도임에 따라, EGFR 돌연변이를 포함하는 개체에서의 감소된 TFH 유사 세포의 수준은 비효율적인 TLS 형성을 초래하여, TLS 발달을 감소시키고, 이에 따라 종양에 대한 면역 세포의 면역 반응을 감소시킬 수 있다.Ultimately, as the main role of T FH cells is the induction of B cells, reduced levels of T FH -like cells in individuals with EGFR mutations result in inefficient TLS formation, resulting in reduced TLS development, thus It can reduce the immune response of immune cells to the tumor.

이에, 도 8d를 참조하면, EGFR 돌연변이 유무에 따른 종양 조직에서의 CD4+T세포 및 CXCL13+CD4+T세포에 대한 면역조직화학법 결과가 도시된다.Accordingly, referring to FIG. 8D , the results of immunohistochemistry for CD4 + T cells and CXCL13 + CD4 + T cells in tumor tissues with or without EGFR mutation are shown.

먼저, 도 8d의 (a)를 참조하면, EGFR 돌연변이를 포함하지 않는 개체(EGFR-WT)의 종양 조직에서 CD4+T세포 및 CXCL13+CD4+T세포가 더 많은 면적을 차지하는 것으로 나타난다. 이에, 단위 면적당 각 세포수를 계수한 결과인 도 8d의 (b)를 참조하면, EGFR-WT에 대한 종양 조직에서의 CD4+T세포의 개수는 약 1300 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CD4+T세포의 개수는 약 750 no/mm2 이하인 것으로 나타난다.First, referring to (a) of FIG. 8D , it appears that CD4 + T cells and CXCL13 + CD4 + T cells occupy a larger area in the tumor tissue of an individual (EGFR-WT) that does not contain an EGFR mutation. Accordingly, referring to FIG. 8d (b), which is the result of counting the number of cells per unit area, the number of CD4 + T cells in the tumor tissue for EGFR-WT is about 1300 no/mm 2 It appears to be abnormal, and the number of CD4 + T cells in the tumor tissue for EGFR-MT is about 750 no/mm 2 or less.

나아가, EGFR-WT에 대한 종양 조직에서의 CXCL13+CD4+T세포의 개수는 약 420 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CXCL13+CD4+T세포의 개수는 약 230 no/mm2 이하인 것으로 나타난다.Furthermore, the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-WT is about 420 no/mm 2 or more, and the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-MT is about 230 no/mm 2 or less.

결국, EGFR 돌연변이를 포함하는 개체는 전술한 바와 같이 B세포를 유도하고, 이에 따른 면역 반응 구조인 TLS를 형성할 수 있는 CXCL13을 생산하는 CD4 T세포인 C9 아형이 감소되어 있음에 따라, 종양에 대한 저항성 및 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다.As a result, as described above, the individual containing the EGFR mutation induces B cells as described above, and the CD4 T cell C9 subtype that produces CXCL13 capable of forming TLS, an immune response structure, is reduced. Resistance and toxicity, that is, the anticancer effect is reduced, and thus the therapeutic responsiveness to the immune anticancer therapy may be reduced.

이에, 종양 조직 및 일반 조직에 대한 CD4+T세포의 수준이 750 no/mm2 이하인 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).Accordingly, the level of CD4 + T cells for tumor tissue and general tissue is 750 no/mm 2 In the case of the following subjects, it can be determined that the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) subjects).

또한, 종양 조직 및 일반 조직에 대한 CXCL13+CD4+T세포의 수준이 230 no/mm2 이하인 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).In addition, if the level of CXCL13 + CD4 + T cells for tumor tissue and normal tissue is 230 no / mm 2 or less, it can be determined that the therapeutic response to immunotherapy is low (therapeutic response negative (-) individual) .

다시, 도 8a를 참조하면, 종양 침윤(tumor-infiltration)에 관여하는 유전자인 FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, 및 GATA3은 C4에서 가장 많이 발현되어 있는 것으로 나타남에 따라, C4는 자가면역억제 및 면역 항상성을 유지하는 Treg 세포의 아형이다.Again, referring to FIG. 8A , genes involved in tumor-infiltration, FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, and GATA3, are shown to be most expressed in C4, C4 is a subtype of Treg cells that maintain autoimmune suppression and immune homeostasis.

결국, EGFR 돌연변이를 포함하는 개체는 자가면역반응을 억제하고 면역 항상성을 유지시킬 수 있는 C4의 아형이 감소되어 있음에 따라, 종양에 대한 저항성 및 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성 및 지속성이 감소될 수 있다.As a result, the individual containing the EGFR mutation suppresses the autoimmune response and the C4 subtype that can maintain immune homeostasis is reduced, so the resistance and toxicity to the tumor, that is, the anticancer effect, is reduced, and thus the immunity Therapeutic responsiveness and persistence to anti-cancer therapy may be reduced.

이에, CD2, CD6, CD82. TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN 및 GATA3 중 적어도 하나, 바람직하게는, FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, 및 GATA3의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는 Treg 세포의 수가 정상 대조군 또는 EGFR 돌연변이를 포함하지 않는 개체보다 감소된 수준을 나타내는 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).Thus, CD2, CD6, CD82. At least one of TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN and GATA3 Specifically, the number of Treg cells expressing mRNA for the marker genes of FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, and GATA3 or a protein thereof is reduced compared to normal controls or individuals not containing the EGFR mutation. For individuals presenting, it can be determined that the therapeutic response to the immuno-cancer therapy is low (therapeutic response negative (-) individuals).

한편, Treg 세포는 전술한 FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, 및 GATA3 유전자 마커 이외의 T세포 활성화에 관여하는 CD27, CD28, ICOS, and TNFRSF4 encoding OX40, TNFRSF9 encoding 41BB, 및 TNFRSF18 encoding GITR 마커 유전자 및 면역 관문 분자인 TIGIT 및 CTLA4 마커 유전자에서도 높은 발현 수준을 가질 수 있다. On the other hand, Treg cells are CD27, CD28, ICOS, and TNFRSF4 encoding OX40, TNFRSF9 encoding 41BB, and TNFRSF18 involved in T cell activation other than the aforementioned FOXP3, IL2RA, IL2RB, IL2RG, IL10RA, MAGEH1, LAYN, and GATA3 gene markers. The encoding GITR marker gene and immune checkpoint molecules TIGIT and CTLA4 marker genes may also have high expression levels.

나아가, 도 8e를 참조하면, EGFR 돌연변이 유무에 따른 Treg 세포의 아형인 C4에서 Treg 세포 활성 촉진 인자와 관련된 유전자 발현 결과가 도시된다. Treg 세포의 아형인 C4에서 TSC22D3 및 NR4A2의 발현은, EGFR-MT에서 증가되어 있는 것으로 나타난다. 이때, TSC22D3는 GILZ 및 NR4A2를 인코딩하는 유전자이며, 특히 GILZ는 수지상 세포 및 T세포에서 면역 억제 효과를 중재하는데, 종양 미세 환경과 같은 면역 억제성 환경에서의 GILZ의 과잉은 T세포의 암 항원에 대한 내성을 유발할 수 있다. Furthermore, referring to FIG. 8E , the results of gene expression related to the Treg cell activation promoting factor in C4, a subtype of Treg cells, are shown according to the presence or absence of EGFR mutations. Expression of TSC22D3 and NR4A2 in C4, a subtype of Treg cells, appears to be increased in EGFR-MT. At this time, TSC22D3 is a gene encoding GILZ and NR4A2, and in particular, GILZ mediates the immunosuppressive effect in dendritic cells and T cells. may lead to tolerance.

이에, EGFR 돌연변이를 포함하는 개체는 Treg 세포의 아형인 C4에 의하여 과잉 분비된 GILZ로 인하여, T세포의 항원에 대한 내성이 유도되어, 종양에 대한 저항성 및 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다.Accordingly, in the subject containing the EGFR mutation, due to GILZ excessively secreted by C4, a subtype of Treg cells, resistance to the antigen of T cells is induced, and resistance and toxicity to the tumor, that is, the anticancer effect is reduced, Accordingly, therapeutic responsiveness to immunotherapy may be reduced.

본 발명의 일 work of the invention 실시예에in the example 따른 폐암에서 면역 항암 Immunocancer in lung cancer following 요법에 대한 치료 반응treatment response to therapy 예측 방법에서 사용되는 면역 세포 검증 Immune Cell Validation Used in Predictive Methods

이하에서는, 도 9a 내지 10d를 참조하여, 본 발명의 일 실시예에 따른 EGFR 돌연변이를 포함하는 개체의 면역 항암 요법에서 사용되는 면역 세포에 대하여 항상성(homeostasis) 및 TLS를 통한 T세포의 활성화를 검증하도록 한다.Hereinafter, with reference to FIGS. 9a to 10d, the activation of T cells through homeostasis and TLS is verified for immune cells used in immune anticancer therapy of an individual containing an EGFR mutation according to an embodiment of the present invention. let it do

먼저, 면역 세포의 항상성에 대한 도 9a를 참조하면, EGFR 돌연변이를 포함하는 특이적인 빈도를 갖는 면역 세포인 CD8 T세포의 아형 간의 전환 확률에 대한 결과가 도시된다.First, referring to FIG. 9a for homeostasis of immune cells, the results for the conversion probability between subtypes of CD8 T cells, which are immune cells with specific frequencies containing EGFR mutations, are shown.

C2에서 C2로 다시 전환될 확률은, EGFR-WT인 경우, 0.57인 것으로 나타나며, EGFR-MT인 경우, 0.41인 것으로 나타나며, EGFR 돌연변이를 포함한 경우, C2에서 C2로 전환 확률이 더 낮은 것으로 나타난다. The probability of converting back to C2 from C2 is shown to be 0.57 in the case of EGFR-WT, and 0.41 in the case of EGFR-MT, and when EGFR mutation is included, the probability of conversion from C2 to C2 is lower.

이와는 대조적으로, C2에서 C0로 전환될 확률은, EGFR-WT인 경우, 0.39인 것으로 나타나며, EGFR-MT인 경우, 0.52인 것으로 나타나며, EGFR 돌연변이를 포함한 경우, C2에서 C0로 전환 확률이 더 높은 것으로 나타난다. In contrast, the probability of C2 to C0 conversion is shown to be 0.39 for EGFR-WT, 0.52 for EGFR-MT, and a higher probability of C2 to C0 conversion when including EGFR mutations appears to be

즉, EGFR 돌연변이는 강한 세포 독성을 나타내는 CD8 T세포에 대한 아형인 C2의 지속성(persistence)이 감소시키고, 낮은 세포 독성을 효과를 갖는 CD8 T세포에 대한 아형인 C0 세포로의 전환을 증가시키는 것으로 나타난다. 이에, EGFR 돌연변이를 포함한 경우, 종양 특이적으로 강한 세포 독성을 갖는 TRM 유사 세포(C2, TRM-like cells)의 항상성이 감소되어, 면역 항암 요법에 대한 항암 효과의 효과 즉, 항암 유지 지속 기간이 감소될 수 있다.That is, the EGFR mutation decreases the persistence of C2, a subtype for CD8 T cells, which exhibits strong cytotoxicity, and increases conversion to C0 cells, which is a subtype for CD8 T cells with low cytotoxicity. appear. Accordingly, when the EGFR mutation is included, the homeostasis of T RM-like cells (C2, T RM - like cells) having a tumor-specific strong cytotoxicity is reduced, so that the effect of the anti-cancer effect on the immune anti-cancer therapy, that is, the maintenance of anti-cancer is maintained. The period may be reduced.

결국, 종양 조직 및 일반 조직에 대한 CD8 T세포에 대한 아형인 C2(TRM 유사 세포, GZMB+CD103+CD8+T세포)에서 C2 세포로의 전환 확률이 41 % 이하인 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).After all, in the case of individuals with a conversion probability of 41% or less from C2 (T RM -like cells, GZMB+CD103+CD8+T cells), which is a subtype of CD8 T cells to tumor tissue and normal tissue, to C2 cells, immuno-cancer therapy can be determined to be low in response to treatment (negative (-) subjects responding to treatment).

나아가, 종양 조직 및 일반 조직에 대한 CD8 T세포에 대한 아형인 C2(TRM 유사 세포, GZMB+CD103+CD8+T세포)에서 C0 세포로의 전환 확률이 52 % 이상인 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(+) 개체).Furthermore, in the case of an individual with a conversion probability of 52% or more from C2 (T RM -like cells, GZMB+CD103+CD8+T cells), which is a subtype of CD8 T cells to tumor tissues and normal tissues, to C0 cells, immunotherapy may be determined to be low in response to treatment (negative (+) subjects responding to treatment).

그 다음, 면역 세포의 전사 조절에 대한 도 9b를 참조하면, CD8 T세포에 대한 아형인 C2(TRM 유사 세포, TRM-like cells)에서 차등적으로 발현되어 있는 전사 인자에 대한 DEGs 결과가 도시된다. 이때, 상향조절(upregulated)되어 있는 전사 인자(transcription factors, TFs)는 점선 테두리로 표시되었다.Next, referring to FIG. 9b for the transcriptional regulation of immune cells, the DEGs results for the transcription factors differentially expressed in C2 (T RM -like cells, T RM -like cells), which are subtypes for CD8 T cells, are is shown At this time, the upregulated (upregulated) transcription factors (transcription factors, TFs) are indicated by a dotted border.

CD8 T세포에 대한 아형인 C2(TRM 유사 세포)에서 상향 조절되어 있는 전사 인자는, SERTAD1, IRF1, ARID5B, ELF1, SLAZ, BHLHE40, FOSL2, RBPJ, ID2 및 HOPX인 것으로 나타나며, 이 중, ID2, RBPJ 및 BHLHE40은 TRM 세포의 발달 및 항상성에 관여하는 할 수 있다. 특히, RBPJ는 노치(NOTCH)와 관련되어 TRM 세포를 분화, 유지 및 활성화하는데 필요한 전사 활성화체(transcriptional activator)를 형성함에 관련되어 있다.Transcription factors up-regulated in C2 (T RM -like cells), a subtype for CD8 T cells, are SERTAD1, IRF1, ARID5B, ELF1, SLAZ, BHLHE40, FOSL2, RBPJ, ID2 and HOPX. Among them, ID2 , RBPJ and BHLHE40 may be involved in the development and homeostasis of T RM cells. In particular, RBPJ is involved in the formation of a transcriptional activator required for differentiation, maintenance and activation of T RM cells in association with Notch.

이에, 도 9c를 참조하면, EGFR 돌연변이 유무에 따른, TRM 세포를 분화, 유지 및 활성화에 관여하는 RBPJ 및 NOTCH1 유전자에 대한 발현 결과가 도시된다. 이때, 발현 결과는 스피어만 상관 계수(Spearman's rank correlation)를 통하여 표현되었으며, 상관 계수가 커질수록 두 유전자가 연관되어 있음을 의미할 수 있다.Accordingly, referring to FIG. 9c , the expression results for the RBPJ and NOTCH1 genes involved in T RM cell differentiation, maintenance and activation are shown according to the presence or absence of EGFR mutation. In this case, the expression result was expressed through Spearman's rank correlation, and as the correlation coefficient increases, it may mean that the two genes are related.

EGFR-WT의 경우, CD8 T세포에 대한 아형인 C2(TRM 유사 세포)에서 RBPJ 및 NOTCH1 모두 TRM 세포의 분화 및 유지에 관련된 다양한 유전자와 높은 양(+)의 상관 관계를 갖는 것으로 나타난다. In the case of EGFR-WT, both RBPJ and NOTCH1 in C2 (T RM -like cells), a subtype for CD8 T cells, have a high positive (+) correlation with various genes involved in the differentiation and maintenance of T RM cells.

이와는 대조적으로, EGFR-MT의 경우, CD8 T세포에 대한 아형인 C2(TRM 유사 세포)에서 RBPJ 및 NOTCH1 모두 TRM 세포의 분화 및 유지에 관련된 다양한 유전자와 낮은 음(-)의 상관 관계를 갖는 것으로 나타난다. In contrast, in the case of EGFR-MT, both RBPJ and NOTCH1 in C2 (T RM -like cells), a subtype for CD8 T cells, showed a low negative correlation with various genes involved in the differentiation and maintenance of T RM cells. appears to have

보다 구체적으로, 전술한 도 9c의 발현 결과를 산점도(scatter plots) 및 회귀 곡선(regression curver)으로 나타낸, 도 9d를 참조하면, EGFR-MT의 경우, EGFR-WT보다 유전자 간의 상관 관계에 대한 계수가 0에 가깝거나 음의 상관 계수를 가짐에 따라, 관계가 소실되거나, 역 상관 관계를 갖는 것으로 나타난다.More specifically, referring to FIG. 9d , which shows the expression result of FIG. 9c described above as scatter plots and a regression curver, in the case of EGFR-MT, the coefficient of correlation between genes rather than EGFR-WT As is close to zero or has a negative correlation coefficient, the relationship disappears or appears to have an inverse correlation.

결국, EGFR 돌연변이는 RBPJ 및 NOTCH1에 의한 TRM 세포 및 TRM 유사 세포의 분화, 유지 및 항상성을 감소시킬 수 있으며, TRM 세포 및 TRM 유사 세포는 RBPJ 및 NOTCH1 네트워크에 의하여 분화, 유지 및 항상성이 조절됨을 의미할 수 있다.Ultimately, EGFR mutations can reduce the differentiation, maintenance and homeostasis of T RM cells and T RM-like cells by RBPJ and NOTCH1, and T RM cells and T RM -like cells differentiate, maintain and homeostasis by RBPJ and NOTCH1 networks. This may mean that it is regulated.

그 다음, TLS를 통한 T세포의 활성화에 대한 도 10a를 참조하면, EGFR 돌연변이 유무에 따른 세포 및 세포간의 상호 작용(cell-cell interaction) 즉, 리간드(ligand) 및 수용체(receptor)에 대한 DEGs 분석 결과 즉, mRAN 및 이의 단백질의 발현량을 도시한 도트 플롯(Dot plot)이 도시된다. 이때, 결과값은 유전자 쌍의 평균 발현 값을 의미할 수 있다.Then, referring to FIG. 10a for the activation of T cells through TLS, cell-cell interaction with or without EGFR mutation, that is, DEGs analysis for ligands and receptors A result, that is, a dot plot showing the expression level of mRNA and its protein is shown. In this case, the result value may mean an average expression value of a gene pair.

EGFR-MT의 경우, CXCL13-CXCR5 축(axis)을 통한 TRM 세포 및 B세포의 상호 작용이 약한 것으로 나타난다. 즉, 각 세포에서 발현되는 리간드 및 수용체에 대한 발현이 낮으며, 이에 따라, 리간드 및 수용체의 결합 및 상호작용으로 발생하는 면역 효과가 감소될 수 있다.In the case of EGFR-MT, the interaction between T RM cells and B cells via the CXCL13-CXCR5 axis appears to be weak. That is, the expression of the ligand and the receptor expressed in each cell is low, and thus, the immune effect generated by the binding and interaction of the ligand and the receptor may be reduced.

이에, 도 10b를 참조하면, CXCL13-CXCR5를 통한 면역 상호 작용을 검증하기 위하여, EGFR 돌연변이 유무에 따른 T세포 및 B세포의 리간드 및 수용체의 결합을 통하여 발생된 TLS 구조에 대한 면역조직화학법 결과가 도시된다. Accordingly, referring to FIG. 10b, in order to verify the immune interaction through CXCL13-CXCR5, immunohistochemistry results for the TLS structure generated through the binding of ligands and receptors of T cells and B cells according to the presence or absence of EGFR mutations is shown

먼저, 도 10b의 (a)를 참조하면, EGFR 돌연변이를 포함하지 않는 개체(EGFR-WT)의 종양 조직에서 CD4+T세포, CD8+T세포 및 B세포에 의하여 형성된 TLS가 더 많은 면적을 차지하는 것으로 나타난다. 이에, 면적을 측정하여, 수치적으로 비교한 결과인 도 10b의 (b)를 참조하면, EGFR-WT에 대한 종양 조직에서의 TLS의 면적은 약 17 mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 TLS의 면적은 약 5 mm2 이하인 것으로 나타난다. 즉, EGFR 돌연변이는 면역 세포의 상호 작용인 TLS 형성을 EGFR-WT에 비하여 약 70 % 감소시킬 수 있다.First, referring to FIG. 10b (a), in the tumor tissue of an individual (EGFR-WT) not containing the EGFR mutation, TLS formed by CD4 + T cells, CD8 + T cells and B cells occupies a larger area. appears to be Accordingly, referring to FIG. 10b (b), which is a numerical comparison result by measuring the area, the area of TLS in the tumor tissue for EGFR-WT is about 17 mm 2 appears to be abnormal, and the area of TLS in the tumor tissue for EGFR-MT is about 5 mm 2 appears to be below. That is, the EGFR mutation can reduce TLS formation, an immune cell interaction, by about 70% compared to EGFR-WT.

나아가, 10c를 참조하면, 전술한 9b의 TLS 영역에서 EGFR 돌연변이 유무에 따른 면역 세포에 대한 면역조직화학법 결과가 도시된다.Furthermore, referring to 10c, immunohistochemistry results for immune cells according to the presence or absence of EGFR mutations in the TLS region of 9b are shown.

CD4, CD20, CD8, CXCL13, CXCR5 및 CD103을 발현하는 세포의 빈도 및 개수는 EGFR-WT의 TLS에서 더 많은 것으로 나타난다. 보다 구체적으로, 도 9c에 대한 결과를 단위 면적당 각 세포수를 계수한 결과인 도 10d를 참조하면, EGFR-WT에 대한 종양 조직에서의 CD4+T세포의 개수는 약 27000 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CD4+T세포의 개수는 약 600 no/mm2 이하인 것으로 나타난다.The frequency and number of cells expressing CD4, CD20, CD8, CXCL13, CXCR5 and CD103 appear to be higher in TLS of EGFR-WT. More specifically, referring to FIG. 10d , which is the result of counting the number of cells per unit area, the number of CD4 + T cells in the tumor tissue for EGFR-WT is about 27000 no/mm 2 appears to be abnormal, and the number of CD4 + T cells in the tumor tissue for EGFR-MT is about 600 no/mm 2 appears to be below.

또한, EGFR-WT에 대한 종양 조직에서의 CXCL13+CD4+T세포의 개수는 약 10000 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CXCL13+CD4+T세포의 개수는 약 1500 no/mm2 이하인 것으로 나타난다.In addition, the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-WT is about 10000 no/mm 2 or more, and the number of CXCL13 + CD4 + T cells in the tumor tissue for EGFR-MT is about 1500 no/mm 2 or less.

또한, EGFR-WT에 대한 종양 조직에서의 CD8+T세포의 개수는 약 7500 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CD8+ CD4+T세포의 개수는 약 1000 no/mm2 이하인 것으로 나타난다.In addition, the number of CD8 + T cells in the tumor tissue for EGFR-WT was about 7500 no/mm 2 appears to be abnormal, and the number of CD8+ CD4+ T cells in the tumor tissue for EGFR-MT is about 1000 no/mm 2 or less.

또한, EGFR-WT에 대한 종양 조직에서의 CD103+CD8+T세포의 개수는 약 2900 no/mm2 이상인 것으로 나타나며, EGFR-MT에 대한 종양 조직에서의 CD103+CD8+ CD4+T세포의 개수는 약 500 no/mm2 이하인 것으로 나타난다.In addition, the number of CD103 + CD8 + T cells in the tumor tissue for EGFR-WT is about 2900 no/mm 2 or more, and the number of CD103 + CD8 + CD4 + T cells in the tumor tissue for EGFR-MT is about It appears to be less than 500 no/mm 2 .

결국, EGFR 돌연변이를 포함하는 개체는 TLS를 형성할 수 있는 B세포, CD4+T세포 및 CD8+T세포 바람직하게는, 및 B세포, B세포와 상호 작용을 야기할 수 있는 리간드인 CXCL12을 발현하는 CD4+T세포(CXCL13+CD4+T세포), 종양 특이적으로 강한 세포 독성을 나타낼 수 있는 CD103+CD8+T세포 및 GZMB+CD103+CD8+T세포가 감소되어 있음에 따라, 종양에 대한 저항성 및 독성 즉, 항암 효과가 감소되어 있으며, 이에 따른 면역 항암 요법에 대한 치료 반응성이 감소될 수 있다.Consequently, an individual containing an EGFR mutation expresses CXCL12, a ligand capable of causing interaction with B cells, CD4 + T cells and CD8 + T cells, preferably, and B cells, capable of forming TLS. CD4+ T cells (CXCL13+CD4+ T cells), CD103+CD8+ T cells and GZMB+CD103+CD8+ T cells that can exhibit strong tumor-specific cytotoxicity are decreased. Resistance and toxicity, that is, the anti-cancer effect is reduced, and thus the therapeutic responsiveness to the immune anti-cancer therapy may be reduced.

이에, 종양 조직 및 일반 조직에 대한 TLS의 면적이 5mm2 이하이거나, 정상 대조의 조직에 대한 TLS 면적의 수준보다 70 %이상 감소된 수준을 나타내는 개체의 경우, 면역 항암 요법에 대한 치료 반응성이 낮은 것으로 결정할 수 있다(치료 반응 음성(-) 개체).Therefore, the area of TLS for tumor tissue and general tissue is 5 mm 2 or less, or for an individual exhibiting a level that is reduced by 70% or more than the level of TLS area for tissue of a normal control, the therapeutic response to immunotherapy is low. (negative (-) subjects responding to treatment).

이상의 결과에 따라, EGFR 돌연변이와 같은 원인에 의하여 면역 항암 요법에 대한 반응성이 낮은 개체의 경우, CD8 T세포의 아형인 C2뿐만 아니라, CXCR5+CD20+B세포 또는 CXCL13+CD4+T세포를 포함하는 다양한 면역 세포들의 수준이 차별적으로 분포되어 있음에 따라, 이를 통하여 면역 항암 요법에 대한 반응성을 예측할 수 있다.According to the above results, in the case of individuals with low reactivity to immunotherapy due to causes such as EGFR mutation, not only C2, which is a subtype of CD8 T cells, but also CXCR5 + CD20 + B cells or CXCL13 + CD4 + T cells. As the levels of various immune cells are differentially distributed, it is possible to predict the responsiveness to immuno-cancer therapy through this.

이상 첨부된 도면을 참조하여 본 발명의 실시 예들을 더욱 상세하게 설명하였으나, 본 발명은 반드시 이러한 실시 예로 국한되는 것은 아니고, 본 발명의 기술사상을 벗어나지 않는 범위 내에서 다양하게 변형 실시될 수 있다. 따라서, 본 발명에 개시된 실시 예들은 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시 예에 의하여 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 그러므로, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 보호 범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.Although the embodiments of the present invention have been described in more detail with reference to the accompanying drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be made within the scope without departing from the technical spirit of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711103770[Project unique number] 1711103770

[과제번호] 2017M3A9E9072669[task number] 2017M3A9E9072669

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 바이오.의료기술개발(R＆D) [Research project name] Bio and medical technology development (R&D)

[연구과제명] 환자유래 순환 종양세포를 이용한 고정밀 전임상 모델 구축을 통한 항암제에 대한 획득 내성 기전 규명과 치료전략 제시[Research project name] Identify the mechanism of acquired resistance to anticancer drugs and present a treatment strategy through the establishment of a high-precision preclinical model using patient-derived circulating tumor cells

[기여율] 2/10 [Contribution rate] 2/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2020.02.01 ~ 2020.12.31 [Research period] 2020.02.01 ~ 2020.12.31

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711104016[Project unique number] 1711104016

[과제번호] 2019M3A9B6065231[task number] 2019M3A9B6065231

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 바이오.의료기술개발(R＆D) [Research project name] Bio and medical technology development (R&D)

[연구과제명] 단일세포 다중오믹스 기반 정밀 면역항암치료에 대한 임상 유용성 검증 [Research project name] Clinical usefulness verification for precision immunotherapy based on single-cell multiomics

[기여율] 2/10 [Contribution rate] 2/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2020.03.01 ~ 2020.12.31 [Research period] 2020.03.01 ~ 2020.12.31

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711104174[Project unique number] 1711104174

[과제번호] 2019M3A9B6065192[task number] 2019M3A9B6065192

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 바이오.의료기술개발(R＆D) [Research project name] Bio and medical technology development (R&D)

[연구과제명] 암미세환경 면역리액톰 및 유도인자 발굴을 위한 단일세포 다중오믹스 통합 분석기술 개발[Research project name] Development of single-cell multiomics integrated analysis technology for the discovery of cancer microenvironmental immunoreactors and inducers

[기여율] 1/10[Contribution rate] 1/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2020.03.01 ~ 2020.12.31 [Research period] 2020.03.01 ~ 2020.12.31

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711108917[Project unique number] 1711108917

[과제번호] 2018R1A5A2025079[Project No.] 2018R1A5A2025079

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 집단연구지원(R＆D) [Research project name] Group research support (R&D)

[연구과제명] 만성난치질환 시스템의학 연구센터 [Research Project Title] Chronic Incurable Disease System Medicine Research Center

[기여율] 1/10 [Contribution rate] 1/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2020.03.01 ~ 2021.02.28 [Research period] 2020.03.01 ~ 2021.02.28

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711104142[Project unique number] 1711104142

[과제번호] 2018M3C9A5064709[task number] 2018M3C9A5064709

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 포스트게놈신산업육성을위한다부처유전체사업(R＆D)(과기정통부) [Research project name] Genome project (R&D) of the Ministry of Science and Technology to foster a new post-genome industry (Ministry of Science and Technology)

[연구과제명] 유전체 빅데이터 활용을 위한 네트워크증강분석 웹서비스 개발 [Research Title] Development of Network Augmented Analysis Web Service for Genome Big Data Utilization

[기여율] 1/10 [Contribution rate] 1/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2020.01.01 ~ 2020.12.31 [Research period] 2020.01.01 ~ 2020.12.31

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711108900[Project unique number] 1711108900

[과제번호] 2018R1A2A1A05076997[Project No.] 2018R1A2A1A05076997

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 개인기초연구(과기정통부)(R＆D) [Research project name] Basic personal research (Ministry of Science and Technology) (R&D)

[연구과제명] 암-면역 분자네트워크를 활용한 환자별 면역억제에 대한 분자기전 규명 및 정밀 면역항암치료 플랫폼 개발 [Research project name] Identification of molecular mechanisms for patient-specific immunosuppression using cancer-immune molecular network and development of precise immunotherapy platform

[기여율] 1/10 [Contribution rate] 1/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2020.03.01 ~ 2021.02.28 [Research period] 2020.03.01 ~ 2021.02.28

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711105688[Assignment number] 1711105688

[과제번호] 2019M3A9B6065221[task number] 2019M3A9B6065221

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 바이오.의료기술개발(R＆D) [Research project name] Bio and medical technology development (R&D)

[연구과제명] 단일세포 다중오믹스 기술을 활용한 면역리액톰 유도기전 규명 [Research Title] Immune Reactome Induction Mechanism Using Single Cell Multiomics Technology

[기여율] 1/10 [Contribution rate] 1/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2020.03.01 ~ 2020.12.31 [Research period] 2020.03.01 ~ 2020.12.31

[이 발명을 지원한 국가연구개발사업][National R&D project supporting this invention]

[과제고유번호] 1711055832[Project unique number] 1711055832

[과제번호] 2017R1A5A1014560[Project No.] 2017R1A5A1014560

[부처명] 과학기술정보통신부[Ministry name] Ministry of Science and Technology Information and Communication

[과제관리(전문)기관명] 한국연구재단[Name of project management (specialized) institution] National Research Foundation of Korea

[연구사업명] 집단연구지원[Research project name] Group research support

[연구과제명] 비임파성 장기 면역 연구센터 [Research project name] Non-Lymphatic Organ Immunization Research Center

[기여율] 1/10 [Contribution rate] 1/10

[과제수행기관명] 연세대학교 산학협력단 [Name of project execution institution] Yonsei University Industry-University Cooperation Foundation

[연구기간] 2017.06.01 ~ 2018.02.28 [Research period] 2017.06.01 ~ 2018.02.28

Claims (26)

개체로부터 분리된 생물학적 시료에 대한, ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준을 측정하는 단계, 및Level of mRNA or protein thereof for at least one marker gene of ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 in a biological sample isolated from an individual measuring, and 측정된 상기 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준을 기초로 상기 개체에 대한 면역 항암 요법의 치료 반응을 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immune anticancer therapy in lung cancer, comprising the step of determining a therapeutic response of the immunotherapy for the subject based on the measured level of mRNA or protein thereof for the marker gene. 제 1항에 있어서,The method of claim 1, 상기 측정하는 단계는,The measuring step is 정상 대조군의 시료에 대한 ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 및 LAG3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준을 측정하는 단계를 포함하고,Measuring the level of mRNA or protein thereof for at least one marker gene of ITGAE, ID2, IFNG, CD7, CXCR6, PRF1, GZMA, GZMB, GNLY, GZMK, GZMH, GZMM, HAVCR2 and LAG3 for a sample of a normal control group comprising steps, 상기 결정하는 단계는,The determining step is 상개 개체에 대한 상기 마커 유전자에 대한 mRNA 또는 이의 단백질의 수준이 상기 정상 대조군의 수준보다 낮은 경우, When the level of mRNA or protein thereof for the marker gene for the normal individual is lower than the level of the normal control, 상기 개체를 면역 항암 요법에 대한 치료 반응성이 음성인 것으로 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to an immune anticancer therapy in lung cancer, comprising the step of determining that the subject has a negative therapeutic response to the immune anticancer therapy. 제 1항 또는 제 2항에 있어서,3. The method of claim 1 or 2, 상기 적어도 하나의 유전자 마커는 ITGAE 및/또는 GZMA인, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.wherein the at least one genetic marker is ITGAE and/or GZMA. 개체로부터 분리된 생물학적 시료에 대한, for a biological sample isolated from a subject; GZMB+CD103+CD8+T세포의 수준을 측정하는 단계, 및 measuring the level of GZMB+CD103+CD8+T cells, and 측정된 상기 GZMB+CD103+CD8+T세포의 수준을 기초로 상기 개체에 대한 면역 항암 요법의 치료 반응을 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.Based on the measured level of the GZMB + CD103 + CD8 + T cells, comprising the step of determining a therapeutic response to the immunotherapy for the subject, a method for predicting a therapeutic response to immunotherapy in lung cancer. 제 4항에 있어서,5. The method of claim 4, 상기 GZMB+CD103+CD8+T세포는,The GZMB + CD103 + CD8 + T cells, ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX 및 TCF7 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.at least one of ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX and TCF7 marker genes A method of predicting a therapeutic response to immunotherapy in lung cancer, which expresses mRNA or a protein thereof. 제 5항에 있어서,6. The method of claim 5, 상기 GNLY의 log FC(fold change)는,1.5 이상인, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.The log FC (fold change) of the GNLY is, 1.5 or more, a method of predicting treatment response to immunotherapy in lung cancer. 제 4항에 있어서,5. The method of claim 4, 상기 GZMB의 log FC(fold change)는,The log FC (fold change) of the GZMB is, 1.2 이상이고,1.2 or higher, 상기 CD103의 log FC는,The log FC of CD103 is, 0.4 이상인, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method of predicting treatment response to immuno-cancer therapy in lung cancer, which is 0.4 or higher. 제 4항에 있어서,5. The method of claim 4, 상기 GZMB+CD103+CD8+T세포에 대한 예측력은, AUC 0.854 인, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.The predictive power for the GZMB + CD103 + CD8 + T cells is, AUC 0.854, a method of predicting a therapeutic response to immunotherapy in lung cancer. 제 4항에 있어서,5. The method of claim 4, 상기 측정하는 단계는,The measuring step is 정상 대조군의 시료에 대한 GZMB+CD103+CD8+T세포를 측정하는 단계를 포함하고,Measuring GZMB + CD103 + CD8 + T cells for a sample of a normal control group, 상기 면역 항암 요법의 치료 반응을 결정하는 단계는,Determining the therapeutic response of the immuno-cancer therapy comprises: 측정된 개체에 대한 상기 GZMB+CD103+CD8+T세포의 수준이 상기 정상 대조군 시료에 대한 GZMB+ITGAE(CD103)+CD8+T세포의 수준보다 감소된 수준을 나타내는 경우,When the measured level of GZMB+CD103+CD8+T cells for the subject indicates a reduced level than the level of GZMB+ITGAE(CD103)+CD8+T cells for the normal control sample, 상기 개체가 면역 항암 요법에 대한 반응성이 음성인 것으로 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to an immune anticancer therapy in lung cancer, comprising the step of determining that the subject is negative for responsiveness to the immune anticancer therapy. 제 4항에 있어서,5. The method of claim 4, 상기 측정하는 단계는,The measuring step is CXCR5+CD20+B세포 또는 CXCL13+CD4+T세포(regulatory T cells, Tregs)를 측정하는 단계를 더 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.CXCR5 + CD20 + B cells or CXCL13 + CD4 + T cells (regulatory T cells, Tregs) further comprising the step of measuring, the treatment response prediction method for immunotherapy in lung cancer. 제 10항에 있어서,11. The method of claim 10, 상기 측정하는 단계는,The measuring step is 정상 대조군의 시료에 대한 CXCL13+CD4+T세포를 측정하는 단계를 더 포함하고,Further comprising the step of measuring CXCL13 + CD4 + T cells for the sample of the normal control, 상기 결정하는 단계는,The determining step is 측정된 개체에 대한 상기 CXCL13+CD4+T세포의 수준이 상기 정상 대조군에 대한 CXCL13+CD4+T세포의 수준보다 감소된 수준을 나타내는 경우, When the measured level of CXCL13 + CD4 + T cells for the individual indicates a reduced level than the level of CXCL13 + CD4 + T cells for the normal control, 상기 개체가 면역 항암 요법에 대한 반응성이 음성인 것으로 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to an immune anticancer therapy in lung cancer, comprising the step of determining that the subject is negative for responsiveness to the immune anticancer therapy. 제 10항에 있어서,11. The method of claim 10, 상기 CXCL13+CD4+T세포는,The CXCL13 + CD4 + T cells, CD2, CD6, CD82. TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN 및 GATA3 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.CD2, CD6, CD82. Marker of at least one of TMFRSF4, TNFRSF9, ICOS, CD27, CD28, TIGIT, PDCD1, CTLA4, CCR6, IL7R, SELL, CD52, ANXA1, ANXA2, CXCR4, FOXP3, IL3RA, IL2RB, IL3RG, IL10RA, MAGEH1, LAYN and GATA3 A method of predicting a therapeutic response to immunotherapy in lung cancer, which expresses mRNA for a gene or a protein thereof. 제 10항에 있어서,11. The method of claim 10, 상기 CXCL13+CD4+T세포는,The CXCL13 + CD4 + T cells, PDCD1, MAF 및 SH2D1A 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 더 발현하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immunocancer therapy in lung cancer, wherein the mRNA for at least one marker gene of PDCD1, MAF, and SH2D1A or a protein thereof is further expressed. 제 10항에 있어서,11. The method of claim 10, 상기 측정하는 단계는,The measuring step is 정상 대조군의 시료에 대한 CXCR5+CD20+B세포의 수준을 측정하는 단계를 더 포함하고,Further comprising the step of measuring the level of CXCR5 + CD20 + B cells relative to the sample of the normal control, 상기 결정하는 단계는,The determining step is 측정된 개체에 대한 상기 CXCR5+CD20+B세포의 수준이 상기 정상 대조군 시료에 대한 CXCR5+CD20+B 세포의 수준보다 감소된 수준을 나타내는 경우, When the measured level of CXCR5 + CD20 + B cells for the subject indicates a reduced level than the level of CXCR5 + CD20 + B cells for the normal control sample, 상기 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immune anticancer therapy in lung cancer, comprising the step of determining that the subject has a negative therapeutic response to immune anticancer therapy. 제 10항에 있어서,11. The method of claim 10, 상기 CXCR5+CD20+B세포는,The CXCR5 + CD20 + B cells, HLA-DPB1, HLA-DRB5, HLA-DQA2 및 HLA-C 중 적어도 하나의 항원 표시 인자 (antigen presentation molecule)을 발현하고,expressing an antigen presentation molecule of at least one of HLA-DPB1, HLA-DRB5, HLA-DQA2 and HLA-C; TAP1 마커 유전자에 대한 mRNA 또는 이의 단백질을 발현하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immuno-cancer therapy in lung cancer, which expresses mRNA for a TAP1 marker gene or a protein thereof. 제 15항에 있어서,16. The method of claim 15, 상기 항원은,The antigen is 정상 대조군에 대한 CXCR5+CD20+B세포의 항원 표지 인자 발현 수준보다 감소된 수준을 나타내는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immuno-cancer therapy in lung cancer, showing a reduced level than the expression level of an antigen marker of CXCR5 + CD20 + B cells relative to a normal control. 제 4항에 있어서,5. The method of claim 4, 상기 측정하는 단계는,The measuring step is 상기 GZMB+CD103+CD8+T세포 수를 측정하는 단계를 더 포함하고,Further comprising the step of measuring the number of GZMB + CD103 + CD8 + T cells, 상기 결정하는 단계는,The determining step is 측정된 상기 GZMB+CD103+CD8+T세포 수가 1 cells/mm2 이하이거나,The measured number of GZMB + CD103 + CD8 + T cells is 1 cells/mm 2 or less, 전체 CD103+CD8+T세포에 대한 상기 GZMB+CD103+CD8+T세포 수의 비율이 30 % 미만인 경우,When the ratio of the number of GZMB+CD103+CD8+T cells to the total CD103+CD8+T cells is less than 30%, 상기 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immune anticancer therapy in lung cancer, comprising the step of determining that the subject has a negative therapeutic response to immune anticancer therapy. 제 4항에 있어서,5. The method of claim 4, 상기 측정하는 단계는,The measuring step is 상기 GZMB+CD103+CD8+T세포의 GZMB+CD103+CD8+T세포로의 전환 확률(transition probability)을 측정하는 단계를 더 포함하고,Further comprising the step of measuring the transition probability (transition probability) of the GZMB + CD103 + CD8 + T cells to GZMB + CD103 + CD8 + T cells, 상기 결정하는 단계는,The determining step is 전체 CD8+T세포로의 전환 확률에 대한 상기GZMB+CD103+CD8+T세포로의 전환 확률이 41%이하인 경우, When the conversion probability of the GZMB+CD103+CD8+T cells with respect to the conversion probability of the total CD8+T cells is 41% or less, 상기 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 더 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immunocancer therapy in lung cancer, further comprising the step of determining that the subject has a negative therapeutic response to immune anticancer therapy. 제 4항에 있어서,5. The method of claim 4, 상기 측정하는 단계는,The measuring step is 상기 개체 및 정상 대조군의 시료에 대한 3 차 림프절 구조 (tertiary lymphoid structures, TLS)의 면적를 측정하는 단계를 더 포함하고,Further comprising the step of measuring the area of tertiary lymphoid structures (TLS) for the sample of the subject and the normal control, 상기 결정하는 단계는,The determining step is 측정된 개체에 대한 상기 TLS의 면적이, 상기 정상 대조군 시료에 대한 TLS 면적의 수준보다 70 %이상 감소된 수준을 나타내거나,The area of the TLS for the measured individual represents a level that is reduced by 70% or more than the level of the TLS area for the normal control sample, or 상기 개체에 대한 TLS의 면적이 5 mm2 미만인 경우,If the area of TLS for the object is less than 5 mm 2 , 상기 개체가 면역 항암 요법에 대한 치료 반응이 음성인 것으로 결정하는 단계를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immune anticancer therapy in lung cancer, comprising the step of determining that the subject has a negative therapeutic response to immune anticancer therapy. 제 4항에 있어서,5. The method of claim 4, 상기 개체는, The object is 폐암 의심 개체이고,a subject suspected of lung cancer, 상기 생물학적 시료는,The biological sample, 조직, 말초 혈액, 혈청 및 혈장으로 이루어진 그룹에서 선택된 적어도 하나를 포함하는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method for predicting a therapeutic response to immuno-cancer therapy in lung cancer, comprising at least one selected from the group consisting of tissue, peripheral blood, serum and plasma. 제 4항에 있어서,5. The method of claim 4, 상기 면역 항암 요법은,The immunotherapy is 항 PD-1 치료인, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.A method of predicting a therapeutic response to immuno-cancer therapy in lung cancer, which is an anti-PD-1 treatment. 제 4항에 있어서,5. The method of claim 4, 상기 GZMB+CD103+CD8+T세포의 수준은,The level of the GZMB + CD103 + CD8 + T cells is, 웨스턴 블롯(western blot), 엘라이자(enzyme linked immunosorbent assay, ELISA), 방사선면역분석(Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(ouchterlony) 면역 확산법, 로켓 면역전기영동, 면역조직화학법(immunohistochemistry, IHC), 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS) 및 단백질 칩(protein chip) 중 적어도 하나의 방법으로 측정되는, 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법.Western blot, enzyme linked immunosorbent assay, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry Measured by at least one method of immunohistochemistry, IHC, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter, FACS, and protein chip, A method for predicting therapeutic response to immunotherapy in lung cancer. 개체로부터 분리된 생물학적 시료에 대한, GZMB+CD103+CD8+T세포의 수준을 측정하도록 구성된 제제를 포함하는, 폐암 개체의 면역 항암 요법에 대한 치료 반응 예측용 키트.A kit for predicting a therapeutic response to immuno-cancer therapy in a lung cancer individual, comprising an agent configured to measure the level of GZMB+CD103+CD8+T cells in a biological sample isolated from the individual. 제 23항에 있어서,24. The method of claim 23, 상기 개체는, The object is 폐암 의심 개체이고,a subject suspected of lung cancer, 상기 생물학적 시료는,The biological sample, 조직, 말초 혈액, 혈청 및 혈장으로 이루어진 그룹에서 선택된 적어도 하나를 포함하는, 폐암 개체의 면역 항암 요법에 대한 치료 반응 예측용 키트.Tissue, peripheral blood, serum and plasma, comprising at least one selected from the group consisting of, a kit for predicting a therapeutic response to immunotherapy of a lung cancer individual. 제 23항에 있어서,24. The method of claim 23, 상기 제제는, The formulation is 상기 GZMB+CD103+CD8+T세포의 ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS, TOX 및 TCF7 중 적어도 하나의 마커 유전자에 대한 mRNA 또는 이의 단백질을 측정하도록 구성된 제제를 더 포함하는, 폐암 개체의 면역 항암 요법에 대한 치료 반응 예측용 키트.ID2, IFNG, CD7, TNFRSF4, CCR7, IL7R, CXCR4, CXCR6, PRF1, GZMA, GNLY, GZMK, GZMH, GZMM, PDCD1, CTAL4, TIGIT, HAVCR2, LAG3, ICOS of the GZMB+CD103+CD8+T cells A kit for predicting a therapeutic response to immuno-cancer therapy in a lung cancer individual, further comprising an agent configured to measure mRNA or a protein thereof for at least one marker gene of TOX and TCF7. 제 23항에 있어서,24. The method of claim 23, 상기 제제는, The formulation is CXCR5+CD20+B세포 또는 CXCL13+CD4+T세포의 수준을 측정하도록 구성된 제제를 더 포함하는, 폐암 개체의 면역 항암 요법에 대한 치료 반응 예측용 키트.CXCR5 + CD20 + B cells or CXCL13 + CD4 + T cells further comprising an agent configured to measure the level of, the kit for predicting the therapeutic response to immunotherapy in lung cancer individuals.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019207030A1 (en) * 2018-04-26 2019-10-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting a response with an immune checkpoint inhibitor in a patient suffering from a lung cancer
US20200123258A1 (en) * 2018-10-23 2020-04-23 Board Of Regents, The University Of Texas System Targeting b cells to enhance response to immune checkpoint blockade
US20200308653A1 (en) * 2017-12-11 2020-10-01 Istituti Fisioterapici Ospitalieri (Ifo) - Istituto Regina Elena Per Lo Studio E La Cura Dei Tumori New onco-immunologic prognostic and theranostic markers

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200308653A1 (en) * 2017-12-11 2020-10-01 Istituti Fisioterapici Ospitalieri (Ifo) - Istituto Regina Elena Per Lo Studio E La Cura Dei Tumori New onco-immunologic prognostic and theranostic markers
WO2019207030A1 (en) * 2018-04-26 2019-10-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting a response with an immune checkpoint inhibitor in a patient suffering from a lung cancer
US20200123258A1 (en) * 2018-10-23 2020-04-23 Board Of Regents, The University Of Texas System Targeting b cells to enhance response to immune checkpoint blockade

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CORGNAC STÉPHANIE, MALENICA INES, MEZQUITA LAURA, AUCLIN EDOUARD, VOILIN ELODIE, KACHER JAMILA, HALSE HELOISE, GRYNSZPAN LAETITIA,: "CD103+CD8+ TRM Cells Accumulate in Tumors of Anti-PD-1-Responder Lung Cancer Patients and Are Tumor-Reactive Lymphocytes Enriched with Tc17", CELL REPORTS MEDICINE, vol. 1, no. 7, 1 October 2020 (2020-10-01), XP055956883, ISSN: 2666-3791, DOI: 10.1016/j.xcrm.2020.100127 *
SINGH D, GANESAN AP, PANWAR B, ESCHWEILER S, HANLEY CJ, MADRIGAL A, RAMÍREZ-SUÁSTEGUI C, WANG A, CLARKE J, WOOD O, GARRIDO-MARTIN : "CD4 + follicular helper-like T cells are key players in anti-tumor immunity", BIORXIV, 9 January 2020 (2020-01-09), XP055956885, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.01.08.898346v1.full.pdf> [retrieved on 20220901], DOI: 10.1101/2020.01.08.898346 *

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