Specific markers for identifying T cells that specifically attack cancer cells

Abstract

The present invention provides a specific marker for identifying T cells that specifically attack cancer cells and its use. In the present invention, a marker with high specificity for identifying T cells that attack cancer cells is provided. In the present invention, a marker using tumor tissue infiltrating CD106 + The cell subpopulation contained in the T cell group or the cell expressing the T cell receptor (TCR) expressed by it is used to treat or prevent cancer in the subject. In another embodiment of the present invention, a method for manufacturing a cell medicine can be provided. In the present invention, a method for using the amount of a cell subpopulation as an indicator of the responsiveness of the subject to cancer immunotherapy can also be provided. In the present invention, a method for obtaining a tumor-specific TCR sequence can also be provided.

Description

Technical Field

[0001] The present invention relates to the medical or pharmaceutical field. Specifically, it relates to a technology for identifying T cells that specifically attack cancer cells, and may relate to the treatment of cancer. For example, the present invention may relate to an adoptive cell therapy or a method for preparing cells for adoptive cell therapy, the effect prediction of cancer immunotherapy, the acquisition of tumor-specific sequences, etc. Background Art

[0002] In cancer treatment, cancer immunotherapy has attracted much attention. In particular, immune checkpoint inhibitors such as the anti-PD-1 antibody Nivolumab have shown a significant improvement in overall survival compared to docetaxel, the previous standard treatment for non-small cell lung cancer, and have become the standard treatment. For cancer tissues, the infiltration of immune cells such as T lymphocytes is believed to be associated with the defense response to cancer cells. However, it is known that not all infiltrating immune cells attack cancer cells. Research on the introduction of immune cells with the ability to attack tumors (adoptive immunotherapy) to treat cancer is underway. Summary of the invention

[0003] Problem Solution

[0004] The inventors have found that CD106-positive tumor-infiltrating T cells specifically attack cancer cells locally in the tumor, and CD106 can be used as a highly specific marker for identifying T cells that attack cancer cells. One embodiment of the present invention may involve using tumor tissue infiltrating CD106 + The cell subpopulations included in the T cell population are used to treat or prevent cancer in a subject. One embodiment of the present invention may involve using tumor tissue infiltrating CD106 + A T cell receptor (TCR) expressed by a cell subpopulation contained in a T cell population or a cell expressing a TCR having the same antigen specificity as the T cell receptor (TCR) is used to treat or prevent cancer in a subject. Another embodiment of the present invention may involve purifying CD106 from a T cell population. + In the present invention, a method for producing a cell medicine by a process of removing a T cell population from a subject can also be provided. + A method for using the amount of a cell subpopulation included in a T cell population as an indicator of the responsiveness of a subject to cancer immunotherapy. The present invention also provides a method for obtaining a sequence of a tumor-specific TCR.

[0005] For example, in a preferred embodiment of the present invention, the following items are provided.

[0006] (Item 1)

[0007] A composition for treating or preventing cancer in a subject, comprising tumor tissue infiltrating CD106+ The cell subsets included in the T cell population.

[0008] (Item 2)

[0009] According to the composition described in the above item, the cell subpopulation is further positive for cell markers selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0010] (Item 3)

[0011] The composition according to any of the above items, wherein the cell subpopulation includes T cells from the subject.

[0012] (Item 4)

[0013] The composition according to any one of the above items is characterized by being used in combination with cancer immunotherapy.

[0014] (Item 5)

[0015] A composition according to any of the above items, wherein the cell subpopulation expresses tumor-specific TCR.

[0016] (Item 6)

[0017] A composition for treating or preventing cancer in a subject, comprising expressing tumor tissue infiltrating CD106 + The T cell population includes a cell subpopulation that expresses a TCR or a TCR with the same antigen specificity.

[0018] (Item 7)

[0019] A composition according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0020] (Item 8)

[0021] The composition according to any of the above items, wherein the cells include T cells from the subject.

[0022] (Item 9)

[0023] The composition according to any one of the above items is characterized by being used in combination with cancer immunotherapy.

[0024] (Item 10)

[0025] The composition according to any one of the above items, wherein the cell is contained in the cell subpopulation, and the TCR is an endogenous TCR.

[0026] (Item 11)

[0027] The composition according to any one of the above items, wherein the TCR is introduced into the cell from the outside.

[0028] (Item 12)

[0029] The composition according to any one of the above items, wherein the cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them.

[0030] (Item 13)

[0031] The composition according to any one of the above items further comprises a pharmaceutically acceptable carrier.

[0032] (Item 14)

[0033] A method for treating tumor tissue infiltrating CD106 + Use of a cell subpopulation comprised of a T cell population in the manufacture of a medicament for treating or preventing cancer in a subject.

[0034] (Item 15)

[0035] The use according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0036] (Item 16)

[0037] The use according to any of the above items, wherein the cell subpopulation includes T cells from the subject.

[0038] (Item 17)

[0039] The use according to any one of the above items is characterized in that the drug is used in combination with cancer immunotherapy.

[0040] (Item 18)

[0041] The use according to any of the above items, wherein the cell subpopulation expresses tumor-specific TCR.

[0042] (Item 19)

[0043] A gene expressing tumor tissue-infiltrating CD106 +Use of a TCR expressed by a cell subpopulation included in a T cell population or a cell having a TCR with the same antigen specificity in the manufacture of a drug for treating or preventing cancer in a subject.

[0044] (Item 20)

[0045] The use according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0046] (Item 21)

[0047] The use according to any of the above items, wherein the cells include T cells from the subject.

[0048] (Item 22)

[0049] The use according to any one of the above items is characterized in that the drug is used in combination with cancer immunotherapy.

[0050] (Item 23)

[0051] The use according to any one of the above items, wherein the cell is contained in the cell subpopulation, and the TCR is an endogenous TCR.

[0052] (Item 24)

[0053] The use according to any one of the above items, wherein the TCR is introduced into the cell from the outside.

[0054] (Item 25)

[0055] The use according to any one of the above items, wherein the cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them.

[0056] (Item 26)

[0057] The use according to any one of the above items, wherein the drug further comprises a pharmaceutically acceptable carrier.

[0058] (Item 27)

[0059] A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a tumor tissue-infiltrating CD106 + The process of identifying cell subsets included in the T cell population.

[0060] (Item 28)

[0061] A method according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0062] (Item 29)

[0063] A method according to any of the above items, wherein the cell subpopulation comprises T cells from the subject.

[0064] (Item 30)

[0065] The method according to any one of the above items is characterized by being used in combination with cancer immunotherapy.

[0066] (Item 31)

[0067] A method according to any of the above items, wherein the cell subpopulation expresses tumor-specific TCR.

[0068] (Item 32)

[0069] A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a tumor tissue-infiltrating CD106 + A step of administering a TCR expressed by a cell subpopulation included in a T cell population, or a cell expressing a TCR having the same antigen specificity as the TCR, in an effective amount.

[0070] (Item 33)

[0071] A method according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0072] (Item 34)

[0073] The method according to any of the above items, wherein the cells include T cells from the subject.

[0074] (Item 35)

[0075] The method according to any one of the above items, further comprising the step of performing cancer immunotherapy.

[0076] (Item 36)

[0077] The method according to any one of the above items, wherein the cell is contained in the cell subpopulation and the TCR is an endogenous TCR.

[0078] (Item 37)

[0079] The method according to any one of the above items, wherein the TCR is introduced into the cell from the outside.

[0080] (Item 38)

[0081] The method according to any one of the above items, wherein the cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them.

[0082] (Item 39)

[0083] A method for treating tumor tissue infiltrating CD106 + A subpopulation of cells comprised of a T cell population is used to treat or prevent cancer in a subject.

[0084] (Item 40)

[0085] A cell subpopulation according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0086] (Item 41)

[0087] The cell subpopulation according to any of the above items, wherein the cell subpopulation comprises T cells from the subject.

[0088] (Item 42)

[0089] The cell subpopulation according to any one of the above items is characterized in that the cell subpopulation is used in combination with cancer immunotherapy.

[0090] (Item 43)

[0091] A cell subpopulation according to any of the above items, wherein the cell subpopulation expresses tumor-specific TCR.

[0092] (Item 44)

[0093] A treatment for tumor tissue infiltrating CD106 + The T cell population comprises a cell subpopulation expressing a TCR or cells having a TCR with the same antigen specificity as the TCR, for use in treating or preventing cancer in a subject.

[0094] (Item 45)

[0095] A TCR or cell according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0096] (Item 46)

[0097] A TCR or cell according to any of the above items, wherein the cell comprises a T cell from the subject.

[0098] (Item 47)

[0099] The TCR or cell according to any one of the above items is characterized in that the TCR or cell is used in combination with cancer immunotherapy.

[0100] (Item 48)

[0101] The TCR or cell according to any one of the above items, wherein the cell is contained in the cell subpopulation and the TCR is an endogenous TCR.

[0102] (Item 49)

[0103] The TCR or cell according to any one of the above items, wherein the TCR is introduced into the cell from the outside.

[0104] (Item 50)

[0105] The TCR or cell according to any of the above 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.

[0106] (Item 51)

[0107] A method for producing a cellular drug for treating or preventing cancer in a subject, comprising purifying CD106 from a T cell population + T cell population process.

[0108] (Item 52)

[0109] The method according to any of the above items further includes a step of purifying a cell population that is positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0110] (Item 53)

[0111] The method according to any one of the above items, wherein the T cell population is isolated from T cells infiltrating into tumor tissue.

[0112] (Item 54)

[0113] The method according to any one of the above items, further comprising the step of providing a cellular medicine comprising the purified T cell population.

[0114] (Item 55)

[0115] The method according to any one of the above items, further comprising the step of modifying the purified T cell population.

[0116] (Item 56)

[0117] The method according to any one of the above items, further comprising the step of introducing the purified TCR possessed by the T cell population or a TCR having the same antigen specificity therewith into cells.

[0118] (Item 57)

[0119] The method according to any of the above items, wherein the cells include T cells from the subject.

[0120] (Item 58)

[0121] The method according to any one of the above items, wherein the cells include induced pluripotent stem cells (iPSCs), embryonic stem cells (ES cells), T cells, or T cells differentiated from any of them.

[0122] (Item 59)

[0123] A method of treating a subject's CD106 + A method for using the amount of a cell subpopulation included in a T cell population as an indicator of the responsiveness of the subject to cancer immunotherapy.

[0124] (Item 60)

[0125] The method according to any of the above items, wherein the cancer immunotherapy comprises administration of an immune checkpoint inhibitor.

[0126] (Item 61)

[0127] A method according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0128] (Item 62)

[0129] The method according to any one of the above items, wherein the cell subpopulation is a tumor-infiltrating T cell subpopulation.

[0130] (Item 63)

[0131] A method for diagnosing responsiveness of a subject to cancer immunotherapy, comprising:

[0132] Obtain CD106 from the subject + The process of T cell population,

[0133] Determination of CD106 in the subject + The process of determining the amount of cell subpopulations included in the T cell population, and

[0134] A step of diagnosing the responsiveness of the subject to cancer immunotherapy based on the amount of the cell subpopulation.

[0135] (Item 64)

[0136] The method according to any of the above items, wherein the cancer immunotherapy comprises administration of an immune checkpoint inhibitor.

[0137] (Item 65)

[0138] A method according to any of the above items, wherein the cell subpopulation is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0139] (Item 66)

[0140] The method according to any one of the above items, wherein the cell subpopulation is a tumor-infiltrating T cell subpopulation.

[0141] (Item 67)

[0142] A method for obtaining tumor-specific T cell receptor (TCR) sequences, comprising

[0143] A step of isolating a CD106-positive T cell population from tumor-infiltrating T cells, and

[0144] A step of obtaining the TCR sequence of T cells in the T cell population.

[0145] (Item 68)

[0146] A method according to any of the above items, wherein the separation step further includes isolating a cell population that is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4 and any combination thereof.

[0147] (Item 69)

[0148] The method according to any one of the above items, wherein the step of obtaining the TCR sequence comprises sequencing the nucleic acid sequence of the T cell.

[0149] (Item 70)

[0150] The method according to any one of the above items, further comprising the step of obtaining a TCR sequence having the same antigen specificity as that of the TCR of the T cells of the T cell population.

[0151] In the present invention, in addition to the explicit combinations, the above one or more features may be further combined to provide. As required, those skilled in the art will understand and appreciate further embodiments and advantages of the present invention after reading and understanding the following detailed description.

[0152] Effects of the Invention

[0153] A highly specific marker can be provided as a marker for T cells that attack tumors. Since CD106 is also a surface marker, it can be used for cell separation, and T cells that specifically attack tumors can be easily extracted. If such T cells can be efficiently increased, or T cell receptor sequences can be analyzed for artificial production, it is possible to obtain tumor-specific cell therapy with very few side effects and good effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0154] [ Figure 1 ] Figure 1 The figure shows the expression of various markers in tumor-infiltrating T cells. In each panel, cells that are positive for the expression of the above markers are colored. Cells are clustered according to their expression patterns, and the circled parts correspond to tumor-killing T cells.

[0155] [ Figure 2 ] Figure 2 This is a diagram showing the secretion of IFNγ when autologous tumors are stimulated in cell subsets sorted by marker expression. IFNγ is produced in CD106-positive parts due to stimulation, and although IFNγ is produced in other marker-positive parts due to stimulation, IFNγ is also produced in other marker-negative parts, which shows that the specificity is poorer than that of CD106. DETAILED DESCRIPTION

[0156] The present invention is described below. Throughout this specification, the expression of the singular form, unless otherwise specified, should be understood as including the concept of its plural form. Therefore, the singular article (for example, "a", "an", "the", etc. in English), unless otherwise specified, should be understood as including the concept of its plural form. In addition, the terms used in this specification, unless otherwise specified, should be understood as the meaning commonly used in the field. Therefore, unless otherwise defined, all professional vocabulary and scientific and technical vocabulary used in this specification have the same meaning as commonly understood by technicians in the field to which this specification belongs. In the event of a conflict, this specification (including definitions) takes precedence.

[0157] (definition)

[0158] In this specification, "about" means ±10% of the following numerical value unless otherwise specified.

[0159] As used herein, "biomarker" refers to a property that is objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions.

[0160] In this specification, "cancer" or "cancer" are used interchangeably and refer to a malignant tumor or a state in which such a malignant tumor exists, which has strong heterogeneity, proliferates faster than normal cells, and can destructively infiltrate or metastasize to surrounding tissues. In the present invention, cancer includes solid cancer and blood tumor, but is not limited thereto.

[0161] In this specification, "cancer immunotherapy" refers to a method of treating cancer using the body's defense mechanism such as the immune mechanism possessed by a living organism.

[0162] In this specification, "cell subset" refers to a collection of any cells with some common characteristics in a cell population containing cells with multiple characteristics. Specific names are well known in the art, and related terms can be used to describe specific cell subsets, or any properties (such as the expression of cell surface markers) can be described to describe specific cell subsets.

[0163] In this specification, the term "relative amount" related to cells can be used interchangeably with "ratio". For example, the terms "relative amount" and "ratio" refer to the amount of cells that form a specific cell population (e.g., CD8 + T cell population) to form the expected cell subsets (e.g., CD106 + CD8 + T cell subsets).

[0164] In this specification, "baseline" refers to an amount used as a comparison object for determining the increase or decrease in the amount or level of a marker described in this specification. When determining the increase or decrease in an amount or level before and after a treatment, for example, the amount or level before the treatment can be used as the "baseline".

[0165] (Division and separation of cells)

[0166] The samples used for cell division and separation can be appropriately collected from the subject by common methods. For example, they can be collected from the subject's peripheral blood, bone marrow, tumor tissue, hematopoietic tissue, spleen, normal tissue, lymph fluid, etc. By isolating the immune cells infiltrating the tumor tissue and further screening as needed, cells that attack cancer can be obtained.

[0167] Those skilled in the art can use common methods to determine the composition of immune cells in a subject's sample. In general, flow cytometry can be used to determine the number of cells that are positive for a marker (e.g., CD4) that defines a target cell subset in a sample. Flow cytometry is generally used to determine the composition of a cell population, but immunostaining of cell-containing samples, methods using antibody arrays, protein expression analysis in cell-containing samples (e.g., Western blot, mass spectrometry, HPLC, etc.), mRNA expression analysis in cell-containing samples (e.g., microarray, next generation sequencing technology), etc. can also be used. In calculating CD106 + CD8 + When determining the number of cells in a cell subpopulation such as a T cell subpopulation, the number can be determined by experimentally removing cells other than those in each cell subpopulation from the total cell population.

[0168] By using single-cell gene expression analysis, the mRNA in each cell is examined to check whether there is mRNA encoding the protein molecule of interest, thereby identifying the cell. Examples of single-cell gene expression analysis include 1) a method of performing next-generation sequencing using Quartz-Seq, 2) a method of separating cells using the Fluidigm C1 System or ICELL8 Slngle-Cell System and preparing a library using SMART-Seq v4, 3) a method of separating cells using a cell sorter and measuring quantitative PCR using the Ambion single Cell-to-CT kit, and 4) CyTOF SYSTEM (Helios). Just in case, the number of cells is calculated from tumor-infiltrating cells, and the cells are separated using a cell sorter, etc. For each separated cell, for example, using the Ambion Single Cell-to-CT kit, the expression level of a specific gene can be measured using a device using the quantitative PCR method. Based on the results, it can be checked whether each cell belongs to which subpopulation, such as CD106. + CD8 + T cell subsets, etc., and calculate the number of cells belonging to each subset.

[0169] In this specification, the expression of a certain marker is "positive", or "(marker)+ Cells” can be confirmed by detecting more than one read when single-cell sequencing is used for confirmation, and by comparing with isotype controls when FCM is used for confirmation.

[0170] (cell)

[0171] In the present invention, a cell subpopulation having a specific marker expression pattern can be provided. In one embodiment, a CD106 + A cell subpopulation contained in a cell population. Such a cell subpopulation may be further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1 (CD39), CTLA-4, and any combination thereof. In the present invention, the cell subpopulations described in the present invention can be used as cells for adoptive cell transfer, as cells for obtaining tumor-specific TCR therefrom, as cells for using their amount as an indicator, and / or as cells for modifying them. The cell population can use a tumor tissue infiltrating cell population or a cell subpopulation contained therein. As a surface marker of T cells that specifically attack tumors, PD-1 or 4-1BB markers have been used in the past, and in recent reports, the latest CD103 or CD39 has attracted much attention. In the present invention, it can be seen from the data of single-cell sequencing technology that as a surface marker, CD106 has the highest specificity, and even based on a comprehensive comparison of reported markers, it is very useful compared to these markers.

[0172] In the present invention, cell or cell group can be immune cell, or its precursor cell, or its group. Immune cell can exemplify dendritic cell, macrophage, lymphocyte (for example, T cell (for example, killer T cell, helper T cell, regulatory T cell, αβT cell, γδT cell, natural killer T cell), B cell, NK (natural killer) cell), neutrophil, eosinophil, basophil, and its combination. In the case of using cell therapy or prevention of cancer treatment, preferably cell is T cell. Cell or cell group can include induced pluripotent stem cell (iPSC), embryonic stem cell (ES cell), peripheral T cell, initial T cell, memory T cell, central memory T cell (TCM), T memory stem cell (TSCM) or T cell differentiated by any one thereof.

[0173] The T cells in the present invention may be CD8 positive T cells. Cytotoxic T cells (CTL) are a type of lymphocyte T cells that recognize and destroy cells that are foreign to the host (cancer cells, etc.). CTLs differentiate from T cells that express CD8 molecules on their surfaces.

[0174] In one embodiment of the present invention, a method comprising tumor tissue infiltrating CD106 + A composition for treating or preventing cancer in a subject comprising a cell subpopulation of a T cell population. Alternatively, a composition comprising administering a tumor tissue-infiltrating CD106 + The present invention relates to a method for treating or preventing cancer in a subject by a process for identifying a cell subset included in a T cell population.

[0175] The cell subpopulation may be further positive for a cell marker selected from 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 from a subject that is the subject of treatment or prevention, and may also include T cells from a subject different from the subject that is the subject of treatment or prevention. The cell subpopulation may express a tumor-specific TCR. Cell subpopulations expressing tumor-specific TCRs can be identified by the markers provided by the present invention. In the present invention, a composition comprising cells or adoptive cell transfer therapy can be used in combination with cancer immunotherapy.

[0176] In one embodiment of the present invention, a method for expressing tumor tissue infiltrating CD106 can be provided. + The T cell population contains a cell subpopulation that expresses a TCR or a cell with a TCR that has the same antigen specificity. The cell can be a tumor tissue infiltrating CD106 + The T cell population may contain cells themselves or cells expressing tumor tissue-infiltrating CD106 + The T cell population contains cell subsets that express different TCRs. Such cells can be obtained by introducing TCRs or screening TCRs. + The cell subpopulation included in the T cell population may be further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1 (CD39), CTLA-4, and any combination thereof. + The cell subpopulation contained in the T cell population, the TCR is the endogenous TCR.

[0177] TCR can be introduced into cells externally. The object cell introduced can include, for example, induced pluripotent stem cells (iPSC), embryonic stem cells (ES cells), peripheral T cells, initial T cells, memory T cells, central memory T cells (TCM), T memory stem cells (TSCM) or T cells differentiated by any one of them. The introduction of TCR can be carried out by methods described elsewhere in this description or methods known in the art by those skilled in the art.

[0178] (How to obtain TCR)

[0179] The body defense mechanism of the immune system depends largely on the specific immunity mainly undertaken by T cells and B cells. T cells and B cells can specifically identify and attack foreign pathogens such as viruses and bacteria without reacting with autologous cells and molecules. Therefore, T cells and B cells have a mechanism that can recognize and identify various antigens from other organisms together with self-antigens through receptor molecules expressed on the cell surface. In T cells, T cell receptors (T ceIIreceptor, TCR) act as antigen receptors. Intracellular signals are conveyed by stimulation from these antigen receptors, the production of inflammatory cytokines and chemokines, etc. is aggravated, cell proliferation is enhanced, and various immune responses begin.

[0180] TCR recognizes peptides (peptide-MHC complex, pMHC) that bind to the peptide binding groove of the major histocompatibility complex (MHC) expressed on antigen presenting cells, while recognizing self and non-self, and antigenic peptides (Cell 1994, 76, 287-299). TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains. There are αβ-type TCRs expressed by general T cells and γδ-type TCRs with special functions. α and β chain TCR molecules form a complex with multiple CD3 molecules (CD3 beg chain, CD3ε chain, CD3γ chain, CD3δ chain), convey intracellular signals after antigen recognition, and initiate various immune responses. CD3 molecules exist in the cell membrane and form a complex with TCR, so they can be used as TCR expression markers. Endogenous antigens such as viral antigens that proliferate in cells following viral infection or cancer antigens from cancer cells are presented as antigenic peptides on MHC class I molecules. In addition, antigens from foreign microorganisms are taken up by antigen presenting cells through endocytosis, processed and presented on MHC class II molecules. These antigens are presented on CD8 + T cells or CD4 + The TCR expressed by the T cell is recognized. It is well known that co-stimulatory molecules such as CD28, ICOS, and OX40 molecules are very important for the stimulation mediated by TCR molecules.

[0181] The TCR gene is composed of multiple V regions (variable region, V), J regions (joining region, J), D regions (diversity region, D) and constant regions C regions (constant region, C) encoded in different regions of the chromosome set. During the differentiation process of T cells, these gene fragments undergo genetic recombination in various combinations, and the α chain and γ chain TCRs express genes composed of VJC, and the β chain and δ chain TCRs express genes composed of VDJC. Diversity is created through the recombination of these gene fragments, and by inserting or deleting more than one base between the V and D or D and J gene fragments to form a random amino acid sequence, a TCR gene sequence with higher diversity can be produced.

[0182] The region (TCR footprint) that TCR molecules and pMHC complex surfaces directly bind is composed of the complementary determining region (complementarity determining region, CDR) CDR1, CDR2 and CDR3 regions rich in diversity in the V region. Among them, the CDR3 region includes a part of the V region, a part of the VDJ region and the J region formed by random sequences, forming the most diverse antigen recognition site. On the other hand, other regions are called FR (framework region), which plays a role in forming the skeleton structure of TCR molecules. During the differentiation and maturation of T cells in the thymus, the β chain TCR first undergoes genetic recombination, associates with pT α molecules, and forms pre-TCR complex molecules. Afterwards, the α chain TCR recombines to form αβTCR molecules, and when functional αβTCR is not formed, recombination occurs in another α chain TCR gene allele. It is known that through positive and negative selection in the thymus, TCR with appropriate affinity is selected to obtain antigen specificity (Annual Review Immunology, 1993, 6, 309-326).

[0183] T cells produce one type of TCR that is highly specific to a particular antigen. Since there are many antigen-specific T cells in an organism, a diverse TCR repertoire is formed, which can effectively exert a defense mechanism against various pathogens.

[0184] In one embodiment of the present invention, a method for obtaining a sequence of a tumor-specific T cell receptor (TCR) can be provided. Although it is desired not to be bound by theory, in this description, it is shown that a cell subset contained in a CD106-expressing positive T cell group specifically attacks cancer, and it is considered that by separating such a cell subset, a tumor-specific TCR can be separated. In the present invention, a method for obtaining a tumor-specific T cell receptor (TCR) sequence can be provided, including a process for separating a CD106-expressing positive T cell group from tumor-infiltrating T cells, and a process for obtaining the TCR sequence of T cells of the T cell group.

[0185] The above-mentioned separation step may further include isolating a cell population that is further positive for any other marker disclosed in the present specification (for example, a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1, ​​CTLA-4 and any combination thereof).

[0186] The acquisition of TCR sequence may include sequencing of T cell nucleic acid sequence. As long as the sequence of nucleic acid sample can be sequenced, the sequencing method is not limited, and any method known in the art can be used, but preferably next generation sequencing technology (NGS). Next generation sequencing technology can exemplify pyrosequencing, sequencing by synthesis, ligation sequencing, ion semiconductor sequencing, etc., but is not limited thereto. TCR sequence can be obtained from mRNA that does not contain introns by amplicon sequencing. In addition, TCR sequence can be obtained by sequencing T cell genome sequence. Alternatively, TCR sequence can also be obtained by directly determining the amino acid sequence of TCR molecule by using mass spectrometry or Edman degradation, etc. In this specification, "TCR sequence" can use any of amino acid sequence and nucleic acid sequence.

[0187] (TCR Clustering)

[0188] In the present invention, a TCR having the same antigen specificity as a tumor-specific TCR can be used. For example, a TCR having the same antigen specificity as CD106 can be used. + The TCRs of the T cell population have the same antigen specificity. "Having the same antigen specificity" can be determined by two TCRs being classified into the same cluster by the following cluster analysis. In the present invention, when obtaining tumor-specific TCRs, a TCR that is similar to CD106 can be obtained. +The TCRs of the T cells of the separated T cell populations such as T cell populations have the same antigen-specific TCR sequence. This can be achieved by clustering one or more TCRs with the TCRs of the separated T cell populations as described below. This type of analysis technique is described in detail in Patent Gazette No. 6500144, the contents of which are cited in this specification as a reference.

[0189] Cluster analysis of TCRs can be performed by an analysis method comprising the following steps: (i) a step of providing at least two TCR feature values, which does not include calculating the feature values ​​from a three-dimensional structural model of the at least two TCRs, (ii) a step of performing machine learning on the analysis of the antigen specificity or binding pattern of the TCR based on the feature values ​​without specifying the antigen specificity or binding pattern, and (iii) a step of classifying the antigen specificity or binding pattern or determining similarities and differences.

[0190] In other embodiments, the TCR set can be analyzed by a method comprising the following steps: (a) a step of extracting feature values ​​for at least one pair of members of the TCR set, which step does not include calculating feature values ​​from a three-dimensional structural model of the at least one pair, (b) a step of calculating the distance between the antigen specificity or binding patterns of the pair, or determining whether the antigen specificity or binding patterns are consistent by machine learning using the feature values, (c) a step of clustering the TCR set based on the distance, and (d) a step of performing analysis based on the classification of the clustering as needed.

[0191] In other embodiments, the TCR set can be analyzed by a method comprising the following steps: (aa) the step of extracting feature values ​​for at least one pair of sequences that are members of the TCR set, respectively, which step does not include calculating feature values ​​from a three-dimensional structural model of the immune entity that is the at least one pair of sequences, (bb) the step of projecting the feature values ​​into a high-dimensional vector space, where the spatial distance of the members reflects the functional similarity of the members, (cc) the step of clustering the set of immune entities based on the distance, and (dd) the step of performing analysis based on the classification of the clusters as needed.

[0192] The feature quantity may include at least one selected from sequence information, the length of CDR1-3 sequences, sequence identity, sequence identity of the framework region, the total charge / hydrophilicity / hydrophobicity / number of aromatic amino acids in the molecule, each CDR, the charge / hydrophilicity / hydrophobicity / number of aromatic amino acids in the framework region, the number of each amino acid, the heavy chain-light chain combination, the number of somatic mutations, the position of mutations, the presence / identity of amino acid motifs, the rarity relative to a reference sequence set, and the probability of the reference sequence binding to HLA.

[0193] The calculation by machine learning can be performed by inputting feature quantities, using random forest or boosting method, and clustering can be performed by simple clustering based on threshold value of combined distance, hierarchical clustering, or non-hierarchical clustering. Machine learning can be selected from machine learning algorithms such as regression method, neural network method, support vector machine and random forest.

[0194] (Method of preparing cells)

[0195] In the present invention, a method for preparing cells, especially cells for use as medicine, can be provided. Although it is not intended to be bound by theory, CD106 in tumors can be used to + CD8 + The cells included in the T cell population express tumor-specific TCRs, and cells that attack tumors can be obtained. Such cells can be used to treat or prevent cancer.

[0196] In one embodiment of the present invention, a method for producing a cellular drug for treating or preventing cancer in a subject may be provided, comprising purifying CD106 from a T cell population. + The method may further include a step of purifying a cell population that is positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1, ​​CTLA-4, and any combination thereof. The T cell population may be separated from T cells that infiltrate tumor tissue.

[0197] A cell medicine comprising a purified T cell population can be provided. It is believed that this T cell population has a tumor-specific TCR and is very useful as a cell medicine. As needed, the purified T cell population can be cultured and / or proliferated and provided as a medicine. The purified T cell population can be further modified as needed. The modification of T cells can be exemplified by stimulation of integrins α4β7 or α4β1 (VLA-4) that interact with CD106, CD3, or IL-2, IL-4, IL-7.

[0198] In some embodiments of the present invention, a method comprising a process of introducing a TCR possessed by a purified T cell population or a TCR having the same antigen specificity into a cell can be provided. The acquisition and / or introduction of TCR can be carried out by the methods described elsewhere in this specification or other methods known to those skilled in the art. Tumor-specific TCR can modify the obtained sequence and import it as needed. As the cell to be introduced, T cells from a subject as a treatment or prevention object can be used, or any other immune cells can be used. As the cell to be introduced, for example, induced pluripotent stem cells (iPSC), embryonic stem cells (ES cells), peripheral T cells, central memory T cells (TCM), T memory stem cells (TSCM), initial T cells, memory T cells, or T cells differentiated by any of them.

[0199] (cancer)

[0200] In the present invention, examples of cancers that can be targeted include lung cancer, kidney (renal cell) cancer, prostate cancer, stomach cancer, testicular cancer, liver (intestinal) cancer, skin cancer, esophageal cancer, melanoma, pancreatic cancer, pancreatic cancer, bone tumors, osteosarcoma, colorectal cancer, bone and soft tissue tumors, 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, thymoma, thyroid cancer, mesothelioma, anal cancer, penile cancer, cancer of unknown primary site, ovarian cancer, breast cancer, etc., but are not limited to these.

[0201] (Cancer Immunotherapy)

[0202] In this specification, "cancer immunotherapy" refers to a method for treating cancer using the immune function possessed by an organism. Cancer immunotherapy is roughly divided into cancer immunotherapy that strengthens the immune function against cancer, and cancer immunotherapy that suppresses the immune avoidance function of cancer. Further, cancer immunotherapy has active immunotherapy that activates the immune function in vivo, and passive immunotherapy that activates the immune function in vitro, or returns the proliferated immune cells to the body. As examples of cancer immunotherapy, nonspecific immunostimulants, cytokine therapy, cancer vaccine therapy, dendritic cell therapy, adoptive immunotherapy, nonspecific lymphocyte therapy, cancer antigen-specific T cell therapy, antibody therapy, immune checkpoint inhibition therapy, CAR-T therapy, etc. can be cited. In the present invention, a method for using the amount of a cell subset that specifically attacks a tumor as an indicator of the responsiveness of a subject to cancer immunotherapy can be provided.

[0203] In one embodiment, a method of treating a subject's CD106 +The amount of the cell subpopulation contained in the T cell group is used as a method for the subject's responsiveness index to cancer immunotherapy. Cancer immunotherapy may include the administration of immune checkpoint inhibitors. The cell subpopulation can use a cell subpopulation that is further positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, TIGIT, ITGAE (CD103), ENTPD1, ​​CTLA-4, and any combination thereof. The cell subpopulation can use the amount of any part, but the amount of the cell subpopulation in the tumor-infiltrating T cell can be preferably used.

[0204] Immune checkpoint (inhibition) therapy using immune checkpoint inhibitors has attracted much attention in recent years (Pardo II 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 is related to avoiding attacks from immune cells such as T cells. Therefore, it is believed that under normal conditions, the immune function of the organism alone cannot eliminate cancer tissue. Immune checkpoint inhibitors enable the immune system of the organism to effectively eliminate cancer by inhibiting the ligand-receptor interaction responsible for transmitting inhibitory signals from such cancer tissues to the immune system. One embodiment of the present invention is to inhibit CD106 + A method for using the amount of a cell subpopulation included in a T cell population as an indicator for predicting the responsiveness of a subject to the following immune checkpoint inhibitor. + A method of administering an immune checkpoint inhibitor as described below to a subject selected by the amount of a cell subpopulation included in a T cell population (responsiveness). In another embodiment, a method of discovering a CD106-based + Methods for interrupting or suspending or avoiding immune checkpoint inhibitors in subjects whose T cell populations contain a subpopulation of cells that is not responsive.

[0205] A representative example of immune checkpoint inhibitors is PD-1 inhibitors. Examples of PD-1 inhibitors include the anti-PD-1 antibody Nivolumab (also known as Opdivo TM Pembrolizumab (marketed as Keytruda TMSales), but not limited to this. Anti-PD-1 antibodies are believed to exert their anti-cancer effects by relieving the inhibition of PD-1 signaling on T cell activation. Through the interaction of PD-1 (programmed death receptor 1, programmed death 1) with PD-L1 or PD-L2, and the recruitment of SHP-2, one of the tyrosine dephosphatases, to the PD-1 cytoplasmic domain, the T cell receptor signaling protein ZAP70 is inactivated, thereby inhibiting the activation of T cells (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 believed to interact with CD80 to inhibit 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 believed to be highly expressed in killer T cells and natural killer cells that infiltrate cancer cells, and the immune response is weakened by PD-L1 on the tumor. The weakening of the immune response caused by the PD-1 signal is inhibited by anti-PD-1 antibodies, and the effect of enhancing the anti-tumor immune response can be obtained.

[0206] Other examples of immune checkpoint inhibitors are PD-L1 inhibitors (e.g., anti-PD-L1 antibodies Avelumab, Durvalumab, or Atezolizumab). PD-L1 inhibitors bind to PD-L1 and block the PD-1 pathway, generating an anti-tumor immune response.

[0207] Other examples of immune checkpoint inhibitors include CTLA-4 inhibitors (such as anti-CTLA-4 antibodies Ipilimumab or Tremelimumab). CTLA-4 inhibitors activate T cells in a different way from PD-1 inhibition to produce an anti-tumor immune response. T cells are activated by the interaction of CD28 on the surface with CD80 or CD86. However, even for temporarily activated T cells, the surface-expressed CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) preferentially interacts with CD80 or CD86 with a higher affinity than CD20, thereby inhibiting its activation. CTLA-4 inhibitors inhibit CTLA-4 and prevent the interaction of CD20 with CD80 or CD86 from being inhibited, thereby producing an anti-tumor immune response.

[0208] In a further embodiment, the immune checkpoint inhibitor can target immune checkpoint proteins such as TIM-3, LAG-3, B7-H3, B7-H4, B7-H5 (VISTA), or TIGIT.

[0209] The above immune checkpoints are believed to suppress the immune response to the autologous tissues, but when antigens such as viruses exist in the body for a long time, immune checkpoints will also increase in T cells. As for tumor tissues, since they become antigens that exist in the body for a long time, they are believed to evade anti-tumor immunity through these immune checkpoints, and the above immune checkpoint inhibitors invalidate the above evasion function and exert anti-tumor effects.

[0210] In one embodiment of the present invention, an index for predicting the responsiveness of a subject with cancer to cancer immunotherapy is provided. The response to cancer immunotherapy can be determined based on RECIST v1.1 (New response evaluation criteria in solid tumors: Revised RECIST guideIine (version 1.1)).

[0211] The effect of cancer treatment can be determined by changes in tumor size, such as complete response (CR), partial response (PR), progressive disease (PD), or stable disease (SD).

[0212] In this specification, "responder" refers to a subject who shows complete remission or partial remission for cancer treatment. In this specification, "non-responder" refers to a subject who shows disease progression or stable disease for cancer treatment.

[0213] The responsiveness of a subject to a cancer treatment includes whether the subject is a "patient in remission" or a "patient in non-remission." Therefore, determining the responsiveness of a subject to a cancer treatment includes determining whether the subject is a patient in remission or a patient in non-remission.

[0214] One aspect of the present invention is the use of CD106 + The amount of cell subpopulations contained in the T cell population can be used to predict or judge whether the subject is a "remitted patient" or a "non-remitted patient". The judgment time is preferably predicted before the start of treatment, but it can also be made after the start of treatment. This is because it is also very useful in medicine to judge whether the current treatment is appropriate. Alternatively, the CD106 of the present invention can also be used + The amount of cell subpopulations contained in the T cell population can be used to determine the prognosis. + The amount of cell subpopulations contained in the T cell population can predict whether a patient who has been in remission will turn into a non-remission, i.e., relapse. Furthermore, the judgment time can be after the implementation of cancer immunotherapy (for example, after the administration of immune checkpoint inhibitors), and the cell subpopulation composition can be analyzed over time to judge the prognosis.

[0215] (Adjunctive use of immune checkpoint inhibitors)

[0216] In other aspects of the present invention, the present invention may provide a composition comprising an immune checkpoint inhibitor for use in CD106 + To treat cancer in a subject with a large number of cell subsets included in the T cell population. + It is believed that the administration of the above-mentioned immune checkpoint inhibitors to subjects with a large number of cell subsets included in the T cell population may be beneficial because more cells attack the tumor. + A subject whose T cell population contains a small amount of a cell subset can be judged as a non-remission patient, and the immune checkpoint inhibitor can be judged not to be administered, or the administration can be interrupted or stopped.

[0217] The composition of the present invention is preferably a pharmaceutical composition, and the immune checkpoint inhibitor contained as its active ingredient can be exemplified by PD-1 inhibitors. PD-1 inhibitors can be exemplified by anti-PD-1 antibodies nivolumab or pembrolizumab. The composition can be made into any dosage form such as aerosol, liquid, extract, elixir, capsule, granule, pill, ointment, powder, tablet, solution, suspension, emulsion, etc. The composition may include any pharmaceutically acceptable additive and / or excipient known in the art. The composition of the present invention can be administered by any appropriate route determined by a person skilled in the art, without limitation, but can be exemplified by intravenous injection, drip, oral, parenteral, transdermal, etc.

[0218] (Composition)

[0219] In the present invention, the composition including the immune checkpoint inhibitor or cell can be appropriately used in combination with other cancer treatments. Other cancer treatments can include other cancer immunotherapies, radiotherapy, chemotherapy, thermotherapy, surgery, etc., but are not limited thereto. The composition including the immune checkpoint inhibitor or cell can be administered in combination with one or more other agents. One or more other agents can be any chemotherapy drug, or can include a second immune checkpoint inhibitor.

[0220] In one embodiment of the present invention, a composition comprising an immune checkpoint inhibitor is provided. Generally speaking, the composition comprising an immune checkpoint inhibitor of the present invention is administered systemically or locally in an oral or parenteral form. The composition comprising an immune checkpoint inhibitor of the present invention is administered to a subject who has shown more cells attacking tumors according to the method described in this specification, thereby achieving a significant therapeutic effect.

[0221] The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but generally, for example, 0.1 mg to 100 mg per adult per time, once or multiple times a day, or 0.01 mg to 30 mg per adult per time, once or multiple times a day, parenteral administration (preferably intravenous administration), or continuous intravenous administration for 1 hour to 24 hours a day. Of course, the dosage varies depending on various conditions, so sometimes less than the above dosage can be sufficient, and sometimes more than the above dosage is required.

[0222] When administering a composition comprising an immune checkpoint inhibitor or a composition comprising cells, a solid preparation for oral administration, a liquid preparation for oral administration, and an injection, an external preparation, a suppository, and the like for parenteral administration can be used. Solid preparations for oral administration include tablets, pills, capsules, powders, granules, and the like. Capsules include hard capsules and soft capsules.

[0223] The composition of the present invention can be used as needed, and one or more active ingredients (for example, antibodies to cells or immune checkpoint proteins) are mixed as is, or with excipients (lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (hydroxypropyl cellulose, polyvinyl pyrrolidone, magnesium aluminum metasilicate, etc.), disintegrants (calcium carboxymethyl cellulose, etc.), lubricants (magnesium stearate, etc.), stabilizers, cosolvents (glutamic acid, aspartic acid), etc., and used in a common method formulation. In addition, it can be coated with a coating agent (white sugar, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate) as needed, and it can also be coated with two or more layers. Further, a capsule made of a substance that can be absorbed like gelatin can also be included.

[0224] Injections for parenteral administration include solutions, suspensions, emulsions, and solid injections that are dissolved or suspended in a solvent when used. Injections are used by dissolving, suspending, or emulsifying one or more active substances in a solvent. Solvents include, for example, alcohols such as distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, ethanol, and combinations thereof. Further, the injection may include stabilizers, cosolvents (glutamic acid, aspartic acid, polysorbate 80 (registered trademark), etc.), suspending agents, emulsifiers, analgesics, buffers, preservatives, and the like. These are prepared by sterilization or aseptic operation in the final process. In addition, sterile solid preparations such as freeze-dried products can also be manufactured and dissolved in sterilized or sterile distilled water for injection or other solvents before use.

[0225] References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as their respective specific descriptions.

[0226] In order to facilitate the understanding of the present invention, the present invention is described by showing preferred embodiments. The present invention is described below based on embodiments, but the above description and the following embodiments are only for the purpose of illustration and do not limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments and embodiments specifically described in this specification, but is only limited to the scope of the claims.

[0227] Example

[0228] (Example 1: Specificity of CD106 expression on tumor-killing T cells)

[0229] (Materials and methods)

[0230] Tumor tissue was obtained from surgical specimens of melanoma patients who had significant efficacy with anti-PD-1 antibodies before the start of anti-PD-1 antibody treatment. Tumor tissue was treated with enzymes by adding collagenase and DNase and stirring at 37°C for 30 minutes to separate cells. CD3-positive cells were sorted from a mixture of tumor tissue cells and tumor-infiltrating immune cells using a BD FACSAria III to obtain T cells expressing TCR.

[0231] For each obtained T cell, 10X Chronium's 5 Prime kit and V(D)J enrichment kit were used to measure both the gene expression of the candidate marker and the TCR sequence by sequencing. The sequencer used was HISEQ3000.

[0232] The obtained fastq files were made into UMAP graphs using Python's umap library, and the expression of each gene was mapped onto the obtained UMAP graph.

[0233] (result)

[0234] Figure 1 The results are shown. Figure 1 In each panel, cells expressing the marker positively are colored. Cells are clustered according to the expression pattern, and the circled part corresponds to tumor-killing T cells. It can be understood that positive cell expression of CD106 (VCAM1) has high specificity for the tumor-killing T cell population. CD106 can be used as a tumor-killing T cell selection marker with higher specificity than other surface markers. TCR sequences can be obtained in cells with more than 90% beta chains and 60% to 90% d chains.

[0235] (Example 2: Tumor aggressiveness of CD106 positive cell population)

[0236] (Materials and methods)

[0237] As in Example 1, cells of tumor tissue were separated, CD3-positive cells were sorted from the mixture using BD FACSAria III, and T cells expressing TCR were obtained as tumor-infiltrating T cells. Tumor-infiltrating T cells were co-cultured with cell lines established from autologous tumors, and the secretion of IFNγ was compared with that without stimulation. The secretion of IFNγ was determined by intracellular staining using anti-IFNγ antibodies. Cells were stained with anti-PD-1 antibodies, anti-TIGIT antibodies, anti-LAG3 antibodies, and anti-CD106 antibodies. As a negative control, an isotype control antibody for anti-CD106 antibodies was used.

[0238] (result)

[0239] Figure 2 The results are shown. IFNγ is secreted in CD106-positive regions upon stimulation, and IFNγ is secreted in other marker-positive regions upon stimulation, but IFNγ is also secreted in other marker-negative regions, indicating that the specificity is poor compared to CD106.

[0240] (Example 3: Preparation of cells)

[0241] Tumor tissues were obtained from patients and cells were isolated by enzyme treatment. Tumor-infiltrating T cells were isolated as CD3-positive fractions by flow cytometry. CD106-positive and CD8-positive fractions were further isolated from the CD3-positive fractions.

[0242] The separated cells are cultured and proliferated to be used as cell medicine.

[0243] The nucleic acid sequence of the isolated part of the cells is sequenced to obtain the TCR sequence. A vector with the obtained TCR sequence is designed and introduced into other cells. As needed, the introduced cells are cultured and proliferated to serve as a cell medicine.

[0244] (Example 4: Prediction of the effect of cancer immunotherapy)

[0245] Obtain information about tumor-infiltrating T cells in patients receiving cancer immunotherapy.

[0246] Obtaining CD106 in tumor-infiltrating T cells + T cell population contains cell subsets (CD106 + T cell subsets, CD106 + CD8 + T cell subsets, LAG-3 + CD106 + CD8 + T cell subsets, PD-1 + CD106 + CD8 + T cell subsets, TIM3 + CD106 + CD8 + T cell subsets, 4-1BB + CD106 + CD8 + T cell subsets, TIGIT + CD106 + CD8 + T cell subsets, ITGAE + CD106 + CD8 + T cell subsets, ENTPD1 + CD106+ CD8 + T cell subsets, CTLA-4 + CD106 + CD8 + T cell subsets).

[0247] By comparing the therapeutic effects (CR, PR, SD, PD) of patients who received cancer immunotherapy with the amounts of cell subpopulations, it was verified that the amount of a specific cell subpopulation can serve as an indicator.

[0248] (Note)

[0249] As described above, the present invention is illustrated using the preferred embodiments of the present invention, but the present invention should be interpreted only according to the scope of the claims. It should be understood that the patents, patent applications and other documents cited in this specification are cited as a whole as a reference to this specification in the same manner as their contents are specifically recorded in this specification. This application claims priority based on Japanese Patent Application No. 2019-128517 filed with the Japan Patent Office on July 10, 2019, and all the contents disclosed are cited herein as references.

[0250] Industrial Application Possibility

[0251] The present invention can be used in the fields of medicine, pharmaceuticals, health care, biology, biochemistry, etc.

Claims

1. A composition for treating cancer in a subject, wherein the tumor tissue is infiltrated with CD106 + The composition of the cell subsets included in the T cell population; The tumor tissue infiltrates CD106 + The cell subsets included in the T cell population are CD106 + T cell subsets selected from CD106 + CD8 + T cell subsets, LAG-3 + CD106 + CD8 + T cell subsets, PD-1 + CD106 + CD8 + T cell subsets, TIM3 + CD106 + CD8 + T cell subsets, 4-1BB + CD106 + CD8 + T cell subsets, TIGIT + CD106 + CD8 + T cell subsets, ITGAE + CD106 + CD8 + T cell subsets, ENTPD1 + CD106 + CD8 + T cell subsets or CTLA-4 + CD106 + CD8 + The invention relates to a T cell subset, wherein the cell subset is from the subject, and the cell subset expresses a tumor-specific TCR.

2. The composition according to claim 1, wherein the cell subpopulation is further positive for a cell marker selected from 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, characterized in that Used in combination with cancer immunotherapy.

4. A method for producing a cellular drug for treating cancer in a subject, comprising purifying CD106 from a T cell population + The process of T cell population; in, The CD106 + T cell population is CD106 + T cell subsets selected from CD106 + CD8 + T cell subsets, LAG-3 + CD106 + CD8 + T cell subsets, PD-1 + CD106 + CD8 + T cell subsets, TIM3 + CD106 + CD8 + T cell subsets, 4-1BB + CD106 + CD8 + T cell subsets, TIGIT + CD106 + CD8 + T cell subsets, ITGAE + CD106 + CD8 + T cell subsets, ENTPD1 + CD106 + CD8 + T cell subsets or CTLA-4 + CD106 + CD8 + The invention relates to a T cell subset, wherein the cell subset is from the subject, and the cell subset expresses a tumor-specific TCR.

5. The method according to claim 4, further comprising the step of purifying a cell population that is positive for a cell marker selected from LAG-3, PD-1, TIM3, 4-1BB, ITGAE (CD103), TIGIT, ENTPD1 (CD39), CTLA-4, and any combination thereof. The method according to claim 4 or 5, wherein the T cell population is isolated from T cells infiltrating into tumor tissue. 7 . The method according to claim 6 , further comprising the step of providing a cellular medicine comprising the purified T cell population.

8. The method according to claim 7, further comprising the step of modifying the purified T cell population. 9 . The method according to claim 4 , further comprising the step of introducing the TCR possessed by the purified T cell population or a TCR having the same antigen specificity therewith into cells.

10. The method of claim 9, wherein the cells comprise T cells from the subject.

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