Tumor-specific immune cell marker and application thereof

Abstract

The invention relates to a tumor specific immune cell marker and application thereof. The immune cell marker combination comprises (1) an immune cell activation marker and (2) an immune cell inhibition marker, and optionally further comprises (3) an intracellular marker and / or (4) a tissue residence memory marker. The marker combination provided by the invention can more accurately identify and enrich tumor specific immune cells, and the enriched cell population has better tumor reactivity and tumor killing function.

Description

[0001] This application is a divisional application of Chinese application 202310242995.1 filed on March 14, 2023. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to tumor-specific immune cell markers and uses thereof. Background Art

[0003] In recent years, tumor infiltrating lymphocyte (TIL) therapy has become an important type of therapy for solid tumors in tumor immune cell therapy technology, and has achieved very significant clinical results for a variety of solid tumors, such as melanoma, cervical cancer, head and neck tumors, and non-small cell lung cancer. TIL is mainly composed of T cells, which is a group of heterogeneous T cells, including tumor-specific T cells and bystander T cells, that is, T cells that specifically recognize tumor antigens and T cells that recognize antigen epitopes unrelated to tumors. Bystander TIL usually includes T cells that recognize viral antigens, such as EBV, HCMV, and influenza virus antigen epitopes. In the context of TIL as adoptive T cell therapy (ACT) for tumor treatment, how to increase the content of tumor-specific T cells in TIL, reduce the proportion of bystander T cells, and obtain TIL cell populations that can persist in the body for a long time have become crucial factors in improving efficacy.

[0004] Published reports mention a variety of different indicators to distinguish tumor-specific T cell populations from bystander T cell populations in TIL. Thomas Duhen et al. Nat Commun. 2018 Jul 13; 9 (1): 2724. It is recorded that in various primary and metastatic tumor CD8+ TIL cells, tumor antigen-specific T cells are highly enriched in the CD39+CD103+ cell population. Kim E Kortekaas et al. Cancer Immunol Res. 2020 Oct; 8 (10): 1311-1321. It is recorded that tumor-specific CD4+ T cells are concentrated in the CD39+ TIL subpopulation. WO2021226085A1 discloses a method for amplifying TIL for clinical therapeutic use, which includes a step of sorting TILs that express positive specific indicators, and the specific indicators include PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3 and / or TIGIT. However, more accurate and effective standards are still needed to identify and / or sort TILs, in order to determine the proportion of tumor-specific and tumor-reactive T cells in the prepared TIL cells, and possibly to further sort and amplify these cells to improve the clinical efficacy of TILs. Summary of the invention

[0005] The first aspect of the present invention provides an immune cell marker combination, comprising: (1) an immune cell activation marker, and (2) an immune cell inhibition marker,

[0006] Optionally, it also includes (3) an intracellular marker, and / or (4) a tissue-resident memory marker.

[0007] In one or more embodiments, the immune cell activation marker is an immune cell surface marker.

[0008] In one or more embodiments, the immune cell activation markers include one or more selected from CD25, CD38, CD69, CD137, CD107a, CD226, CD150 and Ly108.

[0009] In one or more embodiments, the immune cell inhibitory markers include one or more selected from CD39, PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160 and CD161.

[0010] In one or more embodiments, the intracellular markers include: intracellular cytokines and / or immune cell intracellular activation markers. Preferably, the intracellular cytokines include any one or more selected from intracellular IFN-γ, TNF-a, CXCL10, CXCL13, IL-2, IL-4, IL-6, IL-8 and IL-10; preferably, the intracellular activation markers include any one or more selected from intracellular CD137, intracellular CD69 and intracellular CD107a.

[0011] In one or more embodiments, the tissue-resident memory markers include any one or more selected from CD69, CD103 and CD49a.

[0012] In one or more embodiments, the immune cell is a T cell, a NK cell, a NKT cell, or a TIL.

[0013] The present invention also provides a reagent for detecting the marker combination described in any embodiment herein, wherein the reagent is a binding molecule that specifically recognizes each marker.

[0014] In one or more embodiments, the binding molecule is an antibody or an antigen-binding fragment thereof.

[0015] In one or more embodiments, the binding molecule is conjugated to a detectable label, such as biotin or a fluorescent group.

[0016] The present invention also provides a composition comprising the marker combination or reagent described in any embodiment herein.

[0017] The present invention also provides a kit for identifying or preparing tumor-specific immune cells, comprising the marker combination, reagent, or composition described in any embodiment of the present invention.

[0018] In one or more embodiments, the immune cell is a T cell, a NK cell, a NKT cell, or a TIL.

[0019] In one or more embodiments, the kit further comprises an immunoreactive reagent. Preferably, the immunoreactive reagent comprises a blocking solution, a washing solution, and an enzyme-labeled reagent.

[0020] In one or more embodiments, the kit is suitable for use or method as described in any embodiment herein.

[0021] The present invention also provides a method for identifying tumor-specific immune cells, comprising detecting the expression of the immune cell marker combination described herein in immune cells, wherein cells with positive expression are tumor-specific immune cells.

[0022] In one or more embodiments, the immune cell is a T cell, a NK cell, a NKT cell, or a TIL.

[0023] The present invention also provides a method for screening tumor-specific immune cells, comprising screening an immune cell population for cells that positively express the immune cell marker combination described herein.

[0024] In one or more embodiments, the immune cell is a T cell, a NK cell, a NKT cell, or a TIL.

[0025] Use of the marker combination or reagent described in any embodiment of the present invention in the preparation of a product for identifying or preparing tumor-specific immune cells.

[0026] In one or more embodiments, the immune cell is a T cell, a NK cell, a NKT cell, or a TIL.

[0027] In one or more embodiments, the product is a kit or a device.

[0028] The present invention provides a method for preparing tumor-specific immune cells, comprising: screening cells expressing positively the immune cell marker combination described herein from separated tumor-infiltrating lymphocytes.

[0029] In one or more embodiments, the immune cell is a T cell, a NK cell, or a NKT cell.

[0030] In one or more embodiments, the method further comprises the step of obtaining isolated tumor infiltrating lymphocytes from a tumor sample; specifically comprising:

[0031] (1.1) obtaining seed cells from a tumor sample, for example, culturing the tumor sample using a seed cell culture medium to obtain seed cells, and

[0032] (1.2) Cultivate seed cells to obtain isolated tumor infiltrating lymphocytes.

[0033] In one or more embodiments, the isolated tumor infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, surgically removed primary lesion samples, synchronous and metachronous surgically removed metastatic lesion samples, puncture samples, and body fluids of a subject in need thereof. The body fluids include blood, tissue fluid, lymph fluid, and / or body cavity effusions.

[0034] In one or more embodiments, the isolated tumor infiltrating lymphocytes are derived from a tumor selected from the group consisting of melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma and bile duct cancer.

[0035] In one or more embodiments, the isolated tumor infiltrating lymphocytes are from a tissue block after tumor tissue is cut, and the diameter of the tissue block after tumor tissue is cut is about 1 mm to about 10 mm.

[0036] In one or more embodiments, the method further comprises further culturing the obtained tumor-specific immune cells.

[0037] The method for preparing tumor-specific immune cells described in this article is used to prepare tumor-specific immune cells and use them in preparing cancer therapeutic drugs.

[0038] The marker combination of the present invention can more accurately identify and enrich tumor-specific immune cells, and the enriched cell population has better tumor reactivity and tumor killing function. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Killing rate of T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 TIL cell populations on primary melanoma target cells;

[0040] Figure 2 : Killing rate of T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 TIL cell populations on primary cervical cancer target cells;

[0041] Figure 3 : Killing rate of T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20 TIL cell populations on primary gastric cancer target cells;

[0042] Figure 4 : Killing rate of T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19 TIL cell populations on primary ovarian cancer target cells;

[0043] Figure 5 : Killing rate of T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18 TIL cell populations on primary non-small cell lung cancer target cells;

[0044] Figure 6 : Killing rate of T06-REP-16, T06-REP-17 and T06-REP-18 TIL cell populations on primary colon cancer target cells. DETAILED DESCRIPTION

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007); Green et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York 2012).

[0046] After in-depth research, the inventors discovered markers of tumor-specific immune cells. Using these markers, tumor-specific immune cells can be accurately and quickly identified and enriched from tumor-infiltrating lymphocytes, and the enriched cell population has better tumor reactivity and tumor killing function.

[0047] Tumor infiltrating lymphocytes are usually cultured from tumor samples. Herein, tumor samples are any samples containing tumor cells, including but not limited to: ascites, surgical resection of primary lesions, synchronous and metachronous surgical resection of metastatic lesions, puncture samples and body fluids of subjects in need.

[0048] The markers of tumor-specific immune cells of the present invention include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers also include (3) intracellular markers, and / or (4) tissue-resident memory markers. Compositions formed by these markers are also within the scope of the present invention.

[0049] Herein, immune cells include T cells, NK cells, NKT cells or TIL. T cells derived from TIL are preferred.

[0050] The immune cell markers herein are applicable to any tumor. Exemplarily, the tumors include: melanoma, glioma, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma or bile duct cancer; preferably melanoma, cervical cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, colon cancer.

[0051] Immune cell activation markers include immune cell surface markers. Herein, tumor-specific immune cell activation markers include one, two or more selected from CD25, CD38, CD69, CD137, CD107a, CD226, CD150 and Ly108. Preferably, immune cell activation markers include one, two or more selected from CD226, Ly108, CD69, CD107a, CD226, CD150.

[0052] Herein, tumor-specific immune cell inhibition markers include one or more selected from CD39, PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160 and CD161. Preferably, immune cell inhibition markers include one, two or more selected from CD39, LAG3, CD161, TIGIT, TIM3, CD160. In some embodiments, immune cell inhibition markers include CD39 and / or CD161.

[0053] The intracellular markers described herein include: intracellular cytokines and / or intracellular activation markers. The intracellular cytokines include any one or more selected from intracellular IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10 and CXCL13; preferably, the intracellular cytokines include any one or more selected from IFN-γ, CXCL10, CXCL13, TNF-α. The intracellular activation markers include any one or more selected from intracellular CD137, intracellular CD69 and intracellular CD107a; preferably, the intracellular activation markers include intracellular CD137. In some embodiments, the intracellular markers include any one or more selected from IFN-γ, CXCL10, CXCL13, TNF-α, and intracellular CD137. Herein, "intracellular + marker" specifies the marker located in the immune cell, and its structure is different from the marker without the "intracellular" prefix (usually located on the cell membrane surface). For example, "intracellular CD137" designates a CD137 molecule located intracellularly, whereas "CD137" refers to a membrane surface form of the CD137 molecule.

[0054] The tissue-resident memory markers include any one or more selected from CD69, CD103 and CD49a, and preferably include CD49a and / or CD103.

[0055] Herein, each component of the marker of tumor-specific immune cells, i.e., immune cell activation marker, immune cell inhibition marker, intracellular marker and tissue-resident memory marker can be selected from any one or more of the above immune cell activation markers, any one or more of the above immune cell inhibition markers, any one or more of the above intracellular markers and any one or more of the above tissue-resident memory markers. Therefore, the marker of tumor-specific immune cells herein can be any combination of the markers in the above components.

[0056] In some embodiments, the markers of tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers also include (3) intracellular markers. The immune cell activation markers include CD226. In addition, the immune cell activation markers may also include one, two or more selected from CD25, CD38, CD69, CD137, CD107a, CD150 and Ly108. The immune cell inhibition markers include one, two or more selected from CD39, LAG3 and CD161. In addition, the immune cell inhibition markers may also include one, two or more selected from PD-1, TIM3, CTLA-4, TIGIT, CD101, CD160. The intracellular markers include intracellular CD137. In addition, the intracellular markers may also include any one or more selected from IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10, CXCL13, intracellular CD69 and intracellular CD107a; preferably, any one or more selected from IFN-γ, CXCL10, CXCL13, TNF-α. In one or more embodiments, the tumor is melanoma.

[0057] In some embodiments, the markers of tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of tumor-specific immune cells also include (3) intracellular markers. The immune cell activation markers include Ly108. In addition, the immune cell activation markers may also include one, two or more selected from CD25, CD38, CD69, CD137, CD107a, CD226, and CD150. The immune cell inhibition markers include one, two or more selected from CD39, TIGIT, and CD161. In addition, the immune cell inhibition markers may also include one, two or more selected from PD-1, TIM3, LAG3, CTLA-4, CD101, and CD160. The intracellular markers include IFN-γ. In addition, the intracellular markers may also include any one or more selected from TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10, CXCL13, intracellular CD69, intracellular CD137 and intracellular CD107a; preferably, they may also include any one or more selected from CXCL10, CXCL13, TNF-α, and intracellular CD137. In one or more embodiments, the tumor is cervical cancer.

[0058] In some embodiments, the markers of tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of tumor-specific immune cells also include (3) intracellular markers. The immune cell activation markers include CD69. In addition, the immune cell activation markers may also include one, two or more selected from CD25, CD38, CD137, CD107a, CD226, CD150 and Ly108. The immune cell inhibition markers include one, two or more selected from CD39, TIM3, CD161. In addition, the immune cell inhibition markers may also include one, two or more selected from PD-1, LAG3, CTLA-4, TIGIT, CD101, CD160. The immune cell intracellular markers include CXCL13. In addition, the immune cell intracellular markers may also include any one or more selected from IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10, intracellular CD69, intracellular CD137 and intracellular CD107a; preferably, they may also include any one or more selected from IFN-γ, CXCL10, TNF-α, and intracellular CD137. In one or more embodiments, the tumor is gastric cancer.

[0059] In some embodiments, the markers of tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of tumor-specific immune cells also include (3) intracellular markers. The immune cell activation marker is CD107a. In addition, the immune cell activation marker may also include one, two or more selected from CD25, CD38, CD69, CD137, CD226, CD150 and Ly108. The immune cell inhibition marker includes one, two or more selected from CD39, LAG3, CD161. In addition, the immune cell inhibition marker may also include one, two or more selected from PD-1, TIM3, CTLA-4, TIGIT, CD101, CD160. The immune cell intracellular marker includes CXCL10. In addition, the immune cell intracellular markers may also include any one or more selected from IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69, intracellular CD137 and intracellular CD107a; preferably, any one or more selected from IFN-γ, CXCL13, TNF-α, and intracellular CD137. In one or more embodiments, the tumor is ovarian cancer.

[0060] In some embodiments, the markers of tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of tumor-specific immune cells also include (3) intracellular markers and / or (4) tissue-resident memory markers. The immune cell activation markers include one, two or more selected from CD226, Ly108, and CD107a. Preferably, the immune cell activation markers are selected from: (1) CD226, (2) Ly108, or (3) a combination of Ly108 and CD107a. In addition, the immune cell activation markers may also include one, two or more selected from CD25, CD38, CD69, CD137, and CD150. The immune cell inhibition markers include CD39. In addition, the immune cell inhibition markers may also include one, two or more selected from PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160, and CD161. The immune cell intracellular markers include one or more selected from CXCL10, IFN-γ, and intracellular CD137. Preferably, the immune cell intracellular markers are selected from: (1) CXCL10, (2) IFN-γ. In addition, the immune cell intracellular markers may also include any one or more selected from TNF-α, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69, and intracellular CD107a; preferably, CXCL13 and / or TNF-α may also be included. The tissue-resident memory markers of the immune cells include CD103. In addition, the tissue-resident memory markers may also include CD69 and / or CD49a. In one or more embodiments, the tumor is lung cancer, such as non-small cell lung cancer.

[0061] In some embodiments, the markers of tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of tumor-specific immune cells also include (3) intracellular markers and / or (4) tissue-resident memory markers. The immune cell activation markers include one, two or more selected from CD226, Ly108, and CD150. Preferably, the immune cell activation markers are selected from: (1) CD226 and Ly108, or (2) CD226 and CD150. In addition, the immune cell activation markers may also include one, two or more selected from CD25, CD38, CD69, CD137, and CD107a. The immune cell inhibition markers include one, two or more selected from CD39, CD160, and CD161. Preferably, the immune cell inhibition markers are selected from: (1) a combination of CD39 and CD160, or (2) a combination of CD39 and CD161. In addition, the immune cell inhibitory marker may also include one, two or more selected from PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101. The immune cell intracellular marker includes one or two selected from IFN-γ and TNF-α. Preferably, the immune cell intracellular marker is selected from: (1) IFN-γ, or (2) IFN-γ and TNF-α. In addition, the immune cell intracellular marker may also include any one or more selected from IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10, CXCL13, intracellular CD69 and intracellular CD107a; preferably, it may also include CXCL10 and / or CXCL13. The tissue-resident memory marker of the immune cell includes CD49a and / or CD103. Preferably, the tissue-resident memory marker of the immune cell is selected from: (1) CD49a, or (2) CD49a and CD103. In addition, the tissue resident memory marker may further include CD69. In one or more embodiments, the tumor is intestinal cancer, such as colon cancer.

[0062] By detecting whether immune cells express the above markers, the present invention also provides a method for identifying and screening tumor-specific immune cells, wherein the positively expressed immune cells are tumor-specific immune cells.

[0063] Any method that can be used to detect the above-mentioned markers expressed (intracellular or membrane surface) or secreted by cells can be used in the present invention. Preferably, such a method is achieved by incubating cells with binding molecules (such as specific small molecules, nucleic acids, antibodies or their antigen-binding fragments) that specifically recognize each marker and identifying the binding molecules. For ease of detection, the binding molecules are coupled with detectable markers, such as biotin or fluorescent groups. These binding molecules and applicable detectable markers are within the scope of conventional techniques in the art. Exemplarily, the detection is achieved by flow cytometry.

[0064] The reagents used in the method of detecting markers (referred to as detection reagents) are also within the scope of the present invention, such as the binding molecules that specifically recognize each marker as described above.

[0065] The present invention also includes a kit having the markers described herein and / or their detection reagents for identifying or preparing tumor-specific immune cells. The kit may also include immune response reagents, such as blocking solutions, washing solutions, and enzyme-labeled reagents. The kit is suitable for the purposes or methods described herein.

[0066] The markers of the present invention are particularly suitable for a method for screening and obtaining tumor-specific immune cells from an immune cell population, comprising the steps of screening (eg, by flow cytometry) cells positive for the expression of the immune cell marker combination described herein from isolated tumor-infiltrating lymphocytes.

[0067] The method also includes the step of obtaining isolated tumor infiltrating lymphocytes from a tumor sample; specifically, it includes: (1.1) obtaining seed cells from a tumor sample, for example, culturing the tumor sample using a seed cell culture medium to obtain seed cells, and (1.2) culturing the seed cells to obtain isolated tumor infiltrating lymphocytes.

[0068] The seed cell culture medium can be any culture medium used in the art for culturing TIL seed cells, such as RPMI1640 culture medium containing 10% human AB serum, 2mM L-glutamine, 55uM BME, 6000IU / mL IL-2, Glutamax and antibiotics (such as gentamicin).

[0069] In some embodiments, step (1.1) includes: (a) washing the tumor tissue sample (for example, washing with saline containing 100U / mL penicillin, 100μg / mL streptomycin and 50μg / mL gentamicin) and cutting it into small pieces with a diameter of 1mm-10mm, (b) culturing the tumor tissue pieces at 30-42°C and 1-10% CO2 using a seed cell culture medium for 3-20 days. The exemplary step (1.1) is described in Example 2 of WO2021239083A1, and includes the following steps: 1) placing the obtained freshly isolated tumor tissue sample in a 10cm culture dish to which 30mL of saline (containing 100U / mL penicillin, 100μg / mL streptomycin and 50μg / mL gentamicin) has been added in a sterile environment in a secondary biosafety cabinet, and then transferring it to a new 10cm dish to which 30mL of the above-mentioned saline has been added for washing, and the washing is repeated 3 times in total; 2) using a sterile scalpel The fat tissue and necrotic tissue were removed, and the tumor tissue was cut into small pieces with a diameter of 3 mm. Twelve randomly selected tumor tissue pieces were placed in each G-REX10 culture tank (purchased from Wilsonwolf), and TIL seed culture medium was added; 3) Seed cell culture medium was added to different G-REX10 culture tanks, 40 mL per tank, and the tumor tissue pieces were cultured at 37°C and 5% CO2. After the TIL seed cells were harvested on the 12th day, the total number of cells and the viability were counted, and the cell phenotype was detected by flow cytometry.

[0070] The culture described in step (1.2) can use any culture medium for culturing TIL in the art, such as AIM-V culture medium containing 1000 IU / mL IL-2 and 30 ng / mL CD3 antibody (such as OKT3).

[0071] In one or more embodiments, the method further comprises culturing the obtained tumor-specific immune cells (e.g., TIL). The culture can be carried out using any culture medium suitable for immune cells (e.g., TIL, especially tumor-specific TIL) known in the art.

[0072] The tumor-specific immune cells prepared by the method described herein can be used for scientific research or for preparing therapeutic drugs for corresponding cancers. Therefore, the present invention also provides a pharmaceutical composition comprising the tumor-specific immune cells prepared by the method described herein and a pharmaceutically acceptable excipient.

[0073] In the present invention, "pharmaceutically acceptable excipients" are pharmaceutically or food-acceptable carriers, solvents, suspending agents or excipients for delivering the tumor-specific immune cells of the present invention to animals or humans. Herein, pharmaceutically acceptable excipients are non-toxic to the recipient of the composition at the doses and concentrations used. Various types of carriers or excipients commonly used for delivering immune cells in treatments known in the art may be included. Exemplary excipients may be liquid or solid, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers. In some embodiments, pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in combination with pharmacodynamic compounds. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., AR Genrmo, ed., 1990, Mack Publishing Company. The optimal pharmaceutical composition may be determined depending on the intended route of administration, mode of delivery, and desired dosage.

[0074] The pharmaceutical compositions of the present invention may be selected for parenteral delivery, for inhalation or for delivery through the digestive tract (such as oral), for example, for intravenous infusion delivery. The preparation of such compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing immune cells, particularly immune cells (e.g., T cells), in sustained or controlled release delivery formulations.

[0075] Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtering through a sterile filtration membrane. Compositions for parenteral administration can be stored in lyophilized form or in solution (e.g., frozen preparations). Parenteral compositions are usually placed in containers with sterile access holes, such as intravenous solution strips or vials with stoppers that can be pierced by hypodermic needles.

[0076] Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal, frozen material or in a dehydrated or lyophilized powder. The pharmaceutical formulation (e.g., a frozen preparation) can be stored in a ready-to-use form or in a form that is further formulated before administration. For example, suitable for delivering the pharmaceutical composition described herein can be a frozen preparation, which can withstand long-distance transportation without damaging cells. In addition to the cells themselves, the frozen preparation generally includes components such as cell freezing solution and human serum albumin (HSA). Before administration (e.g., intravenous infusion), the frozen pharmaceutical composition needs to be stored at low temperatures (e.g., placed in liquid nitrogen). After thawing, the frozen preparation can be directly or formulated as an infusion composition for infusion to the patient. Those skilled in the art are aware of the components and concentrations of conventional freezing solutions. For example, the freezing solution or infusion composition may also contain dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride or magnesium chloride, etc., and its concentration may be determined by those skilled in the art (e.g., experienced physicians) according to conditions such as cells, diseases, and patients.

[0077] The present invention also provides a device for identifying or preparing tumor-infiltrating lymphocytes, the device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that the processor implements the following steps when executing the program: screening (e.g., by flow cytometry) cells expressing positively the immune cell marker combination described herein from the separated tumor-infiltrating lymphocytes. For example, the device records or contains the markers or reagents of tumor-specific immune cells described herein, and detects or determines whether the cells in the sample contain the markers, thereby identifying and screening the cells positive for the markers.

[0078] The present invention also provides the use of the marker combination or reagent described in any embodiment of the present invention in preparing a product for identifying or preparing tumor-specific immune cells. The product includes the kit or device described herein.

[0079] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend on how the value is measured or determined, for example, the limits of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When describing specific values ​​in the application and claims, unless otherwise indicated, the term "about" is assumed to mean within an acceptable error range for that specific value.

[0080] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0081] Unless expressly stated otherwise, all percentages and ratios / ratios are by weight.

[0082] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0083] Each maximum numerical limitation given throughout this disclosure includes each lower numerical limitation, as if these lower numerical limitations are clearly written in this article. Each minimum numerical limitation given throughout this disclosure includes each higher numerical limitation, as if these higher numerical limitations are clearly written here. Each numerical range given throughout this disclosure includes each narrower numerical range that falls within the described wider numerical range, as if these narrower numerical ranges are clearly written in this article.

[0084] The values ​​described herein should not be understood as being strictly limited to the exact numerical values ​​described. Instead, unless otherwise specifically stated, each of the values ​​is intended to refer to the value described and a functionally equivalent range around that value. For example, a value disclosed as "50 μl" is intended to mean "about 50 μl".

[0085] Unless expressly excluded or otherwise limited, each document cited herein, including any cross-references and related patents or applications, is incorporated herein by reference. The citation of any document is not an admission that it is prior art to any invention disclosed or claimed herein, or that it alone or in any combination with any other reference, or refers to, proposes, suggests, or discloses any such invention. In addition, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0086] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different ways and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the terms used in the specification of the present invention are only for describing specific embodiments and are not intended to limit the present invention. Various other changes and modifications may be made without departing from the spirit and scope of the present disclosure. The scope of the appended claims includes all these changes and modifications within the scope of the present disclosure.

[0087] Example

[0088] Example 1, tumor-specific TIL immune cell marker combination and tumor tissue sample The immune cell marker combination involved in the embodiment is shown in Table 1 below:

[0089] Table 1. Combination of immune cell markers

[0090]

[0091] The tumor tissue samples used in the examples are shown in Table 2 below:

[0092] Table 2. Tumor tissue samples

[0093] Sample No. Cancer T01 Melanoma T02 Cervical cancer T03 Gastric cancer T04 Ovarian cancer T05 Non-small cell lung cancer T06 Colon cancer

[0094] The sources of the related fluorescent group-coupled flow cytometry antibodies used in the examples are shown in Table 3 below:

[0095] Table 3, Flow cytometry fluorescent antibodies

[0096]

[0097]

[0098] Example 2, Cultivation and Sorting of Melanoma-derived TIL

[0099] Fresh melanoma tissue T01 was mechanically cut into 3×3×3mm pieces, and the pieces were mixed as evenly as possible and divided into 2 parts. One part was cultured according to the method described in Robert Suriano et al. Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines J Cancer 2013; 4(5): 371-382. Materials and Methods to obtain primary tumor cells of T01 tissue. The remaining part was cultured according to the method described in Example 2 of WO2021239083A1 to obtain seed cells. The culture medium for culturing seed cells was CM1 medium containing 6000IU / mL IL-2, Glutamax and antibiotics prepared according to the formula and method described in Example 5 of the CN110099998A specification. The obtained seed cells were prepared with an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded and cultured to obtain REP cells, which were further divided into 4 equal parts, and each part was incubated one by one with antibodies coupled to fluorescent groups of each marker in the immune cell marker combination numbered 1, 2, 3 and 18 in Table 1 of Example 1 (except intracellular markers) and flow cytometry (BD FACSAria TMIII, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 1, 2, 3 and 18 positive cell populations were obtained, which were named T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18, respectively.

[0100] Take 10 cells from T01-REP-3 cell population 6 Cells were fixed with PBS containing 2v / v% paraformaldehyde, centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were resuspended and washed with PBS, repeated twice, PBS containing 0.7v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and cells were permeabilized. Centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were washed twice with PBS and resuspended, anti-CD137 antibodies coupled with fluorescent groups were added with appropriate dilutions, incubated at room temperature for 30 minutes, cells were washed twice with PBS, and detected by flow cytometry.

[0101] The results showed that in the T01-REP-3 cell population, the proportion of intracellular CD137-positive cells was 93.2%, indicating that the vast majority of the T01-REP-3 cell population were intracellular CD137-positive cells.

[0102] Example 3, Phenotypic Detection of Sorted Melanoma-derived TILs

[0103] The four cell groups T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 obtained in Example 2 were detected by flow cytometry: 1) lymphocyte phenotype: CD45, CD3, CD4, CD8; 2) exhaustion index: PD-1; 3) activation index CD25; 4) memory T cell index: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion level of cytokine IFN-γ of each of the four cell groups was detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instruction manual.

[0104] The results are shown in Table 4. More than 99% of T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 were CD45+ and CD3+ cells. The proportion of cells positive for exhaustion markers and activation markers in T01-REP-1, T01-REP-2 and T01-REP-3 was higher than that in T01-REP-18. The secretion level of cytokine IFN-γ in T01-REP-1, T01-REP-2 and T01-REP-3 and memory T cells (especially TCM ) ratio is also generally significantly higher than that of T01-REP-18.

[0105] Table 4. Phenotype of TILs derived from T01 tissue

[0106]

[0107] Example 4, Detection of tumor cell killing function of sorted melanoma-derived TIL

[0108] The real-time label-free cell function analyzer (RTCA) of Aisen Company was used to detect the in vitro killing activity of the T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 cell populations obtained in Example 2 against their homologous melanoma primary cells. The specific steps are as follows:

[0109] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place in the instrument, select step 1, zero adjustment;

[0110] (2) Target cell plating: The primary T01 melanoma tissue cells obtained by culture in Example 1 were plated at 10 per well. 4 Cells / 50 μL are spread on the plate containing the detection electrode and left for a few minutes. After the cells are stable, they are placed in the instrument and step 2 is started to culture the cells.

[0111] (3) Adding effector cells: After the target cells are cultured for 18h-24h, the cell index is observed. When the cell index is 1, effector cells T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 are added, 50 μL per well, and the effector-target ratio is 4:1; a control group with only target cells and no effector cells is set up separately, and step (3) is started. After co-culture for more than 48-72h, the target cell killing level is observed and the target cell killing rate is calculated. The target cell killing rate calculation formula is as follows (the target cell killing rate calculation formula in the following embodiments is the same):

[0112]

[0113] A is the cell index of the control group, and B is the cell index of each group with effector cells.

[0114] The results are as follows Figure 1 The results showed that the killing rates of T01-REP-1, T01-REP-2 and T01-REP-3 on primary target melanoma tumor cells were significantly higher than those of T01-REP-18, indicating that compared with T01-REP-18 TILs, T01-REP-1, T01-REP-2 and T01-REP-3 TILs had significantly stronger killing effects on homologous primary melanoma tumor cells.

[0115] Example 5, Cultivation and sorting of cervical cancer-derived TIL

[0116] Fresh cervical cancer tissue T02 was mechanically cut into 3×3×3mm fragments, and the fragments were mixed as evenly as possible and divided into 2 parts. One part of the tissue was cultured according to the method described in AD Santin et al. Induction of human papillomavirus-specific CD4(+)and CD8(+)lymphocytes by E7-pulsed autologous dendritic cellsin patients with human papillomavirus type 16-and 18-positive cervical cancer J Virol. 1999 Jul; 73(7): 5402-10 Materials and Methods to obtain primary tumor cells of T02 tissue. The remaining part was cultured according to the method described in Example 2 of WO2021239083A1 to obtain seed cells. The culture medium for culturing seed cells was CM1 medium containing 6000IU / mL IL-2, Glutamax and antibiotics prepared according to the formula and method described in Example 5 of the CN110099998A specification. The obtained seed cells were prepared with an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded and cultured to obtain REP cells, which were further divided into 4 equal parts, and each part was incubated one by one with antibodies coupled to fluorescent groups of each marker in the immune cell marker combination numbered 4, 5, 6 and 18 in Table 1 of Example 1 (except intracellular markers) and flow cytometry (BD FACS Aria TM III, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 4, 5, 6 and 18 positive cell populations were obtained, which were named T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18, respectively.

[0117] Take 10 cells from T02-REP-4 cell population 6Cells were fixed with PBS containing 2v / v% paraformaldehyde, centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were resuspended and washed with PBS, repeated twice, PBS containing 0.7v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and cells were permeabilized. Centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were washed twice with PBS, resuspended, anti-IFN-γ antibody conjugated with fluorescent group was added with appropriate dilution, incubated at room temperature for 30 minutes, cells were washed twice with PBS, and detected by flow cytometry.

[0118] The results showed that in the T02-REP-4 cell population, IFN-γ positive cells accounted for 97.5%, indicating that the vast majority of the T02-REP-4 cell population were IFN-γ positive cells.

[0119] Example 6, Phenotypic Detection of Sorted Cervical Cancer-derived TILs

[0120] The four cell groups T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 obtained in Example 5 were detected by flow cytometry: 1) lymphocyte phenotype CD45, CD3, CD4, CD8; 2) exhaustion index PD-1; 3) activation index CD25; 4) memory T cell index: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion level of cytokine IFN-γ of each of the four cell groups was detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instruction manual.

[0121] The results are shown in Table 5. More than 90% of T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 cells were CD45+ and CD3+. The percentage of cells positive for exhaustion markers and activation markers in T02-REP-4, T02-REP-5 and T02-REP-6 was higher than that in T02-REP-18. The secretion level of cytokine IFN-γ in T02-REP-4, T02-REP-5 and T02-REP-6 and memory T cells, especially T CM The proportions are also generally significantly higher than those of T01-REP-18.

[0122] Table 5. Phenotype of TILs derived from T02 tissue

[0123]

[0124] Example 7, Detection of tumor cell killing function of sorted cervical cancer-derived TIL

[0125] The real-time label-free cell function analyzer (RTCA) of Aisen Company was used to detect the in vitro killing activity of the T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 cell populations obtained in Example 5 against their homologous cervical cancer primary cells. The specific steps are as follows:

[0126] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place in the instrument, select step 1, zero adjustment;

[0127] (2) Target cell plating: T02 cervical cancer tissue primary cells obtained by culture in Example 5 were plated at 10 per well. 4 Cells / 50 μL are spread on the plate containing the detection electrode and left for a few minutes. After the cells are stable, they are placed in the instrument and step 2 is started to culture the cells.

[0128] (3) Adding effector cells: After the target cells are cultured for 18 h to 24 h, the cell index is observed. When the cell index is 1, effector cells T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 are added, 50 μL per well, with an effector-target ratio of 4:1. In addition, a control group with only target cells but no effector cells is set up separately, and step (3) is started. After co-culture for more than 48 h to 72 h, the target cell killing level is observed and the target cell killing rate is calculated.

[0129] The results are as follows Figure 2 The results showed that the killing rates of T02-REP-4, T02-REP-5, and T02-REP-6 on primary target cells of cervical cancer tumors were significantly higher than those of T02-REP-18, indicating that compared with T02-REP-18 TILs, T02-REP-4, T02-REP-5, and T02-REP-6 TILs had significantly stronger killing effects on homologous primary cervical cancer tumor cells.

[0130] Example 8, Cultivation and Sorting of Gastric Cancer-Derived TIL

[0131] Fresh gastric cancer tissue T03 was mechanically cut into 3×3×3mm fragments, and the fragments were mixed as evenly as possible and divided into 2 parts. One part of the tissue was cultured according to the method described in Jinhua Qin et al. Isolation of Human Gastric Epithelial Cells from Gastric Surgical Tissue and Gastric Biopsies for Primary Culture Methods Mol Biol. 2018; 1817: 115-121. Methods to obtain primary tumor cells of T03 tissue. The remaining part was cultured according to the method described in Example 2 of WO2021239083A1 to obtain seed cells. The culture medium for culturing seed cells was CM1 medium containing 6000IU / mL IL-2, Glutamax and antibiotics prepared according to the formula and method described in Example 5 of the CN110099998A specification. The obtained seed cells were prepared with expansion culture medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded and cultured to obtain REP cells, which were further divided into 4 equal parts, and each part was incubated one by one with antibodies of the fluorescent groups of each marker in the immune cell marker combination numbered 7, 8, 9 and 20 in Table 1 of Example 1 (except intracellular markers) and flow cytometry (BD FACS Aria TM III, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 7, 8, 9 and 20 positive cell populations were obtained, which were named T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20, respectively.

[0132] Take 10 cells from T03-REP-9 cell population 6 Cells were fixed with PBS containing 2v / v% paraformaldehyde, centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were resuspended and washed with PBS, repeated twice, PBS containing 0.7v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and cells were permeabilized. Centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were washed twice with PBS and resuspended, anti-CXCL13 antibodies coupled with fluorescent groups were added with appropriate dilutions, incubated at room temperature for 30 minutes, cells were washed twice with PBS, and detected by flow cytometry.

[0133] The results showed that in the T03-REP-9 cell population, CXCL13-positive cells accounted for 91.3%, indicating that the vast majority of T02-REP-4 cell populations were CXCL13-positive cells.

[0134] Example 9, Phenotypic Detection of Sorted Gastric Cancer-Derived TILs

[0135] The four cell groups T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20 obtained in Example 8 were detected by flow cytometry: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator PD-1; 3) activation indicator CD25; 4) memory T cell indicator: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion level of cytokine IFN-γ of each of the four cell groups was detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instruction manual.

[0136] The results are shown in Table 6. More than 90% of T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20 cells were CD45+ and CD3+. The proportion of cells positive for exhaustion markers in T03-REP-7, T03-REP-8 and T03-REP-9 was generally higher than that in T03-REP-20. The secretion levels of cytokines IFN-γ and memory T cells (T CM , T EM ) ratio is also generally significantly higher than that of T03-REP-20.

[0137] Table 6. Phenotype of TILs from T03 tissue

[0138]

[0139]

[0140] Example 10, Detection of tumor cell killing function of sorted gastric cancer-derived TIL

[0141] The real-time label-free cell function analyzer (RTCA) of Aisen Company was used to detect the in vitro killing activity of the T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20 cell populations obtained in Example 5 against their homologous primary gastric cancer cells. The specific steps are as follows:

[0142] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place in the instrument, select step 1, zero adjustment;

[0143] (2) Target cell plating: The T03 gastric cancer tissue primary cells obtained by culture in Example 5 were plated on a plate containing detection electrodes at a density of 104 cells / 50 μL per well, and left for several minutes. After the cells were stable, they were placed in the instrument and step 2 was started to culture the cells;

[0144] (3) Adding effector cells: After the target cells are cultured for 18 h to 24 h, the cell index is observed. When the cell index is 1, effector cells T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20 are added, 50 μL per well, with an effector-target ratio of 4:1. In addition, a control group with only target cells but no effector cells is set up separately, and step (3) is started. After co-culture for more than 48 h to 72 h, the target cell killing level is observed and the target cell killing rate is calculated.

[0145] The results are as follows Figure 3 The results showed that the killing rates of T03-REP-7, T03-REP-8 and T03-REP-9 on primary target gastric cancer tumor cells were significantly higher than those of T03-REP-20, indicating that compared with T03-REP-20 TILs, T03-REP-7, T03-REP-8 and T03-REP-9 TILs had significantly stronger killing effects on homologous primary gastric cancer tumor cells.

[0146] Example 11, Cultivation and Sorting of Ovarian Cancer-Derived TIL

[0147] Fresh ovarian cancer tissue T04 was mechanically cut into pieces of 3×3×3 mm in size, and the pieces were mixed as evenly as possible and divided into 2 portions. One portion was cultured according to the method described in Lee J.Priby et al. Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens J Vis Exp. 2014; (84): 51581. Protocol to obtain primary tumor cells of T04 tissue. The remaining portion was cultured according to the method described in Example 2 of WO2021239083A1 to obtain seed cells. The culture medium for culturing seed cells was CM1 culture medium containing 6000 IU / mL IL-2, Glutamax and antibiotics prepared according to the formula and method described in Example 5 of CN110099998A specification. The obtained seed cells were prepared with an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded and cultured to obtain REP cells, which were further divided into 4 equal parts, and each part was incubated one by one with antibodies coupled to fluorescent groups of each marker in the immune cell marker combination numbered 10, 11, 12 and 19 in Table 1 of Example 1 (excluding intracellular markers) and flow cytometry (BD FACSAria TM III, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 10, 11, 12 and 19 positive cell populations were obtained, which were named T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19, respectively.

[0148] Take 10 cells from T04-REP-12 cell population 6 Cells were fixed with PBS containing 2v / v% paraformaldehyde, centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were resuspended and washed with PBS, repeated twice, PBS containing 0.7v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and cells were permeabilized. Centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were washed twice with PBS and resuspended, anti-CXCL10 antibodies coupled with fluorescent groups were added with appropriate dilutions, incubated at room temperature for 30 minutes, cells were washed twice with PBS, and detected by flow cytometry.

[0149] The results showed that CXCL10-positive cells accounted for 89.9% of the T04-REP-12 cell population, indicating that the vast majority of the T04-REP-12 cell population were CXCL10-positive cells.

[0150] Example 12, Phenotypic Detection of Sorted Ovarian Cancer-Derived TILs

[0151] The four cell groups T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19 obtained in Example 11 were detected by flow cytometry: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator TIM3; 3) activation indicator CD25; 4) memory T cell indicator: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion level of cytokine IFN-γ of each of the four cell groups was detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instruction manual.

[0152] The results are shown in Table 7. Nearly or more than 95% of T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19 were CD45+ and CD3+ cells. The proportion of cells positive for exhaustion markers in T04-REP-10, T04-REP-11 and T04-REP-12 was generally higher than that in T04-REP-19; the cytokine secretion levels of T04-REP-10, T04-REP-11 and T04-REP-12 and memory T cells (T CM , T EM ) ratio is also generally significantly higher than that of T04-REP-19.

[0153] Table 7. Phenotype of TILs derived from T04 tissue

[0154]

[0155] Example 13, Detection of tumor cell killing function of sorted ovarian cancer-derived TIL

[0156] The real-time label-free cell function analyzer (RTCA) of Aisen Company was used to detect the in vitro killing activity of the T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19 cell populations obtained in Example 11 against their homologous primary ovarian cancer cells. The specific steps are as follows:

[0157] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place in the instrument, select step 1, zero adjustment;

[0158] (2) Target cell plating: The primary T04 ovarian cancer tissue cells obtained by culture in Example 11 were plated at 10 per well. 4 Cells / 50 μL are spread on the plate containing the detection electrode and left for a few minutes. After the cells are stable, they are placed in the instrument and step 2 is started to culture the cells.

[0159] (3) Adding effector cells: After culturing the target cells for 18 h to 24 h, observe the cell index. When the cell index is 1, add effector cells T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19, 50 μL per well, with an effector-target ratio of 4:1. In addition, set up a control group with only target cells but no effector cells, and start step 3. After co-culturing for more than 48 h to 72 h, observe the target cell killing level and calculate the target cell killing rate.

[0160] The results are as follows Figure 4 The results showed that the killing rates of T04-REP-10, T04-REP-11, and T04-REP-12 on primary ovarian cancer target cells were significantly higher than those of T04-REP-19, indicating that compared with T04-REP-19 TILs, T04-REP-10, T04-REP-11, and T04-REP-12 TILs had significantly stronger killing effects on homologous primary ovarian cancer tumor cells.

[0161] Example 14, Cultivation and Sorting of TILs from Non-Small Cell Lung Cancer

[0162] Fresh non-small cell lung cancer tissue T05 was mechanically cut into 3×3×3mm fragments, and the fragments were mixed as evenly as possible and divided into 2 portions. One portion was cultured according to the method described in DP. Kodack et al. Primary Patient-Derived Cancer Cells and Their Potential for Personalized Cancer Patient Care Cell Rep. 2017 Dec 12; 21(11): 3298–3309. EXPERIMENTAL PROCEDURES to obtain primary tumor cells of T05 tissue. The remaining portion was cultured according to the method described in Example 2 of WO2021239083A1 to obtain seed cells. The culture medium for culturing seed cells was CM1 culture medium containing 6000IU / mL IL-2, Glutamax and antibiotics prepared according to the formula and method described in Example 5 of the CN110099998A specification. The obtained seed cells were prepared with an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded and cultured to obtain REP cells, which were further divided into 4 equal parts, and each part was incubated one by one with antibodies coupled to fluorescent groups of each marker in the immune cell marker combination numbered 13, 14, 15 and 18 in Table 1 of Example 1 (excluding intracellular markers) and flow cytometry (BD FACS Aria TMIII, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 13, 14, 15 and 18 positive cell populations were obtained, which were named T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18, respectively.

[0163] Take 10 cells from each of the T05-REP-13, T05-REP-14, and T05-REP-15 cell populations. 6 Cells were fixed with PBS containing 2v / v% paraformaldehyde, centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were resuspended and washed with PBS, repeated twice, PBS containing 0.7v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and cells were permeabilized. Centrifuged at 800g for 5 minutes, supernatant was discarded, cell pellets were washed twice with PBS and resuspended, and appropriately diluted anti-CXCL10 antibody, anti-IFN-γ antibody, and anti-IFN-γ antibody coupled with fluorescent groups were added to T05-REP-13, T05-REP-14, and T05-REP-15 cells, respectively. After incubation at room temperature for 30 minutes, cells were washed twice with PBS and detected by flow cytometry.

[0164] The results showed that in T05-REP-13 cells, CXCL10-positive cells accounted for 90.7%; in T05-REP-14 and T05-REP-15 cells, IFN-γ-positive cells accounted for 87.6% and 88.1%, respectively. The above results show that the vast majority of T05-REP-13 cell populations are CXCL10-positive cells, while the vast majority of T05-REP-14 and T05-REP-15 cell populations are IFN-γ-positive cells.

[0165] Example 15, Phenotypic Detection of Sorted Non-Small Cell Lung Cancer-Derived TILs

[0166] The four cell groups T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18 obtained in Example 14 were detected by flow cytometry: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator PD-1; 3) activation indicator CD25; 4) memory T cell indicator: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion level of cytokine IFN-γ of each of the four cell groups was detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instruction manual.

[0167] The results are shown in Table 8. More than 99% of T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18 were CD45+, and the proportion of CD3+ cells was higher than 80%. The proportion of cells positive for exhaustion markers in T05-REP-13, T05-REP-14 and T05-REP-15 was generally higher than that in T05-REP-18; the secretion level of cytokine IFN-γ and memory T cells (T CM , T EM ) ratio is also generally significantly higher than that of T05-REP-18.

[0168] Table 8. Phenotype of TILs from T05 tissue

[0169]

[0170] Example 16, Detection of tumor cell killing function of sorted non-small cell lung cancer derived TIL

[0171] The real-time label-free cell function analyzer (RTCA) of Aisen Company was used to detect the in vitro killing activity of the T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18 cell populations obtained in Example 14 against their homologous non-small cell lung cancer primary cells. The specific steps are as follows:

[0172] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place in the instrument, select step 1, zero adjustment;

[0173] (2) Target cell plating: The primary T05 non-small cell lung cancer tissue cells obtained by culture in Example 14 were plated at 10 per well. 4 Cells / 50 μL are spread on the plate containing the detection electrode and left for a few minutes. After the cells are stable, they are placed in the instrument and step 2 is started to culture the cells.

[0174] (3) Adding effector cells: After the target cells are cultured for 18h-24h, the cell index is observed. When the cell index is 1, effector cells T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18 are added, 50μL per well, with an effector-target ratio of 4:1. In addition, a control group with only target cells but no effector cells is set up separately, and step (3) is started. After co-culture for more than 48-72h, the target cell killing level is observed and the target cell killing rate is calculated.

[0175] The results are as follows Figure 5The results showed that the killing rates of T05-REP-13, T05-REP-14, and T05-REP-15 on primary target cells of non-small cell lung cancer were significantly higher than those of T05-REP-18, indicating that compared with T05-REP-18 TILs, T05-REP-13, T05-REP-14, and T05-REP-15 TILs had significantly stronger killing effects on homologous primary tumor cells of non-small cell lung cancer.

[0176] Example 17, Culture and sorting of colon cancer-derived TIL

[0177] Fresh colon cancer tissue T06 was mechanically cut into 3×3×3mm pieces, and the pieces were mixed as evenly as possible and divided into 2 parts. One part was cultured according to the method described in S Koshkin et al. Primary cultures of human colon cancer as a model to study cancer stem cells Tumour Biol. 2016 Sep; 37(9): 12833-12842. Materials and methods to obtain primary tumor cells of T06 tissue. The remaining part was cultured according to the method described in Example 2 of WO2021239083A1 to obtain seed cells. The culture medium for culturing seed cells was CM1 medium containing 6000IU / mL IL-2, Glutamax and antibiotics prepared according to the formula and method described in Example 5 of the CN110099998A specification. The obtained seed cells were prepared with an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded and cultured to obtain REP cells, which were further divided into 3 equal parts, and each part was incubated one by one with antibodies coupled to fluorescent groups of each marker in the immune cell marker combination numbered 16, 17 and 18 in Table 1 of Example 1 (except intracellular markers) and flow cytometry (BD FACSAria TM III, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 16, 17 and 18 positive cell populations were obtained, which were named T06-REP-16, T06-REP-17 and T06-REP-18, respectively.

[0178] 2E6 cells were taken from the T06-REP-16 cell population, fixed with PBS containing 2v / v% paraformaldehyde, centrifuged at 800g for 5 minutes, discarded the supernatant, and the cell pellet was resuspended and washed with PBS, repeated twice, and PBS containing 0.7v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and the cells were permeabilized. Centrifuged at 800g for 5 minutes, discarded the supernatant, the cell pellet was washed twice with PBS and resuspended, and anti-IFN-γ antibody and anti-TNF-α antibody conjugated with fluorescent groups were added with appropriate dilutions, incubated at room temperature for 30 minutes, and then washed twice with PBS, and detected by flow cytometry.

[0179] 2E6 cells were taken from the T06-REP-17 cell population, fixed with PBS containing 2v / v% paraformaldehyde, centrifuged at 800g for 5 minutes, discarded the supernatant, and the cell pellet was resuspended and washed with PBS, repeated twice, and PBS containing 0.7v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and the cells were permeabilized. Centrifuged at 800g for 5 minutes, discarded the supernatant, the cell pellet was washed twice with PBS, resuspended, and an appropriately diluted anti-IFN-γ antibody conjugated with a fluorescent group was added. After incubation at room temperature for 30 minutes, the cells were washed twice with PBS and detected by flow cytometry.

[0180] The results showed that 84.6% of the T06-REP-16 cell population were double positive for IFN-γ and TNF-α, and 96.9% of the T06-REP-17 cell population were double positive for IFN-γ. This indicated that the vast majority of the T06-REP-16 cell population were double positive for IFN-γ and TNF-α, and the vast majority of the T06-REP-17 cell population were IFN-γ positive.

[0181] Example 18, Phenotypic Detection of Sorted Colon Cancer-Derived TILs

[0182] The three groups of cells T06-REP-16, T06-REP-17 and T06-REP-18 obtained in Example 2 were detected by flow cytometry: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator PD-1; 3) activation indicator CD25; 4) memory T cell indicator: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion level of cytokine IFN-γ of each of the four cell groups was detected using the HTRF IFN-γ detection kit (CisbioHuman IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instruction manual.

[0183] The results are shown in Table 9. More than 99% of T06-REP-16, T06-REP-17 and T06-REP-18 cells were CD45+ and CD3+. The proportion of cells positive for exhaustion markers in T06-REP-16 and T06-REP-17 was generally higher than that in T06-REP-18. The secretion levels of cytokine IFN-γ in T06-REP-16 and T06-REP-17 and memory T cells (T CM , T EM ) ratio is also generally significantly higher than that of T06-REP-18.

[0184] Table 9. Phenotype of TILs from T06 tissue

[0185]

[0186] Example 19, Detection of Tumor Cell Killing Function of Sorted Colon Cancer-derived TIL

[0187] The in vitro killing activity of the T06-REP-16, T06-REP-17 and T06-REP-18 cell populations obtained in Example 17 against their homologous primary colon cancer cells was detected using the real-time label-free cell function analyzer (RTCA) of Aisen Company. The specific steps are as follows:

[0188] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place in the instrument, select step 1, zero adjustment;

[0189] (2) Target cell plating: The primary T06 colon cancer tissue cells obtained by culture in Example 17 were plated at 10 per well. 4 Cells / 50 μL are spread on the plate containing the detection electrode and left for a few minutes. After the cells are stable, they are placed in the instrument and step 2 is started to culture the cells.

[0190] (3) Adding effector cells: After the target cells are cultured for 18-24 hours, the cell index is observed. When the cell index is 1, effector cells T06-REP-16, T06-REP-17 and T06-REP-18 are added, 50 μL per well, with an effector-target ratio of 4:1. In addition, a control group with only target cells but no effector cells is set up separately, and step 3 is started. After co-culture for more than 48-72 hours, the target cell killing level is observed and the target cell killing rate is calculated.

[0191] The results are as follows Figure 6 The results showed that the killing rates of T06-REP-16 and T06-REP-17 on primary target cells of colon cancer were significantly higher than those of T06-REP-18, indicating that compared with T06-REP-18 TILs, T06-REP-16 and T06-REP-17 TILs had significantly stronger killing effects on homologous primary tumor cells of colon cancer.

[0192] The present invention can be changed and modified to adapt it to various uses and conditions, and such embodiments also fall within the scope of the claims herein. Reference to a list of elements in any definition of a variable herein includes the definition of the variable as any single element or a combination of elements (or subcombinations) listed. Reference to an embodiment herein includes the embodiment as any single embodiment or a combination or part thereof with any other embodiment. All patents and disclosures mentioned in this specification are incorporated herein by reference, as if each separate patent or disclosure is specifically and individually incorporated herein by reference.

Claims

1. An immune cell marker combination for enriching tumor-specific immune cells, comprising: (1) an immune cell activation marker, and (2) an immune cell inhibition marker, the marker combination optionally further comprises (3) an intracellular marker, and / or (4) a tissue-resident memory marker, the immune cell activation marker comprises CD69, the immune cell inhibition marker comprises one, two or more selected from CD39, TIM3, and CD161, the tumor is gastric cancer, Preferably, the intracellular markers include: intracellular cytokines and / or intracellular activation markers of immune cells, More preferably, The immune cell activation markers further include one or more selected from CD25, CD38, CD137, CD107a, CD226, CD150 and Ly108, and / or The immune cell inhibitory markers further include one or more selected from PD-1, LAG3, CTLA-4, TIGIT, CD101 and CD160, and / or The intracellular cytokines include any one or more selected from intracellular IFN-γ, TNF-a, CXCL10, CXCL13, IL-2, IL-4, IL-6, IL-8 and IL-10, and / or The intracellular activation markers include any one or more selected from intracellular CD137, intracellular CD69 and intracellular CD107a, and / or The tissue-resident memory markers include any one or more selected from CD69, CD103 and CD49a.

2. A reagent for detecting the immune cell marker combination according to claim 1, wherein the reagent is a binding molecule that specifically recognizes each marker, Preferably, The binding molecule is an antibody or an antigen-binding fragment thereof, and / or The binding molecule is conjugated to a detectable label, such as biotin or a fluorescent group.

3. A composition for enriching tumor-specific immune cells, comprising the immune cell marker combination of claim 1 or the reagent of claim 2, wherein the tumor is gastric cancer.

4. A kit for identifying or preparing tumor-specific immune cells, comprising the immune cell marker combination of claim 1, the reagent of claim 2, or the composition of claim 3, wherein the tumor is gastric cancer, Preferably, the kit further comprises an immunoreactive reagent.

5. A method for identifying tumor-specific immune cells, comprising detecting the expression of the immune cell marker combination of claim 1 in immune cells, wherein the positively expressed immune cells are tumor-specific immune cells, the immune cells are preferably T cells, NK cells, NKT cells or TILs, and the tumor is gastric cancer.

6. A method for screening tumor-specific immune cells, comprising screening cells in an immune cell population that positively express the immune cell marker combination of claim 1, wherein the immune cells are preferably T cells, NK cells, NKT cells or TILs, and the tumor is gastric cancer.

7. Use of the immune cell marker combination according to claim 1, the reagent according to claim 2 and / or the composition according to claim 3 in preparing a product for identifying or preparing tumor-specific immune cells, wherein the tumor is gastric cancer. Preferably, The immune cells are T cells, NK cells, NKT cells or TIL, and / or The product is a kit or a device.

8. A method for preparing tumor-specific immune cells, comprising: Screening cells that express positively the immune cell marker combination of claim 1 from separated tumor-infiltrating lymphocytes, wherein the tumor is gastric cancer, Preferably, The immune cells are T cells, NK cells or NKT cells, and / or The isolated tumor infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, surgically removed primary lesion samples, synchronous and metachronous surgically removed metastatic lesion samples, puncture samples and body fluids of a subject in need thereof, and / or The isolated tumor infiltrating lymphocytes are derived from gastric cancer.

9. The method according to claim 8, characterized in that The method further comprises the step of obtaining isolated tumor-infiltrating lymphocytes from the tumor sample, and / or the method further comprises the step of further culturing the obtained tumor-specific immune cells, Preferably, the step of obtaining isolated tumor infiltrating lymphocytes from a tumor sample comprises: (1.1) obtaining seed cells from a tumor sample, for example, culturing the tumor sample using a seed cell culture medium to obtain seed cells, and (1.2) culturing the seed cells to obtain isolated tumor infiltrating lymphocytes.

10. Use of tumor-specific immune cells obtained by the method according to claim 8 or 9 in the preparation of cancer therapeutic drugs, wherein the cancer is gastric cancer.

Images