A specific marker for identifying T cells specifically attacking cancer cells

Abstract

The present disclosure provides a specific marker for identifying T cells specifically attacking cancer cells and the utilization of the marker. According to the present disclosure, a marker that has a high specificity for identifying T cells specifically attacking cancer cells is provided. According to the present disclosure, treatment or prevention of cancer in a subject with the use of a cell subpopulation contained in a tumor tissue infiltrating CD106+ T cell population or cells expressing a T cell receptor (TCR) expressed thereby can be provided. According to another embodiment of the present disclosure, a method for producing a cell pharmaceutical can be provided. According to the present disclosure, further, a method that comprises using the amount of a cell subpopulation as an index of the responsiveness of a subject to a cancer immunotherapy can be provided. According to the present disclosure, furthermore, a method for acquiring a tumor specific TCR sequence can be provided.

Description

[Technical field]

[0001] The present disclosure relates to the field of medicine or medicine.More specifically, the present disclosure relates to a technique for identifying T cells that specifically attack cancer cells, and may relate to the treatment of cancer.For example, the present disclosure may relate to adoptive cell therapy or a method for producing cells for such therapy, predicting the effect of cancer immunotherapy, obtaining tumor-specific sequences, etc. [Background technology]

[0002] Cancer immunotherapy has been attracting attention as a treatment for cancer. In particular, immune checkpoint inhibitors, such as nivolumab, an anti-PD-1 antibody, have shown results in terms of overall survival that are significantly better than docetaxel, which was the conventional standard treatment for non-small cell lung cancer, and have become the standard treatment. Immune cells such as T lymphocytes infiltrate cancer tissue, and are thought to be involved in a defense response against cancer cells. However, it is known that not all of the infiltrating immune cells attack cancer cells. The introduction of immune cells capable of attacking tumors (adoptive immunotherapy) is being studied as a treatment for cancer. Summary of the Invention [Means for solving the problem]

[0003] The present inventors have found that CD106-positive tumor-infiltrating T cells specifically attack cancer cells in the tumor local area, and that CD106 can be used as a highly specific marker for identifying T cells attacking cancer cells. + The present disclosure may relate to the treatment or prevention of cancer in a subject using a cell subpopulation contained in a T cell population. + The present disclosure may relate to the treatment or prevention of cancer in a subject using a cell expressing a T cell receptor (TCR) expressed by a cell subpopulation contained in the T cell population, or a TCR having the same antigen specificity as the T cell receptor (TCR).+ The present disclosure may relate to a method for producing a cell medicine, which includes a step of purifying a T cell population. + Also provided may be a method for using the amount of cell subpopulation contained in a T cell population as an indicator of the responsiveness of a subject to cancer immunotherapy. Also provided in the present disclosure may be a method for obtaining the sequence of tumor-specific TCR.

[0004] For example, in a preferred embodiment of the present disclosure, the following items are provided: (Item 1) Tumor tissue infiltrating CD106 + A composition for treating or preventing cancer in a subject, comprising a cell subpopulation within a T cell population. (Item 2) The composition of the preceding paragraph, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 3) The composition of any one of the preceding items, wherein the cell subpopulation comprises T cells derived from the subject. (Item 4) The composition according to any one of the preceding items, wherein the composition is used in combination with cancer immunotherapy. (Item 5) The composition of any one of the preceding items, wherein the cell subpopulation expresses a tumor-specific TCR. (Item 6) Tumor tissue infiltrating CD106 + A composition for treating or preventing cancer in a subject, comprising a cell expressing a TCR expressed by a cell subpopulation contained in a T cell population, or a TCR having the same antigen specificity as that expressed by a cell subpopulation contained in a T cell population. (Item 7) The composition of any one of the preceding items, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 8) The composition of any one of the preceding items, wherein the cells comprise T cells derived from the subject. (Item 9) The composition according to any one of the preceding items, wherein the composition is used in combination with cancer immunotherapy. (Item 10) The composition of any one of the preceding items, wherein the cell is included in the cell subpopulation and the TCR is endogenous. (Item 11) The composition of any one of the preceding items, wherein the TCR is exogenously introduced into the cell. (Item 12) The composition of any one of the preceding items, wherein the cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them. (Item 13) The composition of any of the preceding items, further comprising a pharma- ceutically acceptable carrier. (Item 14) Tumor tissue-infiltrating CD106 in the manufacture of a medicament for treating or preventing cancer in a subject + Use of cell subpopulations within a T cell population. (Item 15) 2. The use of any one of the preceding claims, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 16) The use of any one of the preceding claims, wherein the cell subpopulation comprises T cells derived from the subject. (Item 17) 2. The use according to any one of the preceding items, characterized in that the medicament is used in combination with cancer immunotherapy. (Item 18) The use according to any one of the preceding items, wherein the cell subpopulation expresses a tumor-specific TCR. (Item 19) Tumor tissue-infiltrating CD106 in the manufacture of a medicament for treating or preventing cancer in a subject + The use of cells expressing a TCR expressed by a subpopulation of cells within a T cell population, or a TCR having the same antigen specificity as that expressed by a subpopulation of cells within a T cell population. (Item 20) 2. The use of any one of the preceding claims, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 21) The use of any one of the preceding items, wherein the cells comprise T cells derived from the subject. (Item 22) The use according to any one of the preceding items, characterized in that the medicament is used in combination with cancer immunotherapy. (Item 23) The use according to any one of the preceding items, wherein the cell is included in the cell subpopulation and the TCR is endogenous. (Item 24) The use according to any one of the preceding items, wherein the TCR is exogenously introduced into the cell. (Item 25) The use according to any one of the preceding items, wherein the cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them. (Item 26) The use according to any one of the preceding items, wherein the medicament further comprises a pharma- ceutically acceptable carrier. (Item 27) A method for treating or preventing cancer in a subject, comprising: + administering to the subject an effective amount of a cell subpopulation contained in a T cell population. (Item 28) The method of any one of the preceding paragraphs, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 29) The method of any one of the preceding paragraphs, wherein the cell subpopulation comprises T cells derived from the subject. (Item 30) The method according to any one of the preceding items, characterized in that it is used in combination with cancer immunotherapy. (Item 31) The method of any one of the preceding paragraphs, wherein the cell subpopulation expresses a tumor-specific TCR. (Item 32) A method for treating or preventing cancer in a subject, comprising: + A method comprising the step of administering to a subject an effective amount of a TCR expressed by a cell subpopulation contained in a T cell population, or an effective amount of a cell expressing a TCR having the same antigen specificity. (Item 33) The method of any one of the preceding paragraphs, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 34) The method of any one of the preceding items, wherein the cells comprise T cells derived from the subject. (Item 35) The method according to any one of the preceding items, further comprising administering cancer immunotherapy. (Item 36) The method of any one of the preceding items, wherein the cell is included in the cell subpopulation and the TCR is endogenous. (Item 37) The method according to any one of the preceding items, wherein the TCR is exogenously introduced into the cell. (Item 38) The method of any one of the preceding items, wherein the cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them. (Item 39) Tumor tissue infiltrating CD106 for the treatment or prevention of cancer in a subject + A subpopulation of cells within the T cell population. (Item 40) The cell subpopulation of any one of the preceding paragraphs, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 41) The cell subpopulation of any one of the preceding paragraphs, wherein the cell subpopulation comprises T cells derived from the subject. (Item 42) 2. The cell subpopulation of any one of the preceding items, wherein the cell subpopulation is in combination with cancer immunotherapy. (Item 43) The cell subpopulation of any one of the preceding items, wherein the cell subpopulation expresses a tumor-specific TCR. (Item 44) Tumor tissue infiltrating CD106 for the treatment or prevention of cancer in a subject + A cell expressing a TCR expressed by a subpopulation of cells within a T cell population, or a TCR having the same antigen specificity as that expressed by a subpopulation of cells within a T cell population. (Item 45) The TCR or cell of any one of the preceding paragraphs, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 46) The TCR or cell of any one of the preceding items, wherein the cell comprises a T cell derived from the subject. (Item 47) The TCR or cell according to any one of the preceding items, characterized in that the TCR or cell is used in combination with cancer immunotherapy. (Item 48) The TCR or cell of any one of the preceding items, wherein the cell is included in the cell subpopulation and the TCR is endogenous. (Item 49) The TCR or cell described in any one of the preceding items, wherein the TCR is exogenously introduced into the cell. (Item 50) The TCR or cell of any one of the preceding items, wherein the cell comprises an induced pluripotent stem cell (iPSC), an embryonic stem cell (ES cell), a T cell, or a T cell differentiated from any of them. (Item 51) A method for producing a cellular medicine for treating or preventing cancer in a subject, comprising: + A method comprising the step of purifying a T cell population. (Item 52) The method of any one of the preceding paragraphs, further comprising the step of purifying the cell population positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 53) The method of any one of the preceding paragraphs, wherein the T cell population is isolated from T cells that have infiltrated tumor tissue. (Item 54) The method according to any one of the preceding items, further comprising the step of providing a cell medicine comprising the purified T cell population. (Item 55) The method of any one of the preceding paragraphs, further comprising modifying the purified T cell population. (Item 56) The method according to any one of the preceding items, further comprising a step of introducing into cells a TCR possessed by the purified T cell population or a TCR having the same antigen specificity as that of the purified T cell population. (Item 57) The method of any one of the preceding items, wherein the cells comprise T cells derived from the subject. (Item 58) The method of any one of the preceding items, wherein the cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them. (Item 59) Subject's CD106 + A method in which the amount of a cell subpopulation contained in a T cell population is used as an indicator of the subject's responsiveness to cancer immunotherapy. (Item 60) The method of any one of the preceding paragraphs, wherein the cancer immunotherapy comprises administration of an immune checkpoint inhibitor. (Item 61) The method of any one of the preceding paragraphs, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 62) The method of any one of the preceding paragraphs, wherein the cell subpopulation is a tumor-infiltrating T cell subpopulation. (Item 63) A method for diagnosing a subject's responsiveness to cancer immunotherapy, comprising: CD106 from the subject + Obtaining a T cell population CD106 of the subject +measuring the amount of a cell subpopulation in the T cell population; and Diagnosing the responsiveness of the subject to cancer immunotherapy based on the amount of the cell subpopulation. A method comprising: (Item 64) The method of any one of the preceding paragraphs, wherein the cancer immunotherapy comprises administration of an immune checkpoint inhibitor. (Item 65) The method of any one of the preceding paragraphs, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 66) The method of any one of the preceding paragraphs, wherein the cell subpopulation is a tumor-infiltrating T cell subpopulation. (Item 67) A method for obtaining a tumor-specific T cell receptor (TCR) sequence, comprising: isolating a population of T cells positive for CD106 expression from the tumor-infiltrating T cells; and obtaining the TCR sequence of the T cells in the T cell population; A method comprising: (Item 68) The method of any one of the preceding paragraphs, wherein the isolating step further comprises isolating a cell population that is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. (Item 69) The method of any one of the preceding items, wherein the step of obtaining the sequence of the TCR comprises sequencing the nucleic acid sequence of the T cell. (Item 70) The method according to any one of the preceding items, further comprising a step of obtaining a TCR sequence having the same antigen specificity as the TCR of a T cell in the T cell population.

[0005] It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. Effect of the Invention

[0006] This can provide a highly specific marker for T cells attacking tumors. Because CD106 is also a surface marker, it can be used to separate cells, making it easy to extract T cells that specifically attack tumors. If we could efficiently increase such T cells or artificially create them by analyzing the T cell receptor sequence, this would lead to highly effective tumor-specific cell therapy with very few side effects. [Brief description of the drawings]

[0007] [Figure 1] Figure 1 shows the expression of various markers in tumor-infiltrating T cells. In each panel, cells positive for the expression of the markers described above are colored. The cells are clustered according to the expression pattern, and the circled areas correspond to tumor-toxic T cells.

[0008] [Diagram 2] Figure 2 shows the secretion of IFNγ when autologous tumor stimulation was performed on cell subpopulations sorted by marker expression. It can be seen that the specificity is inferior to CD106 because IFNγ was secreted by stimulation in the CD106-positive fraction, and IFNγ was secreted by stimulation in the other marker-positive fractions, but also in the other marker-negative fractions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present disclosure will be described below. Throughout this specification, the singular expression should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) shall prevail.

[0010] (definition) In this specification, unless otherwise specified, the term "about" means ±10% of the numerical value that follows.

[0011] As used herein, the term "biomarker" refers to a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to a therapeutic intervention.

[0012] As used herein, "cancer" or "carcinoma" are used interchangeably and refer to a malignant tumor or the condition in which such a malignant tumor exists that is highly atypical, grows faster than normal cells, and can destructively invade surrounding tissues or cause metastasis. In this disclosure, cancer includes, but is not limited to, solid cancers and hematopoietic tumors.

[0013] As used herein, the term "cancer immunotherapy" refers to a method for treating cancer using a living organism's defense mechanisms, such as the immune system.

[0014] As used herein, the term "cell subpopulation" refers to any collection of cells that share some common characteristic in a cell population that contains cells with diverse characteristics. If a specific name is known in the art, such a term can be used to refer to a specific cell subpopulation, or any property (e.g., expression of a cell surface marker) can be described to refer to a specific cell subpopulation.

[0015] As used herein, the term "relative amount" with respect to cells is used interchangeably with "proportion." Typically, the terms "relative amount" and "proportion" refer to the amount of a particular cell population (e.g., CD8 + The number of cells forming a desired cell subpopulation (e.g., CD106 + CD8 + This refers to the number of cells that form a T cell subpopulation.

[0016] As used herein, the term "baseline" refers to a comparative amount for determining an increase or decrease in the amount or level of a marker described herein. When determining an increase or decrease in an amount or level before and after a treatment, for example, the "baseline" includes the amount or level before the treatment.

[0017] (Cell fractionation and separation) Samples for cell fractionation and separation can be appropriately collected from a subject by a conventional method. For example, samples can be collected from the subject's peripheral blood, bone marrow, tumor tissue, hematopoietic tissue, spleen, normal tissue, lymph, etc. It is believed that cells that attack cancer can be obtained by isolating immune cells that have infiltrated into tumor tissue and further selecting them as necessary.

[0018] The composition of immune cells in a subject sample can be measured by a person skilled in the art using a conventional method. Usually, the number of cells positive for a marker (e.g., CD4) that defines a target cell subpopulation in a sample can be measured using flow cytometry or the like. The composition of a cell population is generally measured using flow cytometry, but may also be measured using other methods such as immunostaining of a sample containing cells, a method using an antibody array, protein expression analysis in a sample containing cells (e.g., Western blot, mass spectrometry, HPLC, etc.), and mRNA expression analysis in a sample containing cells (e.g., microarray, next-generation sequencing, etc.). CD106 + CD8 + To count the number of cells in a cell subpopulation such as a T cell subpopulation, the number may be determined by experimentally removing cells other than the respective cell subpopulation from the total cells.

[0019] Single cell gene expression analysis can also identify cells by examining the mRNA in individual cells and examining the presence or absence of mRNA that codes for a protein molecule of interest. Examples of single cell gene expression analysis include: 1) a method of performing next-generation sequencing using Quartz-Seq; 2) a method of isolating cells using Fluidigm C1 System or ICELL8 Single-Cell System and preparing a library using SMART-Seq v4; 3) a method of separating cells using a cell sorter and measuring by quantitative PCR using the Ambion Single Cell-to-CT kit; and 4) a method using the CyTOF SYSTEM (Helios). To be on the safe side, the number of cells can be counted from tumor-infiltrating cells, the cells can be separated using a cell sorter, etc., and the expression level of a specific gene can be measured for each separated cell using, for example, the Ambion Single Cell-to-CT kit with a quantitative PCR device. Based on the results, it can be determined whether each cell expresses CD106 + CD8 + It is possible to determine which subpopulation, such as a T cell subpopulation, the cells fall into and count the number of cells that fall into each subpopulation.

[0020] As used herein, expression of a marker is referred to as being "positive" or "positive for (the marker) + When confirming by single cell sequencing, the identity of a "cell" can be confirmed by detecting one or more reads, and when confirming by FCM, it is possible to determine positivity by comparison with an isotype control.

[0021] (cell) In the present disclosure, cell subpopulations having specific marker expression patterns may be provided. In one embodiment, CD106 + A cell subpopulation contained in the cell population may be provided. Such a cell subpopulation may further be positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1 (CD39), CTLA-4, and any combination thereof. The cell subpopulation described in the present disclosure may be used in the present disclosure as a cell for adoptive cell transfer, a cell for obtaining a tumor-specific TCR therefrom, a cell for using the amount as an index, and / or a cell for modifying it. A tumor tissue infiltrating cell population or a cell subpopulation contained therein may be used as the cell population. As a surface marker for T cells specifically attacking tumors, markers such as PD-1 and 4-1BB have been used in the past, and a very recent report has focused on CD103 and CD39. In the present disclosure, from the data of single cell sequencing, CD106 is the most specific surface marker, and based on a comprehensive comparison of reported markers, it is considered to be more useful than those markers.

[0022] In the present disclosure, the cell or cell population may be an immune cell or its precursor cell or a population thereof. Immune cells include dendritic cells, macrophages, lymphocytes (e.g., T cells (e.g., killer T cells, helper T cells, regulatory T cells, αβT cells, γδT cells, natural killer T cells), B cells, NK (natural killer) cells), neutrophils, eosinophils, and basophils, and combinations thereof. In the context of treating or preventing cancer using cells, preferably, the cell may be a T cell. The cell or cell population may include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), peripheral T cells, naive T cells, memory T cells, central memory T cells (TCMs), T memory stem cells (TSCMs), or T cells differentiated from any of them.

[0023] The T cells in the present disclosure may be CD8 positive T cells. Cytotoxic T cells (CTLs) are a type of lymphocyte T cell that recognizes and destroys cells (such as cancer cells) that are foreign to the host. CTLs are differentiated from T cells that express CD8 molecules on their surface.

[0024] In one embodiment of the present disclosure, tumor tissue infiltrating CD106 + A composition for treating or preventing cancer in a subject may be provided, comprising a cell subpopulation contained in a T cell population. Alternatively, tumor tissue-infiltrating CD106 + A method may be provided for treating or preventing cancer in a subject comprising administering a subpopulation of cells contained in the T cell population.

[0025] The cell subpopulation may further be positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1 (CD39), CTLA-4, and any combination thereof. The cell subpopulation may include T cells derived from a subject to be treated or prevented, or may include T cells derived from a subject different from the subject to be treated or prevented. The cell subpopulation may express a tumor-specific TCR. The markers provided in the present disclosure may identify a cell subpopulation expressing a tumor-specific TCR. In the present disclosure, the composition comprising the cells or adoptive cell transfer therapy may be combined with cancer immunotherapy.

[0026] In one embodiment of the present disclosure, tumor tissue infiltrating CD106 + A cell expressing a TCR expressed by a cell subpopulation contained in the T cell population or a TCR having the same antigen specificity as that expressed by the cell subpopulation can be provided. The cell is a tumor tissue-infiltrating CD106 + These may be cells contained in the T cell population itself, or tumor tissue-infiltrating CD106 + The T cell population may be a different cell expressing a TCR expressed by a cell subpopulation. Such cells can be obtained by TCR introduction or by TCR screening. Tumor tissue infiltration CD106 + The cell subpopulations contained within the T cell population may further be positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1 (CD39), CTLA-4, and any combination thereof. The cells may be positive for tumor tissue infiltrating CD106 + It may be contained in a cell subpopulation contained in a T cell population, and the TCR may be endogenous.

[0027] TCR can be introduced into cells from the outside. The cells to be introduced can include, for example, induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), peripheral T cells, naive T cells, memory T cells, central memory T cells (TCMs), T memory stem cells (TSCMs), or T cells differentiated from any of them. TCR can be introduced by the methods described elsewhere in this disclosure and known to those skilled in the art.

[0028] (How to obtain TCR) The immune system's defense mechanism is largely dependent on specific immunity, which is mainly provided by T cells and B cells. T cells and B cells do not react to self-cells or molecules, but can specifically recognize and attack foreign pathogens such as viruses and bacteria. To achieve this, T cells and B cells have a mechanism that allows them to recognize and distinguish a variety of antigens from other organisms as well as self-antigens using receptor molecules expressed on their cell surface. In T cells, the T cell receptor (TCR) acts as an antigen receptor. Stimulation from these antigen receptors transmits intracellular signals, which enhance the production of inflammatory cytokines and chemokines, promote cell proliferation, and initiate various immune responses.

[0029] TCR recognizes peptides (peptide-MHC complex, pMHC) bound to the peptide-binding groove of the major histocompatibility complex (MHC) expressed on antigen-presenting cells, thereby distinguishing between self and non-self and recognizing antigen peptides (Cell 1994, 76, 287-299). TCR is a heterodimer receptor molecule consisting of two TCR polypeptide chains, and there are αβ-type TCRs expressed by normal T cells and γδ-type TCRs with special functions. α and β chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, CD3δ chain), transmit intracellular signals after antigen recognition, and initiate various immune responses. CD3 molecules are present on the cell membrane and form complexes with TCR, so they can be used as a marker for TCR expression. Endogenous antigens such as viral antigens proliferated in cells following virus infection and cancer antigens derived from cancer cells are presented as antigen peptides on MHC class I molecules. In addition, antigens derived from foreign microorganisms are taken up by antigen-presenting cells by endocytosis, processed, and then presented on MHC class II molecules. These antigens are expressed by CD8 and CD9. + T cells or CD4 + It is recognized by the TCR expressed on T cells. It is also known that costimulatory molecules such as CD28, ICOS, and OX40 molecules are important for stimulation via TCR molecules.

[0030] TCR genes consist of multiple V regions (variable regions, V), J regions (joining regions, J), D regions (diversity regions, D), and constant regions (C) encoded in different regions on the genome. During the differentiation process of T cells, these gene fragments are rearranged in various combinations, and α- and γ-chain TCRs express genes consisting of VJC, while β- and δ-chain TCRs express genes consisting of VDJC. The rearrangement of these gene fragments creates diversity, and the insertion or deletion of one or more bases between the V and D or D and J gene fragments forms random amino acid sequences, creating a more diverse TCR gene sequence.

[0031] The region where the TCR molecule directly binds to the surface of the pMHC complex (TCR footprint) is composed of the highly diverse complementarity determining regions (CDRs) CDR1, CDR2, and CDR3 in the V region. The CDR3 region contains a part of the V region, a part of the VDJ region formed by random sequences, and a part of the J region, and forms the most diverse antigen recognition site. On the other hand, the other regions are called FR (framework region) and play a role in forming the structure that forms the skeleton of the TCR molecule. During the differentiation and maturation process of T cells in the thymus, the β chain TCR is first genetically rearranged and associates with the pTα molecule to form a pre-TCR complex molecule. Then, the α chain TCR is rearranged to form an αβTCR molecule, and if a functional αβTCR is not formed, rearrangement occurs in the other α chain TCR gene allele. It is known that TCRs with appropriate affinity are selected through positive and negative selection in the thymus, and acquire antigen specificity (Annual Review Immunology, 1993, 6,309-326).

[0032] T cells produce one type of TCR with high specificity for a particular antigen. The existence of a large number of antigen-specific T cells in the body results in the formation of a diverse TCR repertoire, which can function effectively as a defense mechanism against various pathogens.

[0033] In one embodiment of the present disclosure, a method for obtaining a sequence of a tumor-specific T cell receptor (TCR) may be provided. Although not wishing to be bound by theory, the present disclosure shows that a cell subpopulation contained in a CD106 expression positive T cell population specifically attacks cancer, and it is believed that a tumor-specific TCR can be isolated by isolating such a cell subpopulation. The present disclosure may provide a method for obtaining a sequence of a tumor-specific T cell receptor (TCR), comprising the steps of isolating a CD106 expression positive T cell population from tumor-infiltrating T cells; and obtaining the sequence of the TCR of the T cell of the T cell population.

[0034] The isolating step may further include isolating a cell population that is further positive for any other marker disclosed herein (e.g., a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1, ​​CTLA-4, and any combination thereof).

[0035] The acquisition of the sequence of the TCR may include sequencing the nucleic acid sequence of the T cell. The method of sequencing is not limited as long as it can determine the sequence of the nucleic acid sample, and any method known in the art can be used, but it is preferable to use next-generation sequencing (NGS). Next-generation sequencing includes, but is not limited to, pyrosequencing, sequencing by synthesis (sequencing by synthesis), sequencing by ligation, ion semiconductor sequencing, and the like. The TCR sequence can be obtained by amplicon sequencing from intron-free mRNA. In addition, the TCR sequence can be obtained by sequencing the genomic sequence of the T cell. Alternatively, the TCR sequence may be obtained by directly determining the amino acid sequence of the TCR molecule using mass spectrometry or Edman degradation, and the like. In this specification, both amino acid sequence and nucleic acid sequence can be used as the "TCR sequence".

[0036] (TCR cluster) In the present disclosure, a TCR having the same antigen specificity as a TCR having tumor specificity can be used. For example, CD106 + A TCR having the same antigen specificity as the TCR of the T cell population can be used. "Having the same antigen specificity" can be determined by classifying two TCRs into the same cluster by cluster analysis as shown below. In the present disclosure, in obtaining a tumor-specific TCR, CD106 + It is possible to obtain a sequence of a TCR having the same antigen specificity as the TCR of a T cell of an isolated T cell population such as a T cell population. This can be achieved by subjecting one or more TCRs to cluster analysis as shown below with the TCR of a T cell of the isolated T cell population. Such an analysis method is described in detail in Japanese Patent No. 6500144, the contents of which are incorporated herein by reference.

[0037] TCR cluster analysis may be performed by an analysis method including the steps of: (i) providing features of at least two TCRs, excluding calculating features from a three-dimensional structural model of the at least two TCRs; (ii) performing machine learning to analyze the antigen specificity or binding mode of the TCR based on the features, without identifying the antigen specificity or binding mode; and (iii) classifying or determining whether the antigen specificity or binding mode is the same or different.

[0038] In other embodiments, a collection of TCRs may be analyzed by a method including the steps of: (a) extracting features for at least one pair of members of the collection of TCRs, excluding calculating features from a three-dimensional structural model of the at least one pair; (b) calculating a distance between the antigen specificities or binding modes of the pair by machine learning using the features, or determining whether the antigen specificities or binding modes match; (c) clustering the collection of TCRs based on the distance; and (d) analyzing based on classification by the clustering, if necessary.

[0039] In another embodiment, a set of TCRs may be analyzed by a method comprising the steps of: (aa) extracting features for each of at least one paired sequence of members of the set of TCRs, excluding calculating features from a three-dimensional structural model of an immune entity of the at least one paired sequence; (bb) projecting the features into a high dimensional vector space, where the spatial distance of the members reflects the functional similarity of the members; (cc) clustering the set of immune entities based on the distance; and (dd) optionally analyzing based on the classification by the clustering.

[0040] The features may include at least one selected from the group consisting of sequence information, length of CDR1-3 sequence, sequence identity, sequence identity of framework regions, total number of charged / hydrophilic / hydrophobic / aromatic amino acids of the molecule, each CDR, number of charged / hydrophilic / hydrophobic / aromatic amino acids in framework regions, number of each amino acid, heavy chain-light chain combination, number of somatic mutations, position of mutation, presence / identity of amino acid motif, rarity against a reference sequence set, and odds ratio of binding HLA with a reference sequence.

[0041] The machine learning calculation is performed using random forest or boosting with the feature amount as input, and the clustering can be performed based on a simple threshold based on the linkage distance, hierarchical clustering, or non-hierarchical clustering. The machine learning can be selected from the group consisting of machine learning algorithms such as regression methods, neural network methods, support vector machines, and random forests.

[0042] (Method of producing cells) In the present disclosure, a method for producing cells, particularly cells for use as a medicament, can be provided. Without wishing to be bound by theory, CD106 in tumors + CD8 + By utilizing the expression of tumor-specific TCR in cells contained in a T cell population, it is possible to obtain cells that attack tumors, and such cells are thought to be useful for the treatment or prevention of cancer.

[0043] In one embodiment of the present disclosure, a method for producing a cellular medicine for the treatment or prevention of cancer in a subject, comprising: + A method may be provided that includes purifying a T cell population. The method may further include purifying a cell population that is positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1, ​​CTLA-4, and any combination thereof. The T cell population may be isolated from T cells that have infiltrated tumor tissue.

[0044] A cell medicine comprising a purified T cell population can be provided. Such a T cell population is considered to have a tumor-specific TCR and can be useful as a cell medicine. If necessary, the purified T cell population can be cultured and / or expanded to provide a medicine. If necessary, the purified T cell population can be further modified. Modifications of T cells include stimulation with integrins α4β7 and α4β1 (VLA-4) that interact with CD106, as well as CD3, IL-2, IL-4, and IL-7.

[0045] In some embodiments of the present disclosure, a method may be provided that includes a step of introducing a TCR or a TCR having the same antigen specificity as that of a purified T cell population into a cell. The TCR may be obtained and / or introduced by a method described elsewhere herein or by other methods known to those skilled in the art. The tumor-specific TCR may be introduced by modifying the obtained sequence, as necessary. The cell to be introduced may be a T cell derived from a subject to be treated or prevented, or any other immune cell. The cell to be introduced may include, for example, an induced pluripotent stem cell (iPSC), an embryonic stem cell (ES cell), a peripheral T cell, a central memory T cell (TCM), a T memory stem cell (TSCM), a naive T cell, a memory T cell, or a T cell differentiated from any of them.

[0046] (cancer) Cancers that are targeted in this disclosure include, but are not limited to, lung cancer, kidney (renal cell) cancer, prostate cancer, gastric cancer, testicular cancer, liver cancer, skin cancer, esophageal cancer, melanoma, pancreatic cancer, bone tumor / osteosarcoma, colorectal cancer, bone and soft tissue tumor, biliary tract cancer, multiple myeloma, malignant lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma), urothelial carcinoma, uterine cancer (body / neck), head and neck cancer, brain tumor, thymus tumor, thyroid cancer, mesothelioma, anal cancer, penile cancer, cancer of unknown primary site, ovarian cancer, and breast cancer.

[0047] (Cancer immunotherapy) In this specification, "cancer immunotherapy" refers to a method of treating cancer using the immune function of an organism. Cancer immunotherapy can be broadly divided into cancer immunotherapy by strengthening the immune function against cancer and cancer immunotherapy by inhibiting the immune evasion function of cancer. Furthermore, cancer immunotherapy can be divided into active immunotherapy, which activates the immune function in the body, and passive immunotherapy, which returns immune cells whose immune function has been activated or proliferated outside the body to the body. Examples of cancer immunotherapy include non-specific immunostimulants, cytokine therapy, cancer vaccine therapy, dendritic cell therapy, adoptive immunotherapy, non-specific lymphocyte therapy, cancer antigen-specific T cell therapy, antibody therapy, immune checkpoint inhibitor therapy, and CAR-T therapy. In the present disclosure, a method can be provided in which the amount of a cell subpopulation that specifically attacks a tumor is used as an index of a subject's responsiveness to cancer immunotherapy.

[0048] In one embodiment, the subject's CD106 + A method may be provided in which the amount of a cell subpopulation contained in a T cell population is used as an indicator of the responsiveness of a subject to cancer immunotherapy. The cancer immunotherapy may include administration of an immune checkpoint inhibitor. The cell subpopulation may further be positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1, ​​CTLA-4, and any combination thereof. The amount of the cell subpopulation at any site may be used, but the amount of the cell subpopulation in tumor-infiltrating T cells may be used preferably.

[0049] Immune checkpoint therapy using immune checkpoint inhibitors has attracted much attention in recent years (Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012 Mar 22;12(4):252-64.). Cancer cells express various proteins on their surface, which allow them to avoid attacks by immune cells such as T cells. It is therefore believed that under normal conditions, the body's immune function alone is unable to eliminate cancer tissues. Immune checkpoint inhibitors enable the body's immune function to efficiently eliminate cancer by inhibiting ligand-receptor interactions, etc., which are responsible for the transmission of inhibitory signals from such cancer tissues to the immune function. One embodiment of the present disclosure relates to a method for the efficient elimination of cancers by the immune function of the body. + Another embodiment of the present disclosure is a method for using the amount of a cell subpopulation contained in a T cell population as an indicator for predicting the responsiveness of a subject to an immune checkpoint inhibitor as described below. + In another embodiment, a method of administering an immune checkpoint inhibitor to a (responsive) subject selected based on the amount of a cell subpopulation in a T cell population, such as CD106. + Methods are provided for interrupting, discontinuing or avoiding immune checkpoint inhibitors in subjects who are found to be non-responsive based on the amount of cell subpopulations in a T cell population.

[0050] A representative example of an immune checkpoint inhibitor is a PD-1 inhibitor. PD-1 inhibitors include the anti-PD-1 antibody nivolumab (Opdivo). TM (sold as pembrolizumab; Keytruda) TMAnti-PD-1 antibodies include, but are not limited to, those sold under the name PD-1. Anti-PD-1 antibodies are believed to exert an anticancer effect by relieving the inhibition of T cell activation by PD-1 signals. When PD-1 (programmed death 1) interacts with PD-L1 or PD-L2, SHP-2, a type of tyrosine phosphatase, is recruited to the cytoplasmic domain of PD-1, which inactivates ZAP70, a T cell receptor signaling protein, thereby suppressing T cell activation (Okazaki, T., Chikuma, S., Iwai, Y. et al.: A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat. Immunol., 14, 1212-1218 (2013)). In addition, PD-L1 is also thought to interact with CD80 and suppress T cell activation (Butte, MJ, Keir, ME, Phamduy, TB et al.: PD-L1 interacts specifically with B7-1 to inhibit T cell proliferation. Immunity, 27, 111-122 (2007)). PD-1 is highly expressed in killer T cells and natural killer cells infiltrating cancer tissue, and it is thought that PD-L1 on tumors attenuates immune responses. When the attenuation of immune responses caused by this PD-1 signal is inhibited by anti-PD-1 antibodies, the anti-tumor immune response is enhanced.

[0051] Other examples of immune checkpoint inhibitors include PD-L1 inhibitors (e.g., anti-PD-L1 antibodies avelumab, durvalumab, or atezolizumab). PD-L1 inhibitors bind to the PD-L1 side to inhibit the above-mentioned PD-1 pathway, resulting in an anti-tumor immune response.

[0052] Other examples of immune checkpoint inhibitors include CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies ipilimumab or tremelilumab). CTLA-4 inhibitors activate T cells through a pathway different from PD-1 inhibition, resulting in antitumor immune responses. T cells are activated by the interaction of CD28 on the surface with CD80 or CD86. However, even once activated, it is believed that CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) expressed on the surface of T cells preferentially interacts with CD80 or CD86 with a higher affinity than CD20, thereby suppressing activation. CTLA-4 inhibitors inhibit CTLA-4, thereby preventing the inhibition of the interaction between CD20 and CD80 or CD86, thereby generating antitumor immune responses.

[0053] In further embodiments, immune checkpoint inhibitors may target immune checkpoint proteins such as TIM-3, LAG-3, B7-H3, B7-H4, B7-H5 (VISTA), or TIGIT.

[0054] The immune checkpoints described above are thought to suppress immune responses to self-tissues, but when antigens such as viruses are present in the body for a long period of time, the immune checkpoints in T cells also increase. As for tumor tissues, they are also antigens that exist in the body for a long period of time, so it is thought that these immune checkpoints allow them to evade anti-tumor immunity. The immune checkpoint inhibitors described above disable such evasive functions and exert anti-tumor effects.

[0055] In one embodiment of the present disclosure, an index is provided for predicting the responsiveness of a subject with cancer to cancer immunotherapy.The response to cancer immunotherapy can be determined based on RECIST v1.1 (New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)).

[0056] The effectiveness of cancer treatment can be determined based on changes in tumor size, etc., as complete response (CR), partial response (PR), progressive disease (PD), or stable disease (SD).

[0057] As used herein, a "responder" refers to a subject who shows complete or partial response to cancer treatment. As used herein, a "non-responder" refers to a subject who shows progression or stable disease to cancer treatment.

[0058] The responsiveness of a subject to a cancer treatment includes whether the subject is a "responder" or whether the subject is a "non-responder." Thus, determining the responsiveness of a subject to a cancer treatment includes determining whether the subject is a responder or non-responder.

[0059] One aspect of the present disclosure is to + The amount of a cell subpopulation contained in a T cell population is used to predict or determine whether a subject is a "responder" or a "non-responder". The timing of the judgment is preferably before the start of treatment, but may be after the start of treatment. This is because it is medically useful to determine whether the current treatment is appropriate. Alternatively, the CD106 of the present disclosure may be used. + The amount of a cell subpopulation contained in a T cell population can also be used to determine prognosis. + Using the amount of cell subpopulations contained in the T cell population, it is possible to predict whether a responding patient will become non-responding, i.e., relapse. Furthermore, in terms of the timing of judgment, it is possible to analyze the composition of cell subpopulations over time after cancer immunotherapy (e.g., after administration of immune checkpoint inhibitors) and determine the prognosis.

[0060] (Companion use for immune checkpoint inhibitors) In a further aspect of the present disclosure, the present disclosure provides a composition comprising an immune checkpoint inhibitor, + The present invention provides a composition for treating cancer in a subject having a high amount of a cell subpopulation contained in a T cell population. + Administration to subjects with a high amount of a cell subpopulation contained in a T cell population may be advantageous because it is believed that there are more cells that attack tumors. + A subject having a low amount of a cell subpopulation contained in a T cell population can be determined to be a non-responder, and a decision can be made not to administer the immune checkpoint inhibitor or to interrupt or discontinue administration.

[0061] The composition of the present disclosure is preferably a pharmaceutical composition, and examples of immune checkpoint inhibitors contained as active ingredients thereof include PD-1 inhibitors. PD-1 inhibitors include anti-PD-1 antibodies, such as nivolumab or pembrolizumab. The composition can be formulated into any dosage form, such as an aerosol, liquid, extract, elixir, capsule, granule, pill, ointment, powder, tablet, solution, suspension, emulsion, etc. The composition may include any pharma- ceutically acceptable additive and / or excipient known in the art. The composition of the present disclosure can be administered by any suitable route, as determined by the skilled artisan, including, but not limited to, intravenous injection, infusion, oral, parenteral, transdermal, etc.

[0062] (composition) In the present disclosure, the composition comprising immune checkpoint inhibitor or cells may be used in combination with other cancer treatments as appropriate.Other cancer treatments include, but are not limited to, other cancer immunotherapy, radiation therapy, chemotherapy, hyperthermia, surgical procedures, etc.The composition comprising immune checkpoint inhibitor or cells may be administered in combination with one or more additional agents.The one or more additional agents may be any chemotherapy drug or may include a second immune checkpoint inhibitor.

[0063] In one embodiment of the present disclosure, a composition comprising immune checkpoint inhibitor is provided.The composition comprising immune checkpoint inhibitor of the present disclosure is usually administered systemically or locally, orally or parenterally.It is believed that the composition comprising immune checkpoint inhibitor of the present disclosure can exert a remarkable therapeutic effect by being administered to a subject who is shown to have a large number of cells attacking tumors by the method described herein.

[0064] The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually, for example, orally administered once to several times a day in the range of 0.1 mg to 100 mg per dose per adult, or parenterally administered (preferably intravenously) once to several times a day in the range of 0.01 mg to 30 mg per dose per adult, or continuously administered intravenously for 1 to 24 hours a day. Of course, the dosage varies depending on various conditions, so in some cases a smaller dosage than the above-mentioned dosage is sufficient, and in other cases a dosage exceeding the range is necessary.

[0065] The composition containing an immune checkpoint inhibitor or the composition containing cells may be administered in the form of a solid preparation for oral administration, a liquid preparation for oral administration, and an injection, an external preparation, a suppository, etc. for parenteral administration. The solid preparation for oral administration includes tablets, pills, capsules, powders, granules, etc. The capsule includes hard capsules and soft capsules.

[0066] The composition of the present disclosure may be used by mixing one or more active ingredients (e.g., cells or antibodies against immune checkpoint proteins) as is or with excipients (lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropylcellulose, polyvinylpyrrolidone, magnesium aluminometasilicate, etc.), disintegrants (calcium cellulose glycolate, etc.), lubricants (magnesium stearate, etc.), stabilizers, solubilizers (glutamic acid, aspartic acid, etc.), etc., according to a conventional method. In addition, it may be coated with a coating agent (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) or with two or more layers, if necessary. Furthermore, capsules of absorbable materials such as gelatin are also included.

[0067] Injectables for parenteral administration include solutions, suspensions, emulsions, and solid injectables that are dissolved or suspended in a solvent when used. Injectables are used by dissolving, suspending, or emulsifying one or more active substances in a solvent. As the solvent, for example, distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof are used. Furthermore, the injectables may contain stabilizers, dissolving aids (glutamic acid, aspartic acid, polysorbate 80 (registered trademark), etc.), suspending agents, emulsifying agents, soothing agents, buffering agents, preservatives, etc. These are sterilized in the final process or prepared by an aseptic operation method. Also, a sterile solid preparation, for example a lyophilized product, can be prepared and dissolved in sterilized or sterile distilled water for injection or other solvent before use.

[0068] All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0069] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0070] (Example 1: Specificity of CD106 expression to tumor-toxic T cells) (material and method) Tumor tissue was obtained from a surgical specimen of a melanoma patient who had responded well to anti-PD-1 antibody therapy before the start of anti-PD-1 antibody therapy. The tumor tissue was enzymatically treated with collagenase and DNase by stirring at 37°C for 30 minutes to separate cells. CD3-positive cells were sorted from the mixture of tumor tissue cells and tumor-infiltrating immune cells using a BD FACSAria III to obtain TCR-expressing T cells.

[0071] For each of the obtained T cells, both the gene expression of marker candidates and the TCR sequence were measured by sequencing using 10X Chronium 5 Prime Kit and V(D)J Enrichment Kit. The sequencer used was HISEQ3000.

[0072] The fastq files were used to create a UMAP plot using the umap library in Python, and the expression of each gene was mapped onto the resulting UMAP plot.

[0073] (result) The results are shown in Figure 1. In each panel of Figure 1, cells positive for the expression of the markers described are shown in color. The cells are clustered according to their expression patterns, and the circled areas correspond to tumor-toxic T cells. It is understood that CD106 (VCAM1) positive cells are expressed with high specificity in the tumor-toxic T cell population. CD106 can be used as a selection marker for tumor-toxic T cells with higher specificity than other surface markers. The TCR sequence was obtained for β chains in more than 90% of the cells and α chains in 60-90% of the cells.

[0074] Example 2: Tumor aggressiveness of CD106 positive cell population (material and method) As in Example 1, cells from tumor tissue were separated, and CD3-positive cells were sorted from the mixture using BD FACSAria III to obtain TCR-expressing T cells as tumor-infiltrating T cells. The tumor-infiltrating T cells were co-cultured with a cell line established from an autologous tumor, and IFNγ secretion was compared with that in the unstimulated case. IFNγ secretion was measured by intracellular staining using anti-IFNγ antibody. Cell staining was performed with anti-PD-1 antibody, anti-TIGIT antibody, anti-LAG3 antibody, and anti-CD106 antibody. As a negative control, an isotype control antibody against the anti-CD106 antibody was used.

[0075] (result) The results are shown in Figure 2. IFNγ was released by stimulation in the CD106-positive fraction, and IFNγ was released by stimulation in the other marker-positive fractions, but also in the other marker-negative fractions, indicating that the specificity is inferior to that of CD106.

[0076] Example 3: Cell Production Tumor tissue from a patient is obtained and cells are separated by enzymatic digestion. Tumor-infiltrating T cells are isolated as a CD3-positive fraction by flow cytometry. CD106-positive and CD8-positive fractions are further isolated from the CD3-positive fraction.

[0077] The cells of the isolated fraction are cultured and expanded to produce a cell medicine.

[0078] The nucleic acid sequence of the isolated fraction of cells is sequenced to obtain the TCR sequence. A vector carrying the obtained TCR sequence is designed and introduced into other cells. The introduced cells are cultured and expanded as necessary to produce a cell medicine.

[0079] (Example 4: Prediction of the effect of cancer immunotherapy) Obtain information about tumor-infiltrating T cells in patients receiving cancer immunotherapy.

[0080] CD106 on tumor-infiltrating T cells + A subpopulation of cells within the T cell population (CD106 + T cell subpopulation, CD106 + CD8 + T cell subpopulation, LAG-3 + CD106 + CD8 + T cell subpopulations, PD-1 + CD106 + CD8 + T cell subpopulation, TIM3 + CD106 + CD8 + T cell subpopulation, 4-1BB + CD106 + CD8 + T cell subpopulation, TIGIT + CD106 + CD8 + T cell subpopulation, ITGAE + CD106 + CD8 + T cell subpopulation, ENTPD1 + CD106 + CD8 + T cell subpopulation, CTLA-4 + CD106 + CD8 + Obtain the amount of T cell subpopulations.

[0081] By comparing the therapeutic effect (CR, PR, SD, PD) of patients receiving cancer immunotherapy with the amount of cell subpopulations, we will verify that the amount of specific cell subpopulations can be used as an indicator.

[0082] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the claims. It is understood that the patents, patent applications and other documents cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically described in this specification. This application claims priority to Japanese Patent Application No. 2019-128517 filed on July 10, 2019 at the Japan Patent Office, and all of the contents of that application are incorporated by reference in this application. [Industrial Applicability]

[0083] The present disclosure can be used in the fields of medicine, pharmaceuticals, healthcare, biology, biochemistry, and the like.

Claims

1. CD106 from tumor tissue-infiltrating T cell populations ＋ A composition for treating cancer in a subject, comprising a purified subpopulation of T cells, the subpopulation comprising CD106 ＋ T cell subpopulation, CD106 ＋ CD8 ＋ T cell subpopulation, LAG-3 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, PD-1 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, TIM3 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, 4-1BB ＋ CD106 ＋ CD8 ＋ T cell subpopulation, TIGIT ＋ CD106 ＋ CD8 ＋ T cell subpopulations, ITGAE ＋ CD106 ＋ CD8 ＋ T cell subpopulation, ENTPD1 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, or CTLA-4 ＋ CD106 ＋ CD8 ＋ A composition comprising a T cell subpopulation, said cell subpopulation being derived from said subject, said cell subpopulation expressing a tumor-specific TCR.

2. 2. The composition of claim 1, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof.

3. The composition according to claim 1 or 2, which is used in combination with cancer immunotherapy.

4. CD106 from tumor tissue-infiltrating T cell populations ＋ A composition for treating cancer in a subject, comprising cells expressing a TCR expressed only by a purified subpopulation of cells from a T cell population, or a TCR having the same antigen specificity as the purified subpopulation of cells, wherein the subpopulation of cells expresses CD106 ＋ T cell subpopulation, CD106 ＋ CD8 ＋ T cell subpopulation, LAG-3 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, PD-1 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, TIM3 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, 4-1BB ＋ CD106 ＋ CD8 ＋ T cell subpopulation, TIGIT ＋ CD106 ＋ CD8 ＋ T cell subpopulations, ITGAE ＋ CD106 ＋ CD8 ＋ T cell subpopulation, ENTPD1 ＋ CD106 ＋ CD8 ＋ T cell subpopulation, or CTLA-4 ＋ CD106 ＋ CD8 ＋ A composition comprising a T cell subpopulation, said cell subpopulation being derived from said subject, said cell subpopulation expressing a tumor-specific TCR.

5. 5. The composition of claim 4, wherein the cell subpopulation is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof.

6. The composition according to claim 4 or 5, which is used in combination with cancer immunotherapy.

7. A composition described in any one of claims 4 to 6, wherein the TCR is endogenous.

8. The composition according to any one of claims 4 to 6, wherein the TCR is exogenously introduced into the cell.

9. 6. The composition of claim 4 or 5, wherein the cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them.

10. CD106 from T cell population ＋ A method for producing the composition according to any one of claims 1 to 9, comprising the step of purifying a T cell population.

11. The method of claim 10, further comprising the step of purifying a cell population positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof.

12. The method of claim 10 or 11, wherein the T cell population is isolated from T cells that have infiltrated tumor tissue.

13. A method for obtaining a tumor-specific T cell receptor (TCR) sequence in vitro, the method comprising the steps of: isolating a CD106 expression positive T cell population from an isolated tumor-infiltrating T cell population; and obtaining a TCR sequence of a T cell in the T cell population.

14. 14. The method of claim 13, wherein said isolating step further comprises isolating a cell population that is further positive for a cell marker selected from the group consisting of LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof.

15. 15. The method of claim 13 or 14, wherein obtaining the sequence of the TCR comprises sequencing the nucleic acid sequence of the T cell.

16. The method according to any one of claims 13 to 15, further comprising the step of obtaining a TCR sequence having the same antigen specificity as the TCR of a T cell in the T cell population.

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