Method for providing information on immunotherapy in HPV-induced cancer

Abstract

The present invention provides: a marker composition for predicting a response to immunotherapy in HPV-induced cancer; and a method for providing information on immunotherapy in HPV-induced cancer using the marker composition. The marker composition contains a tissue-resident memory T cell-specific KLRB1 mRNA or a protein encoded by the mRNA or CD161+ tissue-resident memory T cells as a marker for predicting a response to immunotherapy in HPV-induced cancer.

Description

How to provide information on immunotherapy in HPV-induced cancers

[0001] The present invention relates to a method for providing information on immunotherapy in HPV-induced cancer.

[0002] Human papillomavirus (HPV) is a family of small, non-enveloped DNA viruses that are primarily transmitted through sexual contact. HPV can be divided into over 170 types, some of which can cause warts or benign papillomas. High-risk types of HPV have been linked to head and neck cancer and oropharyngeal cancer (e.g., cancer of the throat, base of the tongue, and tonsils), cervical cancer, and anogenital cancer (e.g., cancer of the anus, penis, vagina, and vulva).

[0003] More specifically, HPV can induce head and neck squamous cell carcinoma (HNSCC) in the oropharynx, also known as oropharyngeal squamous cell carcinoma (OPSCC).

[0004] At this time, HPV-induced oropharyngeal squamous cell carcinoma has distinct clinical features in contrast to non-virus-induced squamous cell carcinoma (HPV-OPSCC), which is characterized by a relatively high mutation burden.

[0005] That is, in the case of HPV-induced cancer, a different approach may be required compared to non-HPV-induced cancer in setting the treatment direction.

[0006] For this reason, there is a continuous need to develop methods to provide information that can predict treatment responses to existing treatments for HPV-induced cancer and suggest early diagnosis and treatment directions.

[0007] The background technology of the invention has been prepared to facilitate a better understanding of the present invention. It should not be construed as an admission that the matters described in the background technology of the invention constitute prior art.

[0008] Meanwhile, PD-1 immunotherapies such as pembrolizumab and nivolumab may be applied in the treatment of HPV-induced cancers, especially HPV+ squamous cell carcinoma.

[0009] At this time, the efficacy of immunotherapy in HPV+ patients may be similar to that in HPV- patients, contrary to expectations that the treatment response would be superior to that in HPV- cancers, as the existing immune system may be strengthened by promoting anti-tumor immune responses according to viral antigens.

[0010] The inventors of the present invention recognized that treatment outcomes for immunotherapy in HPV+ patients are heterogeneous and that uncharacterized factors exist that impede HPV-related clinical benefit from immunotherapy.

[0011] In particular, the inventors of the present invention sought to identify cellular and molecular factors that could explain the discrepancy in clinical benefits due to HPV infection, and were able to recognize that distinct tumor microenvironment (TME) characteristics could offset benefits such as enhanced anti-tumor activity due to HPV infection.

[0012] As a result, the inventors of the present invention discovered that intratumoral immune cells, including CD4+ T cells and CD8+ T cells, and further B cells, are regulated by HPV infection.

[0013] In particular, the inventors of the present invention were able to find that tissue-resident memory T cells (Trm) showing high expression of KLRB1 (encoding CD161 receptor) in HPV+ head and neck cancer subjects are associated with TME features for reduced anti-tumor activity.

[0014] In this regard, the inventors of the present invention found an inverse correlation between the density of CD161+ tissue-resident memory T cells and changes in tumor size, and were able to recognize that CD161+ tissue-resident memory T cells offset the clinical benefit of immunotherapy of HPV-induced cancers, particularly immune HPV infection in HPV+ head and neck cancer subjects.

[0015] Moreover, the inventors of the present invention were able to further note that when KLRB1 is expressed at high levels in tissue-resident memory T cells of HPV+ head and neck cancer, the expression level of CLEC2D (encoding CD161 ligand) in B cells is relatively low.

[0016] As a result, the inventors of the present invention have developed a new information providing method through the combination of KLRB1 and / or KLRB1 of tissue-resident memory T cells and CLEC2D of B cells as a biomarker capable of predicting an anti-tumor environment in HPV+ head and neck cancer.

[0017] The inventors of the present invention expected that the development of a novel information providing method could provide information related to the anti-tumor environment, particularly information that can predict the therapeutic response to immune checkpoint inhibitors in HPV-induced cancer entities.

[0018] Accordingly, the problem to be solved by the present invention is to provide a marker composition comprising tissue-resident memory T cell-specific KLRB1 mRNA and its encoding protein, or CD161+ tissue-resident memory T cells.

[0019] Another problem to be solved by the present invention is to provide a method for providing information that can measure the biomarker level of tissue-resident memory T cell-specific KLRB1 or the level of CD161+ tissue-resident memory T cells, and determine and provide the treatment response of immunotherapy for an individual based on the biomarker level, so as to increase the treatment effect by predicting the responsiveness of immunotherapy for an individual at an early stage.

[0020] Furthermore, another problem to be solved by the present invention is to provide an information providing kit including a preparation for measuring the expression level of tissue-resident memory T cell-specific KLRB1 capable of predicting a therapeutic response to immunotherapy based on the information providing method described above.

[0021] The tasks of the present invention are not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those skilled in the art from the description below.

[0022] To solve the above-described problem, the present invention provides a marker composition for predicting response to immunotherapy in HPV-induced cancer, comprising mRNA of tissue-resident memory T cell-specific KLRB1 or a protein encoded by the mRNA, as a marker for predicting response to immunotherapy in HPV-induced cancer.

[0023] According to a feature of the present invention, markers of tissue-resident memory T cells, such as CD103, CD69, CD49a (Integrin α1), CD44, CD101, CD127, CXCR6 and CD8, can be selectively used as biomarkers.

[0024] According to a feature of the present invention, the HPV-induced cancer may be at least one selected from oropharyngeal cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, and vulvar cancer caused by HPV infection.

[0025] In order to solve the problem described above, the present invention provides a method for providing information on immunotherapy of HPV-induced cancer, comprising the steps of measuring the expression level of tissue-resident memory T cell-specific KLRB1 mRNA or a protein encoded by the mRNA for a biological sample isolated from an HPV-infected individual, and determining a characteristic associated with immunotherapy for the individual based on the measured expression level of KLRB1 mRNA or a protein encoded by the mRNA.

[0026] According to a feature of the present invention, the determining step may include determining that the subject is negative for treatment response to immunotherapy if the expression level of tissue-resident memory T cell-specific KLRB1 mRNA or a protein encoded by the mRNA is above a predetermined level, and that the subject is positive for treatment response otherwise.

[0027] According to another feature of the present invention, the predetermined level may be a normalized RNA average of 2 to 3 [mean(exp(x)-1].

[0028] According to another feature of the present invention, the measuring step may further include a step of measuring the expression level of mRNA of tissue-resident memory T cell-specific KLRB1 or a protein encoded by the mRNA and the expression level of mRNA of B cell-specific CLEC2D or a protein encoded by the mRNA.

[0029] According to another feature of the present invention, the HPV-induced cancer may be oropharyngeal cancer caused by HPV infection.

[0030] To solve the above-described problem, the present invention provides a method for providing information on immunotherapy in HPV-induced cancer, comprising the steps of measuring the level of CD161+ tissue-resident memory T cells in a biological sample isolated from an HPV-infected subject, and determining a characteristic associated with immunotherapy for the subject based on the measured level of CD161+ tissue-resident memory T cells.

[0031] According to a feature of the present invention, the measuring step may include a step of measuring the cell level of at least one of CD161+CD8+ T cells, CD161+CD103+ T cells and CD161+CD8+CD103+ T cells, and the determining step may include a step of determining the characteristic based on the cell level of at least one of the cells.

[0032] According to another feature of the present invention, the measuring step may further comprise measuring the levels of CD161+ tissue-resident memory T cells and CLEC2D+ B cells.

[0033] According to another feature of the present invention, the step of determining comprises: determining whether the level of CD161+ tissue-resident memory T cells for the biological sample is 15 cells / mm; 2 If the above is true, the step may include determining that the subject is negative for treatment response to immunotherapy, and if not, that the subject is positive for treatment response.

[0034] According to another feature of the present invention, the step of determining comprises: determining whether the level of CD161+ tissue-resident memory T cells for the biological sample is 15 cells / mm; 2 In this case, the step of determining blockade of CD161 based on inhibitory immune checkpoint molecule of CD161 as a treatment for the subject may be included.

[0035] To solve the above-mentioned problem, the present invention provides a kit for providing information on immunotherapy in HPV-induced cancer, comprising a formulation for measuring the expression level of mRNA of tissue-resident memory T cell-specific KLRB1 or a protein encoded by the mRNA for a biological sample isolated from an HPV-infected subject.

[0036] According to a feature of the present invention, the preparation may include an antibody targeting the CD161 surface protein of tissue-resident memory T cells (Trm).

[0037] According to another feature of the present invention, the formulation may comprise a bispecific monoclonal antibody targeting CD161 and CD103 surface proteins of tissue-resident memory T cells.

[0038] According to another feature of the present invention, the expression level of mRNA can be measured through at least one method selected from fluorescence in situ hybridization (FISH), NanoString nCounter analysis, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase-6 protection assay (RPA), microarray, and northern blotting.

[0039] According to another feature of the present invention, the protein expression level can be measured by at least one method selected from among western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and protein chip.

[0040] Hereinafter, the present invention will be described in more detail through examples. However, these examples are provided merely to illustrate the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

[0041] The present invention has the effect of providing a new biomarker capable of predicting a therapeutic response to an immune checkpoint inhibitor in an HPV-infected subject.

[0042] The present invention has the effect of determining and providing information on the anti-tumor environment, such as treatment responsiveness to immunotherapy, based on the expression level of biomarkers in various samples within an HPV-infected subject.

[0043] Moreover, the present invention can provide information on determining a blocking agent, such as a specific antibody targeting the surface protein of a biomarker, based on the expression pattern of the biomarker in a sample, thereby contributing to enhancing the effectiveness of immunotherapy.

[0044] The present invention can provide information to quickly determine whether to proceed with a selected immunotherapy by predicting an early treatment response to immunotherapy for an individual using a biomarker.

[0045] Accordingly, by early screening of patients who may respond effectively to treatment and those who are non-responsive, side effects and unnecessary cost consumption can be reduced, thereby maximizing the effectiveness of treatment in clinical settings.

[0046] The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0047] FIG. 1 illustrates an exemplary procedure for a method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention.

[0048] FIGS. 2A to 2K illustrate the results of gene expression analysis of CD4+ T cells for biomarker selection provided in a method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention.

[0049] Figures 3a to 3i illustrate the results of analysis of differentiation and anticancer activity of CD8+ Trm by HPV infection for biomarker selection provided in a method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention.

[0050] FIGS. 4A to 4I illustrate biomarker selection results provided in a method for providing information on immunotherapy in HPV-induced cancer according to various embodiments of the present invention.

[0051] FIGS. 5A to 5G illustrate the results of biomarker-based immunotherapy analysis provided in a method for providing information on immunotherapy in HPV-induced cancer according to various embodiments of the present invention.

[0052] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined solely by the scope of the claims.

[0053] The term "marker composition" or "biomarker" as used herein is an indicator that can identify biological changes using proteins, DNA, RNA (ribonucleic acid), metabolites, etc.

[0054] In the present specification, a “biomarker” may be a marker for diagnosing cancer progressed by HPV infection, and further for predicting treatment response to immunotherapy.

[0055] At this time, HPV infection may be indicated as “HPV positive” or “HPV+” within the specification, and HPV non-infection may be indicated as “HPV negative” or “HPV-”.

[0056] In various embodiments of the present invention, tissue-resident memory T cell-specific KLRB1 mRNA or its protein is presented as a marker for predicting therapeutic response to immunotherapy.

[0057] Here, "tissue-resident memory T cells" are a type of T cell that resides in tissues and is responsible for immune memory. They exist uniquely within tissues and can play a role in inducing rapid and strong immune responses.

[0058] In this specification, “tissue-resident memory T cells” means “T rm " can be used interchangeably.

[0059] As used herein, the term "tissue-resident memory T cell specific" may refer to a characteristic or property that is unique to tissue-resident memory T cells.

[0060] That is, “tissue-resident memory T cell-specific KLRB1 mRNA or protein thereof” can be interpreted as KLRB1 mRNA or protein thereof expressed in tissue-resident memory T cells.

[0061] T cells with high expression levels of the KLRB1 gene encoding CD161 (or KLRB1 protein) in HPV-infected individuals, especially in HPV-infected head and neck cancer individuals rm cells (resident memory T cells) appear, which are "CD161+ T rm can be defined as "cells". At this time, CD161+ T rm The cells may be associated with anticancer activity.

[0062] In particular, CD161+ T rm The cells may be associated with tertiary lymphoid structures (TLS), which may enhance the persistence and aggressiveness of immune cells.

[0063] More specifically, CD161+ T rm Cells can be made less responsive to immunotherapy, i.e., immune checkpoint inhibitors that can suppress the cytotoxic effects of immune cells via the tumor microenvironment, by weakening the formation of TLS.

[0064] That is, in the present specification, the biomarker is the KLRB1 gene or its protein encoding the CD161 receptor specifically for tissue-resident memory T cells, preferably CD161+ T rm It can mean the cell itself.

[0065] In various embodiments of the present invention, the biomarker is T rmSpecific CD103, CD69, CD49a (Integrin α1), CD44, CD101, CD127, CXCR6 and CD8 can be used selectively.

[0066] For example, the biomarker is CD161+CD103+ T rm cells, CD161+CD8+ T rm cells and CD161+CD103+CD8+ T rm At least one of the cells, but not limited to, CD161 and the aforementioned T rm It could be one of many combinations of specific biomarkers.

[0067] Meanwhile, the receptor protein CD161 may be associated with its ligand CLEC2D, which can bind to CD161 on NK cells and tissue-resident memory T cells and inhibit their cytotoxic function.

[0068] At this time, T with high expression of KLRB1 rm Cells may have reduced interaction with B cells associated with antitumor activity within cancer tissues, which may be due to CD161+ T rm This may suggest that TLS activity can be reduced by suppressing CLEC2D expression in these B cells.

[0069] Therefore, when the expression of CLEC2D, a protein that mediates the interaction between tissue-resident memory T cells and B cells expressed on the surface of CD161, is below a predetermined level, TLS activity is reduced, which may suppress the immune efficacy of the tumor and negatively affect the elimination of tumor cells.

[0070] Accordingly, in various embodiments of the present invention, optionally, in an HPV-positive head and neck cancer subject, mRNA or protein of CLEC2D, and further, immune cells expressing CLEC2D positively and CD20 positively, a B cell-specific marker, i.e., CLEC2D+CD20+ B cells, can be presented as markers for predicting the treatment response to immunotherapy.

[0071] In a specific example, if the level of CLEC2D+CD20+ B cells is below a predetermined level, the subject may be determined to have a negative treatment response to immunotherapy.

[0072] The term "HPV-induced cancer" as used herein refers to cancer that progresses due to HPV infection. The HPV-induced cancer may be at least one of oropharyngeal cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, and vulvar cancer caused by HPV infection.

[0073] Preferably, the HPV-induced cancer may be, but is not limited to, oropharyngeal cancer caused by HPV infection.

[0074] Immunotherapy may be applied to treat these HPV-induced cancers.

[0075] The term "biological sample" as used herein may include, but is not limited to, at least one selected from the group consisting of tissue, peripheral blood, serum, and plasma.

[0076] As used herein, the term "immunotherapy" may refer to a treatment designed to block the mechanisms that prevent immune cells from attacking cancer cells. In various embodiments, immunotherapy may be neoadjuvant immunotherapy. However, it is not limited thereto, and immunotherapy may include various therapies that alter the tumor microenvironment to enhance the immune response, thereby reducing tumor size or eliminating cancer cells prior to surgery.

[0077] For example, immunotherapy may include anti-PD-1 therapy, which blocks the binding of PD-L1 and PD-L2, proteins on the surface of cancer cells, to PD-1, a protein on the surface of T cells. When the immunotherapy agent binds to a specific receptor on a T cell, it can inhibit the T cell's ability to evade cancer cells.

[0078] Accordingly, in this specification, “immunotherapy” may be interpreted to have the same meaning as “immuno-oncology therapy” or “immune checkpoint inhibition.”

[0079] The term "positive therapeutic response" as used herein may mean that the binding reaction of receptors and ligands on the surface of immune cells is blocked by immunotherapy, i.e., an immune checkpoint inhibitor, or that the secretion ability for cytotoxic substances such as cytokines is enhanced due to the blocking.

[0080] More specifically, a positive treatment response to immunotherapy in HPV-induced cancers may include any response associated with remission or good prognosis of HPV-induced cancers, such as enhanced immune response by immunotherapy altering the tumor microenvironment, reduction in tumor size, or removal of cancer cells before surgery.

[0081] Conversely, "treatment-negative" may mean that immunotherapy, i.e., immune checkpoint inhibitors, fail to block the binding of receptors and ligands on the surface of immune cells, or that the resulting blockade does not enhance the secretion of cytotoxic substances such as cytokines. However, without limitation, a treatment-negative response may also include a decrease in any response associated with the worsening of HPV-induced cancer or a poor prognosis following immunotherapy.

[0082] The term "positive or +" as used herein may mean that a marker that can characterize the cell is expressed. For example, in the case of tissue-resident memory T cells in an HPV-infected individual, CD161 is expressed on the cell surface, and accordingly, CD161-expressing tissue-resident memory T cells are referred to as CD161+ tissue-resident memory T cells or CD161+ T rm can be denoted as; however, it is not limited thereto, and excluding the notation for '+', CD161 tissue-resident memory T cells or CD161 T rm It can also be written as .

[0083] The term "about" as used herein refers to a typical error range for each value, which is readily known to those skilled in the art. References herein to "about" a value or parameter include embodiments relating to that value or parameter itself.

[0084] The term "patient or subject" as used herein is used interchangeably and refers to any single animal, more preferably a mammal (including non-human animals such as cats, dogs, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) in need of treatment. In certain embodiments, the patient herein is a human. The patient may be a "cancer patient," i.e., a person suffering from one or more symptoms of cancer, at risk of suffering from cancer, or suffering from cancer.

[0085] As used herein, the terms "level of expression" and "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 (e.g., genetically encoded and / or epigenetic information) is converted into a structure that exists and functions in said cell.

[0086] Thus, as used herein, "expression" refers to the transcription of a polynucleotide, translation of a polypeptide, or further a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, a translated polypeptide, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing (alternative splicing) or from degraded transcripts, or from post-translational processing of a polypeptide, e.g., by proteolysis.

[0087] Meanwhile, an "expressed gene" includes one that is transcribed into a polynucleotide as mRNA and then translated into a polypeptide, as well as one that is transcribed into RNA but not translated into a polypeptide (e.g., transfer and ribosomal RNAs). The expression level for more than one gene of interest can be determined by aggregation methods known to those skilled in the art and also disclosed herein, including, for example, by calculating the median or average of the expression levels of all genes of interest. Prior to aggregation, the expression level of each gene of interest can be normalized using statistical methods known to those skilled in the art and also disclosed herein, including, for example, normalizing to the expression levels of one or more housekeeping genes, or normalizing to the total library size, or normalizing to the median or average expression level value across all genes measured. In some cases, prior to aggregation across multiple genes of interest, the normalized expression levels of each gene of interest may be normalized using statistical methods known to those skilled in the art and also disclosed herein, including, for example, by calculating a Z-score of the normalized expression levels of each gene of interest.

[0088] In various embodiments, the therapeutic response to immunotherapy can be determined by the normalized RNA average of KLRB1 based on the expression level of the housekeeping gene.

[0089] Here, the predetermined level for the normalized RNA mean value of KLRB1 may be, but is not limited to, 2 to 3 [mean(exp(x)-1].

[0090] For example, if the RNA mean value of KLRB1 is 3 [mean(exp(x)-1] or more, the treatment response to immunotherapy may be determined to be negative, and if the RNA mean value is less than 3 [mean(exp(x)-1], the treatment response may be determined to be positive.

[0091]

[0092] Hereinafter, with reference to FIG. 1, a method for providing information on immunotherapy in HPV-induced cancer according to various embodiments of the present invention will be described in detail.

[0093] Referring to FIG. 1, a method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention includes the steps of measuring the level of a biomarker of KLRB1 and the level of a tissue-resident memory T cell-specific biomarker for a biological sample isolated from an individual (S110) and determining a characteristic associated with immunotherapy based on the measured expression level (S120).

[0094] In the step of measuring the expression level (S110), the mRNA level of tissue-resident memory T cells-specific KLRB1 or the expression level of their proteins can be measured to predict the therapeutic response to immunotherapy of HPV-induced cancer, as high expression of KLRB1 in tissue-resident memory T cells can reduce the interaction between immune cells in the tumor microenvironment.

[0095] According to a feature of the present invention, in the step (S110) of measuring the expression level, at least one T among CD103, CD69, CD49a (Integrin α1), CD44, CD101, CD127, CXCR6 and CD8 rm Expression levels for specific markers can be measured.

[0096] In various embodiments, in the step (S110) of measuring the expression level, CD161+ T rm Cells can be established as biomarkers for predicting HPV+ treatment response.

[0097] For example, in the step (S110) of measuring the expression level, various CD161+ T cells such as CD161+CD8+ T cells, CD161+CD103+ T cells and CD161+CD8+CD103+ T cells are measured. rm The level of cells can be measured.

[0098] According to another feature of the present invention, in the step of measuring the expression level (S110), the level of CLEC2D+CD20+ B cells may be measured.

[0099] At this time, in the method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention, CD161+CD8+ T cells, CD161+CD103+ T cells and CD161+CD8+CD103+ T cells, and further CLEC2D+ B cells and CLEC2D+CD20+ B cells provided as biomarkers may be immune cells that control an immune response to a tumor, i.e., an anticancer effect, through interaction in a tumor microenvironment.

[0100] According to another feature of the present invention, in the step of measuring the expression level (S110), the number of CD161+CD8+ T cells, CD161+CD103+ T cells and CD161+CD8+CD103+ T cells, and further CLEC2D+ B cells and CLEC2D+CD20+ B cells, i.e., the number of CD161+ tissue-resident memory T cells or CLEC2D+ B cells per unit area in a biological sample isolated from an individual can be measured.

[0101] According to another feature of the present invention, in the step of measuring the expression level (S110), the transition probability into CD161+ tissue-resident memory T cells may also be measured.

[0102] According to another feature of the present invention, in the step of measuring the expression level (S110), the area of ​​the TLS for the sample of the individual and the normal control may be measured.

[0103] The subject may have at least one cancer caused by HPV infection selected from among oropharyngeal cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, and vulvar cancer.

[0104] Preferably, the entity may be, but is not limited to, oropharyngeal cancer due to HPV infection.

[0105] Furthermore, the biological sample may include at least one selected from the group consisting of tissue, peripheral blood, serum, and plasma, and the immunotherapy may be neoadjuvant immunotherapy. However, the immunotherapy may not be limited thereto, and may include various therapies that alter the tumor microenvironment to enhance the immune response, thereby reducing tumor size, eliminating cancer cells prior to surgery, or causing changes in the tumor microenvironment that activate the immune response or inhibit tumor growth.

[0106] Meanwhile, in the step of measuring the expression level (S110), the expression level of the mRNA of the biomarker gene can be measured by any one method selected from the group consisting of fluorescence in situ hybridization (FISH), droplet digital PCR (dd-PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chip.

[0107] Preferably, in the step of measuring the expression level (S110), two genes (KLRB1 and T rm The FISH method may be selected to confirm that marker genes) are expressed simultaneously in the same cell, but is not limited thereto.

[0108] For example, the levels of various immune cells, including CD161+ tissue-resident memory T cells, can be measured by at least one of western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry (IHC), immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), and protein chip.

[0109] Furthermore, the expression level of the biomarker protein can be measured by at least one method selected from the group consisting of western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry (IHC), immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), and protein chip, but is not limited thereto, and can include various methods by which proteins and mRNAs can be measured.

[0110] At the step (S120) where a characteristic associated with immunotherapy is determined, if the level of mRNA or protein encoded by the mRNA of tissue-resident memory T cells specific for the individual is equal to or greater than a predetermined level, the individual may be determined to have a negative therapeutic response to immunotherapy, otherwise, the individual may be determined to have a positive therapeutic response.

[0111] For example, in the step (S120) where a characteristic associated with immunotherapy is determined, if the mean RNA value of tissue-resident memory T cell-specific KLRB1 is 3 [mean(exp(x)-1] or more, the treatment response to immunotherapy may be determined to be negative, and if the mean RNA value is less than 3 [mean(exp(x)-1], the treatment response may be determined to be positive.

[0112] According to another embodiment of the present invention, whether a treatment response to immunotherapy will occur in HPV-induced cancer can be predicted based on the level of immune cells.

[0113] According to a feature of the present invention, in the step (S120) where the characteristic associated with immunotherapy is determined, the level of CD161+ tissue-resident memory T cells for the biological sample is 15cells / mm 2 If this is the case, the subject can be determined to have a negative response to immunotherapy, otherwise it can be determined to have a positive response to treatment.

[0114] According to another feature of the present invention, in the step (S120) where a characteristic associated with immunotherapy is determined, the level of CD161+ tissue-resident memory T cells for the biological sample is 15 cells / mm 2 In this case, blockade of CD161 based on the inhibitory immune checkpoint molecule CD161 may be determined as an immunotherapy.

[0115] According to another feature of the present invention, in the step (S120) where a characteristic associated with immunotherapy is determined, if the level of CD161+ tissue-resident memory T cells for the measured individual shows an increased level compared to the level of CD161+ tissue-resident memory T cells for a normal control sample, the individual can be determined to have a negative responsiveness to immunotherapy.

[0116] According to another feature of the present invention, in the step (S120) where a characteristic associated with immunotherapy is determined, if the level of CLEC2D+ B cells for the measured individual is reduced compared to the level of CLEC2D+ B cells for a normal control sample, the individual can be determined to have a negative response to immunotherapy.

[0117] According to the above procedure, the method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention can provide information to enable early prediction of treatment response to immunotherapy, particularly neoadjuvant immunotherapy, for an individual by measuring the levels of various markers. That is, the method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention can determine and provide treatment response with high predictive power by using tissue-resident memory T cell-specific KLRB1, preferably CD161+ tissue-resident memory T cells, and optionally CLEC2D, CLEC2D+ B cells.

[0118] Meanwhile, the present invention can provide a kit for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention, based on the method for providing information on immunotherapy in HPV-induced cancer described above.

[0119] At this time, the kit for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention may include a formulation configured to measure the expression level of mRNA of tissue-resident memory T cell-specific KLRB1 or a protein encoded by the mRNA, or the level of CD161+CD8+ T cells, CD161+CD103+ T cells, and CD161+CD8+CD103+ T cells, for a biological sample isolated from an individual.

[0120] According to a feature of the present invention, the formulation comprises: T rm Antibodies targeting the CD161 surface protein of cells, or T rm It may be a bispecific antibody comprising the CD161 and CD103 surface proteins of the cell, but is not limited thereto.

[0121] Example 1: Gene expression analysis of CD4+ T cells

[0122] Hereinafter, the results of gene expression analysis of CD4+ T cells for biomarker selection provided in a method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention will be described with reference to FIGS. 2a to 2k.

[0123] First, a total of 89,339 immune / stromal cells were clustered in the tumor samples based on single-cell transcriptome profiles.

[0124] Referring to Figure 2a, the integrated data through sample-wise normalization and cell cycle effect regression is depicted, and a total of 20 distinct immune cell clusters were identified and annotated based on canonical marker expression.

[0125] At this time, clusters showed a balanced distribution across HPV infection status (HPV+ vs. HPV-) or scRNA-seq platform technology (3' vs. 5' end sequencing), which may indicate that technical bias was properly compensated for by the absence of patient bias.

[0126] Referring to Figure 2b, the composition of immune / stromal cells in HPV+ tumors and HPV- tumors was compared, and T fh The proportion of CD20+ B cells and TLS B cells was found to be significantly higher in HPV+ tumors than in HPV- tumors (p < 0.05, two-tailed Mann-Whitney U test). At this time, CD20+ B cells and TLS B cells also showed higher proportions in HPV+ tumors than in HPV- tumors, but this was not statistically significant.

[0127] At this time T fh , activated B cells, and TLS were increased in HPV+ tumors compared to HPV- tumors, which may positively correlate with prognosis. Therefore, these results may suggest that immune regulation by HPV infection in oropharyngeal cancer may differ from that in other HNSCC subtypes.

[0128] In particular, the above results indicate that intratumoral Tfh and a high proportion of B cells may contribute to a favorable clinical outcome in HPV+ oropharyngeal cancer. Here, T cells in HPV+ tumors fh A high proportion of CD4+ T naive to T following viral infection fh This may be due to the enhanced differentiation of the .

[0129] Referring to Figure 2c, CD4+ T cells were classified into sub-clusters: CD4+ naive T cells (CD4 Tn), CD4+ memory T cells (CD4 Tm) 25, and CD4+ effector T fh Cells (T fh eff), and CD4+ exhausted T fh Cells (T fh ex) Four subgroups were determined.

[0130] Referring to Figure 2d, trajectory analysis using Slingshot identified two lineages of CD4+ T cell differentiation starting from CD4 Tn. More specifically, T fh Subpopulation (CD4 Tn-> T fh eff-> T fh ex) and the results show differentiation into Tm subgroups (CD4 Tn-> CD4 Tm).

[0131] Referring to Figure 2e, T fh The results of differential expression gene analysis of eff cells are shown. At this time, T fh T in eff fh Expression of the KLRB1 gene appears to be upregulated along the trajectory. KLRB1 in CD4+ T eff cells may be associated with favorable clinical outcomes in HPV+.

[0132] Referring to Figures 2f and 2g together, NK cells and T fh High expression of KLRB1 was observed in .

[0133] Referring further to Figures 2h and 2i, expression analysis of CLEC2D (LLT1), which encodes a ligand for CD161, shows that it is expressed in various immune cell types, and in particular, for CD20+ B cells and TLS B cells, expression of CLEC2D appears to be restricted to HPV+ tumors.

[0134] Furthermore, KLRB1 is a T-cell in HPV+ tumors. rm In HPV- tumors, the CD161 receptor on T cells was upregulated compared to HPV- tumors, and these results suggest that the CD161 receptor on T cells is required for TLS activity in HPV+ oropharyngeal cancer patients. rm -BT fh It may contribute to cooperation and suggest that it is associated with strong anticancer responses and favorable clinical outcomes.

[0135] Referring to Figure 2j, differences in cell differentiation or homeostasis of cell types identified using RNA velocity analysis are depicted. More specifically, HPV+ tumors are characterized by a higher number of T cells in CD4 Tn fh It appears to have a higher RNA velocity.

[0136] Referring to (a) and (b) of Fig. 2k, the results of the comparison of transition probabilities between cell states show that CD4 Tn has a lower persistence probability and T in HPV+ tumors compared to HPV- tumors. fh It appears that eff has a higher probability of transition. At this time, T fh may be associated with anticancer activity, and T fh A higher probability of conversion to eff could imply clinical benefit in patients with HPV-induced diseases, particularly oropharyngeal cancer.

[0137]

[0138] Example 2: CD8+ T cells induced by HPV infection rm Differentiation and anticancer activity

[0139] Hereinafter, with reference to FIGS. 3a to 3i, CD8+ T cells due to HPV infection for biomarker selection provided in a method for providing information on immunotherapy in HPV-induced cancer according to one embodiment of the present invention rm The results of the differentiation and anticancer activity analysis are explained.

[0140] Referring to Figure 3a, three CD8+ T cell subpopulations among 20 immune cell populations were selected to investigate the modulation of tumor-infiltrating CD8+ T cells by HPV infection and their impact on clinical outcomes.

[0141] Next, referring to Figure 3b, four new subpopulation clusters representing different cell states were generated. More specifically, clusters of stem-like CD8+ T cells (CD8 Tstem), effector-like CD8+ T cells (CD8 Teff), interferon-alpha-stimulated CD8+ T cells (CD8 Tstim), and exhausted CD8+ T cells (CD8 Texh) were generated.

[0142] Referring to Figure 3c, the progenitor module score decreases and the terminal differentiation score increases as one moves from CD8 Tstem to CD8 Texh.

[0143] More specifically, in the HPV+ group, the precursor scores for CD8 Teff and CD8 Texh were significantly decreased, whereas the terminal differentiation scores for CD8 Tstem, CD8 Tstim, and CD8 Texh were significantly increased. These results may indicate that CD8+ T cell differentiation is more rapid in the HPV+ group than in the HPV- group.

[0144] That is, this may suggest that HPV infection accelerates the differentiation of CD8+ T cells, which may enhance the immune response against tumors and lead to better clinical outcomes.

[0145] Referring to Figure 3D, the results of RNA velocity analysis to quantify the tendency of CD8 Tstem to CD8 Texh to transition are depicted. More specifically, a distinct difference in cell state transition is revealed between the HPV+ and -negative groups.

[0146] In particular, referring to (a) and (b) of Fig. 3e together, CD8+ T cells in the HPV+ group appear to have a significantly higher probability of transitioning from CD8 Tstem to CD8 Teff and to CD8 Texh than those in the HPV- group.

[0147] CD8 Texh are mostly terminally differentiated cytotoxic T cells rm Considering that HPV infection is composed of cytotoxic T cells, these results suggest that HPV infection is caused by cytotoxic T cells. rm This may suggest that accelerating differentiation into induced favorable clinical outcomes.

[0148] Referring further to Figure 3f, the comparison of clonal expansion of T cells between the HPV+ and negative groups shows that the majority of CD8 Teffs and CD8 Texhs were clonally expanded.

[0149] Referring to Figure 3g, CD8+ T cells for all four subpopulations appear to exhibit greater clonal expansion in the HPV+ group.

[0150] This may suggest that the presence of expanded infiltrating T cells recognizing HPV antigens provides additional antitumor activity in HPV+.

[0151] In particular, referring to Figure 3h, the clone-extended T rmCells in the HPV+ group exhibited higher signature scores for PD-L1 signaling, IFN-gamma, MHC class 2, and T-cell-mediated immunity. This may be attributed to their stronger antitumor activity and favorable treatment outcomes.

[0152] Meanwhile, better clinical outcomes due to HPV infection may also be associated with changes in cell-cell interactions (CCIs) that are important for the anti-tumor activity of immune cells. For example, BT fh Wow BT rm Interactions may be important for TLS formation and activity.

[0153] The number of detected ligand-receptor pairs can correspond to the probability of cell-to-cell interactions, and the number of estimated significant ligand-receptor pairs is shown in Fig. 3i.

[0154] More specifically, B cell subpopulations (TLS B cells and CD20+ B cells) and T cell subpopulations (T fh , a sharp increase in the interaction between CD8 Tstem, CD8 Teff, and CD8 Texh is observed.

[0155] These results suggest that some B cells and T cells may have favorable immuno-oncological outcomes in HPV+ fh and T rm This may suggest that increased interaction is associated with this.

[0156] Example 3: Establishment of the KLRB1 biomarker

[0157] Hereinafter, the biomarker selection results provided in the method for providing information on immunotherapy in HPV-induced cancer according to various embodiments of the present invention will be described with reference to FIGS. 4a to 4i.

[0158] Referring to Figures 4a and 4b, the results of DEG analysis between the HPV+ group and the negative group for each cell subpopulation are first shown.

[0159] More specifically, this identified key molecules in CD8+ T cells that modulate HPV-associated clinical outcomes, with the NK and MAIT cell marker KLRB1 being upregulated in three major CD8+ T cell subpopulations: CD8 Tstem, CD8 Teff, and CD8 Texh.

[0160] Referring to Figure 4c, the results of an analysis of dynamic changes in gene expression according to CD8+ T cell differentiation are depicted. More specifically, trajectory analysis was performed using Slingshot, and the proportional changes in each CD8+ T cell subpopulation over pseudo time were investigated.

[0161] Next, changes in the expression of genes involved in the regulation of cytotoxic CD8+ T cells were investigated.

[0162] Figure 4d shows the comparison of the expression levels of the following genes between HPV-positive and -negative tumors at three stages of CD8+ T cell differentiation. These genes are comprised of co-suppressive (PDCD1, LAG3, HAVCR2, TIGIT) and co-stimulatory (CD27, CD82, TNFRSF4, TNFRSF9, CXCR3) immune checkpoint molecules, effector molecules (GZMA, GZMB, PRF1, IFNG, FASLG), CD8+ T cell stemness regulation (TCF7, IL7R, STAT4, FOXP1, GPR183) and exhaustion (TOX, ENTPD1, ​​BATF, PRDM1), and cell signaling (CCL3, CCL5, CXCL13).

[0163] More specifically, co-inhibitory immune checkpoint molecules and factors that promote T cell exhaustion are shown to be upregulated in CD8 Tstem, CD8 Teff, and CD8 Texh.

[0164] In particular, the immune checkpoint molecule KLRB1 was found to increase in expression over time in the HPV+ group, but not in the HPV-negative group.

[0165] These results indicate that CD8+ T cells, particularly T rm This may suggest that CD161 expression in is induced by HPV infection.

[0166] Referring to Figure 4e together, for the HPV-positive group, along with the mock time of CD8+ T cells, KLRB1-low T rm showed higher expression of genes involved in antiviral activity (IRF1, IRF7, OASL, IFI27, IFI6 IFIT1 and ISG20), of which IFIT1 and ISG20 have not yet been reported to be associated with HPV infection.

[0167] These results are T rm The low expression level of CD161 indicates the immunogenicity of the TME, which may be partially associated with the favorable prognostic effect in HPV+.

[0168] Referring to Figure 4f, KLRB1 expression induced by HPV infection induces cytotoxic T in tumors. rm To analyze whether differentiation can be transformed into , the results comparing RNA velocities between KLRB1-high and KLRB1-low groups of CD8+ T cells from HPV+ patients are shown.

[0169] Specifically, referring to Figure 4g, among HPV+ patients, the KLRB1-low group showed a significantly higher probability of transitioning from CD8 T stem to CD8 T eff and then to CD8 T exh compared to the KLRB1-high group. This result is consistent with the results comparing the RNA kinetics of CD8+ T cells in the HPV-infected and -uninfected groups. Therefore, it suggests that further dividing HPV-infected CD8 T cells by the level of KLRB1 expression may serve as a criterion for inducing meaningful clinical differences.

[0170] CD8 Texh are mostly terminally differentiated cytotoxic T cells rm Considering that the CD161 receptor is composed of cytotoxic T cells, these results suggest that in HPV+, the CD161 receptor is cytotoxic T rm This may suggest that it inhibits differentiation and reduces anticancer activity.

[0171] That is, expression of the KLRB1 gene may be associated with immunotherapy in HPV+, especially HPV+ oropharyngeal cancer individuals.

[0172] Referring to Figure 4h, the results comparing cell-cell interactions between the KLRB1-high group and the KLRB1-low group for HPV+ tumors are shown.

[0173] More specifically, the interaction between T and B cell subpopulations in the KLRB1-high group was significantly reduced compared to the HPV+ group in the KLRB1-low group. This result is also consistent with previous comparisons of HPV-positive and -negative cell interactions, suggesting that further dividing the HPV-positive group based on KLRB1 expression in Trm may be clinically meaningful.

[0174] Referring to Figure 4i, clonal expansion analysis using single-cell TCR sequencing data illustrates immune differences between the KLRB1-high and KLRB1-low groups of HPV+ patients. More specifically, in HPV+ patients, the KLRB1-low group exhibits a higher number of clonally expanded CD8+ T cells compared to the KLRB1-high group.

[0175] This result is T rm This may suggest that CD161 may reduce anticancer activity in HPV+.

[0176] Referring to Figure 4j, the three major cellular components of TLS are T fh , T rm , and the results of expression analysis of ligands KLRB1 and CLEC2D, CD161, in B cells (CD20+ B and TLS B) are shown.

[0177] More specifically, KLRB1 is HPV+ KLRB1-low T rm and KLRB1-high T rm T of the tumor fh While CLEC2D expression in B cells is highly expressed in KLRB1-low T rm appears to be limited to tumors with HPV infection. These results suggest that HPV infection is T rm Induces CD161 expression in some of these CD161+ T cells, and then rm This may suggest that by suppressing CLEC2D expression in B cells, it may inhibit the interaction with B cells and consequently reduce TLS activity associated with anticancer activity.

[0178] Therefore, CD161 may be established as a biomarker for predicting treatment response, as it plays a dominant role in regulating antitumor activity in HPV+ individuals, especially in HPV+ oropharyngeal cancer individuals.

[0179] [Correction under Rule 91 19.07.2024] Referring further to Figures 4k and 4l, the top 7 out of 14 in the violinplot for HPV+ individuals are T rm They were classified into the KLRB1-high group, and these groups were shown to have a normalized RNA average value of 2.4 [mean(exp(x)-1] or higher.

[0180] Accordingly, the T of the object rm If the normalized RNA mean value of KLRB1 is 2 to 3 [mean(exp(x)-1] or higher, the TLS activity may be reduced due to high expression of KLRB1, resulting in a lower therapeutic response than individuals without such a high expression.

[0181] That is, in various embodiments, T rm Based on the normalized RNA mean value of KLRB1, a positive or negative response to immunotherapy may be determined.

[0182] However, this is not limited to this, and therapeutic responsiveness to immunotherapy can be determined based on the level of CLEC2D in B cells.

[0183]

[0184] Example 4: Predicting the Efficacy of Tissue-Residual Memory T Cell-Specific KLRB1 Biomarker and Immunotherapy

[0185] Hereinafter, the results of biomarker-based immunotherapy analysis provided in the method for providing information on immunotherapy in HPV-induced cancer according to various embodiments of the present invention are described with reference to FIGS. 5a to 5g.

[0186] First, in an independent cohort of patients with oropharyngeal cancer, T fh and T rm To validate HPV-induced KLRB1 expression in HNSCC samples from the TCGA cohort, 70 tumor samples derived from the oropharynx were selected.

[0187] Finally, 50 HPV+ and 20 HPV- oropharyngeal cancer samples were identified from the TCGA cohort.

[0188] Referring to Figure 5a, T in HPV+ tumor samples compared to HPV- tumor samples fh The expression correlation between markers (CD4 and CXCR5) and KLRB1 was found to be significantly high.

[0189] Referring to Figure 5b, T was higher in HPV+ tumors than in HPV- tumors. rm The expression correlation between markers (CD8 and ITGAE) and KLRB1 appears to have higher statistical significance.

[0190] These results suggest that intratumoral T cells are involved in HPV-induced cancers, particularly HPV-induced oropharyngeal cancers. fh and T rm This may suggest that HPV-induced KLRB1 expression occurs in .

[0191] Referring to Figure 5c, immunofluorescence analysis results based on tissue samples obtained from 13 HPV+ oropharyngeal cancer patients, including 8 patients who received neoadjuvant immunotherapy, are shown. More specifically, common fluorescence reactions of CD161+, CD103+, and CD8+ cells are confirmed. This indicates that T rm may suggest CD161 expression.

[0192] In particular, referring to Figure 5d, T in HPV+ tumors compared to HPV- tumors fh The expression correlation between the marker and the B cell marker (CD79A) was found to be significantly higher, and with further reference to Figure 7e, T in HPV+ tumors compared to HPV- tumors rm The expression correlation between the marker and the B cell marker was found to be significant.

[0193] These results indicate that T in HPV+ tumors fh and T rmThis suggests that B cell recruitment mediated by this CD161-CLEC2D interaction may increase the possibility of TLS formation, thereby enhancing antitumor activity.

[0194] In other words, B cells and T cells in HPV-positive cancer rm Cells and T fh Increased cell-cell interactions may lead to increased antitumor activity.

[0195] Meanwhile, gene expression analysis results showed that KLRB1-high T rm These HPV+ tumors have TLS B cells that express only CLEC2D at low levels, which may suggest reduced TLS activity.

[0196] At this time, considering that TLS plays an important role in immune checkpoint blockade treatment response, CD161+ T rm This may negatively affect the efficacy of immunotherapy.

[0197] Meanwhile, referring to Figures 5f and 5g, CD161+ T rm To verify the impact of this immunotherapy, we compared the number of intratumoral CD161+ T cells in eight HPV+ oropharyngeal cancer patients who received neoadjuvant immunotherapy. rm The results of analyzing the relationship between density (expressed as CD161+CD103+CD8+ cells) and changes in tumor size are shown.

[0198] More specifically, intratumoral CD161+ T rm A significant inverse correlation was observed, with higher density leading to smaller tumor size.

[0199] These results indicate that CD161+ T cells are present in the TME of patients with HPV+ oropharyngeal cancer. rm This may suggest that density could be used as a biomarker of immunotherapy efficacy.

[0200] That is, in patients with HPV+, especially HPV-induced oropharyngeal cancer, CD161+ T rmTreatment responsiveness to immunotherapy can be determined based on the levels of cellular biomarkers.

[0201] Accordingly, at least one cell among CD161+CD8+ T cells, CD161+CD103+ T cells and CD161+CD8+CD103+ T cells, preferably CD161+CD8+ T rm cells, CD161+CD103+ T rm cells and CD161+CD8+CD103+ T rm At least one of the cells may serve as a biomarker for predicting immunotherapy treatment response of HPV-induced cancer.

[0202] However, it is not limited to this, and the biomarker of CD161 (or KLRB1) and at least one of CD103, CD69, CD49a (Integrin α1), CD44, CD101, CD127, CXCR6 and CD8 rm A more diverse combination of biomarkers for cells may serve as biomarkers for predicting treatment response in HPV-induced cancers.

[0203] That is, the present invention can provide information to quickly determine whether to proceed with a selected immunotherapy by predicting an early treatment response of an immunotherapy for an individual using a biomarker.

[0204] Accordingly, by early screening of patients who may respond effectively to treatment and those who are non-responsive, side effects and unnecessary cost consumption can be reduced, thereby maximizing the effectiveness of treatment in clinical settings.

[0205] Although the embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be implemented 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 it, 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 exemplary in all aspects and not restrictive. The protection scope of the present invention should be interpreted by the claims below, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

[0206] [National Research and Development Project Supporting This Invention]

[0207] [Project ID]1711154178

[0208] [Assignment Number] 2019M3A9B6065231

[0209] [Ministry Name] Ministry of Science and ICT

[0210] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0211] [Research Project Name] Bio-Medical Technology Development (R&D)

[0212] [Research Project Name] Clinical Trial of Precision Immuno-Oncology Treatment Based on Single-Cell Multi-Omics

[0213] Usability verification

[0214] [Name of Project Performing Organization] Yonsei University

[0215] [Research Period] January 1, 2023 - December 31, 2023

[0216] [National Research and Development Project Supporting This Invention]

[0217] [Project ID]1711162819

[0218] [Assignment Number] 2021R1A2C2094629

[0219] [Ministry Name] Ministry of Science and ICT

[0220] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0221] [Research Project Name] Basic Research Project in Science and Engineering

[0222] [Research Project Title] Identification of T-cell exhaustion-inducing regulons using a rapid interactive screening (RIS) platform and discovery of lead compounds for overcoming immune checkpoint inhibitor-refractory head and neck cancer.

[0223] [Name of Project Performing Organization] Yonsei University

[0224] [Research Period] March 1, 2023 - February 29, 2024

[0225] [National Research and Development Project Supporting This Invention]

[0226] [Project ID]20022947

[0227] [Assignment Number] 20022947

[0228] [Ministry Name] Ministry of Trade, Industry and Energy

[0229] [Name of Project Management (Specialist) Institution] Korea Institute of Industrial Technology Evaluation and Planning

[0230] [Research Project Name] Bioindustry Core Technology Development Project

[0231] [Research Project Name] Human Microbiome for Enhancing the Efficacy of Immunotherapy in Cancer Patients

[0232] Development of therapeutic technology

[0233] [Name of the project performing organization] Yonsei University Industry-Academic Cooperation Foundation

[0234] Research Period: April 1, 2023 - December 31, 2023

[0235] [National Research and Development Project Supporting This Invention]

[0236] [Project ID]1711133032

[0237] [Assignment Number] 2019M3A9B6065192

[0238] [Ministry Name] Ministry of Science and ICT

[0239] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0240] [Research Project Name] Bio-Medical Technology Development (R&D)

[0241] [Research Project Name] Single-cell Immune Reactome and Inducer Discovery in the Cancer Microenvironment

[0242] Development of multi-omics integrated analysis technology

[0243] [Name of Project Performing Organization] Yonsei University

[0244] Research Period: January 1, 2021 - December 31, 2021

[0245] [National Research and Development Project Supporting This Invention]

[0246] [Project ID]1711108917

[0247] [Assignment Number] 2018R1A5A2025079

[0248] [Ministry Name] Ministry of Science and ICT

[0249] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0250] [Research Project Name] Group Research Support (R&D)

[0251] [Research Project Name] Center for Systems Medicine for Chronic and Intractable Diseases

[0252] [Name of Project Performing Organization] Yonsei University

[0253] [Research Period] March 1, 2020 - February 28, 2021

Claims

1. A marker composition for predicting response to immunotherapy in HPV-induced cancer, comprising mRNA of tissue-resident memory T cell (Trm)-specific KLRB1 or a protein encoded by the mRNA, as a marker for predicting response to immunotherapy in HPV-induced cancer.

2. In paragraph 1, A marker composition for predicting response to immunotherapy in HPV-induced cancer, wherein the HPV-induced cancer is at least one selected from oropharyngeal cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, and vulvar cancer caused by HPV infection.

3. Method for providing information on immunotherapy for HPV-induced cancer. A step of measuring the expression level of mRNA of tissue-resident memory T cell-specific KLRB1 or a protein encoded by said mRNA for a biological sample isolated from an HPV-infected subject, and A method for providing information on immunotherapy of HPV-induced cancer, comprising the step of determining a characteristic associated with immunotherapy for said subject based on the expression level of measured KLRB1 mRNA or a protein encoded by said mRNA.

4. In paragraph 3, The above decision step is, A method for providing information on immunotherapy in HPV-induced cancer, comprising the step of determining that the subject is negative for treatment response to immunotherapy if the expression level of the mRNA of the tissue-resident memory T cell-specific KLRB1 or the protein encoded by the mRNA is above a predetermined level, and otherwise, determining that the subject is positive for treatment response.

5. In paragraph 4, The above predetermined level is, A method for providing information on immunotherapy in HPV-induced cancers, wherein the normalized RNA average is 2 to 3 [mean(exp(x)-1].

6. In paragraph 3, The above measuring steps are: A method for providing information on immunotherapy in HPV-induced cancer, further comprising the step of measuring the expression level of mRNA of the tissue-resident memory T cell-specific KLRB1 or the protein encoded by the mRNA of KLRB1 and the expression level of mRNA of the B cell-specific CLEC2D or the protein encoded by the mRNA of CLEC2D.

7. In paragraph 3, A method for providing information on immunotherapy in HPV-induced cancer, wherein the HPV-induced cancer is at least one selected from oropharyngeal cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, and vulvar cancer caused by HPV infection.

8. In paragraph 7, The above HPV-induced cancer is an oropharyngeal cancer caused by HPV infection, and provides information on immunotherapy for HPV-induced cancer.

9. A step of measuring the level of CD161+ tissue-resident memory T cells in a biological sample isolated from an HPV-infected subject, and A method for providing information on immunotherapy in HPV-induced cancer, comprising the step of determining a characteristic associated with immunotherapy for said individual based on the level of said CD161+ tissue-resident memory T cells measured.

10. In paragraph 9, The above measuring steps are: Comprising a step of measuring the cell level of at least one of CD161+CD8+ T cells, CD161+CD103+ T cells and CD161+CD8+CD103+ T cells, The above decision step is, A method for providing information on immunotherapy in HPV-induced cancer, comprising the step of determining said characteristic based on at least one cell level.

11. In paragraph 9, The above measuring steps are: A method for providing information about immunotherapy in HPV-induced cancer, further comprising the step of measuring the levels of CD161+ tissue-resident memory T cells and CLEC2D+ B cells.

12. In paragraph 9, The above decision step is, The level of CD161+ tissue-resident memory T cells in the above biological sample was 15 cells / mm 2 A method for providing information on immunotherapy in HPV-induced cancer, comprising the step of determining that the subject is negative for treatment response to immunotherapy if the above is true, and positive for treatment response if the above is not true.

13. In paragraph 9, The above decision step is, The level of CD161+ tissue-resident memory T cells in the above biological sample was 15 cells / mm 2 If this is abnormal, A method for providing information on immunotherapy in HPV-induced cancer, comprising the step of determining that blockade of CD161 based on an inhibitory immune checkpoint molecule of CD161 is a therapeutic regimen for said entity.

14. In paragraph 9, A method for providing information on immunotherapy in HPV-induced cancer, wherein the HPV-induced cancer is at least one selected from oropharyngeal cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, and vulvar cancer caused by HPV infection.

15. In paragraph 14, The above HPV-induced cancer is an oropharyngeal cancer caused by HPV infection, and provides information on immunotherapy for HPV-induced cancer.

16. As a kit for providing information on immunotherapy in HPV-induced cancer, A kit for providing information on immunotherapy in HPV-induced cancer, comprising a formulation for measuring the expression level of mRNA of tissue-resident memory T cells specific for KLRB1 or a protein encoded by said mRNA in a biological sample isolated from an HPV-infected subject.

17. In paragraph 16, The above formulation is, A kit for providing information on immunotherapy in HPV-induced cancer, comprising an antibody targeting the CD161 surface protein of tissue-resident memory T cells (Trm).

18. In paragraph 16 or 17, The above formulation is, A kit for providing information on immunotherapy in HPV-induced cancer, comprising a bispecific monoclonal antibody targeting CD161 and CD103 surface proteins of tissue-resident memory T cells.

19. In paragraph 16, A kit for providing information on immunotherapy in HPV-induced cancer, wherein the HPV-induced cancer is at least one selected from oropharyngeal cancer, head and neck cancer, oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, cervical cancer, vaginal cancer, anal cancer, and vulvar cancer caused by HPV infection.

20. In paragraph 16, A kit for providing information on immunotherapy in HPV-induced cancer, wherein the expression level of the mRNA is measured by at least one method selected from among FISH (Fluorescence In Situ Hybridization), NanoString nCounter analysis, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, RNase-6 protection assay (RPA), microarray, and northern blotting.

21. In paragraph 16, A kit for providing information on immunotherapy in HPV-induced cancer, wherein the protein expression level is measured by at least one method selected from among western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and protein chip.

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