Tumor-specific immune cell markers and their uses

Abstract

The present invention relates to tumor-specific immune cell markers and their uses. Specifically, an immune cell marker combination is provided, comprising: (1) an immune cell activation marker, and (2) an immune cell inhibition marker, optionally further comprising (3) an intracellular marker, and / or (4) a tissue-resident memory marker. 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.

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 form of cancer immune cell therapy for solid tumors, achieving remarkable clinical results in a variety of solid tumors, including melanoma, cervical cancer, head and neck cancer, and non-small cell lung cancer. TILs are primarily composed of T cells, a heterogeneous group of T cells that includes both tumor-specific T cells and bystander T cells—those that specifically recognize tumor antigens and those that recognize antigenic epitopes unrelated to the tumor. Bystander TILs typically include T cells that recognize viral antigens, such as EBV, HCMV, and influenza virus epitopes. In the context of adoptive T cell therapy (ACT) for cancer treatment using TILs, increasing the content of tumor-specific T cells in TILs, reducing the proportion of bystander T cells, and obtaining a TIL population that can persist long-term in vivo are crucial factors in improving efficacy.

[0004] Published reports have mentioned a variety of different indicators for distinguishing tumor-specific T cell populations from bystander T cell populations in TILs. Thomas Duhen et al. Nat Commun. 2018 Jul 13; 9(1): 2724. It was recorded that among CD8+ TIL cells in various primary and metastatic tumors, 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 was recorded that tumor-specific CD4+ T cells are concentrated in the CD39+ TIL subpopulation. WO2021226085A1 discloses a method for expanding TILs for clinical therapeutic use, which includes a step of sorting TILs that express positive specific indicators, including 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 to determine the proportion of tumor-specific and tumor-reactive T cells in the prepared TIL cells, and possibly to further sort and expand 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 intracellular immune cell activation markers. Preferably, the intracellular cytokines include any one or more selected from intracellular IFN-γ, TNF-α, 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 positive for 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 herein 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 isolated 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 the 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 resected primary lesion samples, synchronous and metachronous surgically resected metastatic lesion samples, puncture samples, and body fluids of a subject in need thereof, including blood, tissue fluid, lymphatic fluid, and / or body cavity fluid.

[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 dissected tumor tissue block having a diameter of 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 invention relates to the use of the tumor-specific immune cells prepared by the method for preparing tumor-specific immune cells described in this article in the preparation of 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 rates 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 rates 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 rates 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 rates 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 rates 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, for example, 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 for tumor-specific immune cells. Using these markers, tumor-specific immune cells can be accurately and rapidly identified and enriched from tumor-infiltrating lymphocytes. The enriched cell populations exhibit enhanced tumor reactivity and tumor-killing capabilities.

[0047] Tumor infiltrating lymphocytes are usually cultured from tumor samples. Herein, a tumor sample is any sample 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 a subject in need thereof.

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

[0049] Herein, immune cells include T cells, NK cells, NKT cells or TILs. T cells derived from TILs 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, and 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 suppression markers include one or more selected from CD39, PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160, and CD161. Preferably, the immune cell suppression markers include one, two, or more selected from CD39, LAG3, CD161, TIGIT, TIM3, and CD160. In some embodiments, the immune cell suppression 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 the membrane surface form of the CD137 molecule.

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

[0055] Herein, the components of the tumor-specific immune cell markers, i.e., immune cell activation markers, immune cell inhibition markers, intracellular markers, and tissue-resident memory markers, can be selected from any one or more of the aforementioned immune cell activation markers, any one or more of the aforementioned immune cell inhibition markers, any one or more of the aforementioned intracellular markers, and any one or more of the aforementioned tissue-resident memory markers. Therefore, the tumor-specific immune cell markers herein can be any combination of the markers in the aforementioned 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 further 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 further 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, they may further include any one or more selected from IFN-γ, CXCL10, CXCL13, and 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 further 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 further 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 further 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 further 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 further 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 further 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 further 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 further 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, they may further include 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 further 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 marker includes 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 further 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] The present invention also provides a method for identifying and screening tumor-specific immune cells by detecting whether immune cells express the above markers, wherein the cells expressing positive markers 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 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 a detectable label, such as biotin or a fluorescent group. These binding molecules and applicable detectable labels are within the scope of conventional techniques in this area. Exemplarily, the detection is achieved by flow cytometry.

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

[0065] The present invention also includes a kit comprising the markers described herein and / or detection reagents thereof for use in identifying or preparing tumor-specific immune cells. The kit may also include immunoreactive reagents, such as blocking solutions, washing solutions, and enzyme-labeled reagents. The kit is suitable for the uses 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 (e.g., by flow cytometry) cells positive for 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 the tumor sample; specifically, it includes: (1.1) obtaining seed cells from the 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 (e.g., gentamicin).

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

[0070] The culture described in step (1.2) can use any culture medium for culturing TILs 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 known in the art suitable for immune cells (e.g., TIL, especially tumor-specific TIL).

[0072] The tumor-specific immune cells produced 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 produced by the method described herein and a pharmaceutically acceptable excipient.

[0073] In the present invention, a "pharmaceutically acceptable excipient" is a pharmaceutically or food-acceptable carrier, solvent, suspending agent or excipient used to deliver the tumor-specific immune cells of the present invention to animals or humans. Herein, a pharmaceutically acceptable excipient is non-toxic to the recipient of the composition at the dosage and concentration used. It may include various types of carriers or excipients commonly used for delivering immune cells in treatments known in the art. 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, a pharmaceutically acceptable excipient 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 the pharmacological compound. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., AR Genrmo, ed., 1990, Mack Publishing Company. The optimal pharmaceutical composition can 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 orally), for example, for intravenous 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 that contain immune cells, particularly immune cells (e.g., T cells), in sustained or controlled release delivery formulations.

[0075] Pharmaceutical compositions for in vivo administration are typically provided in the form of sterile formulations. Sterilization is achieved by filtration 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 typically placed in containers with sterile access ports, such as intravenous solution strips or bottles with stoppers pierceable by hypodermic needles.

[0076] Once formulated, the pharmaceutical composition is stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid, crystal, frozen product, or dehydrated or lyophilized powder. The pharmaceutical formulation (e.g., 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 delivery of the pharmaceutical composition described herein can be a frozen preparation that can withstand long-distance transportation without damaging the cells. In addition to the cells themselves, the frozen preparation typically also 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 infused into the patient directly or formulated as an infusion composition. 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 can be determined by those skilled in the art (e.g., experienced physicians) based on the conditions of the cells, disease, and patient.

[0077] The present invention also provides a device for identifying or preparing tumor-infiltrating lymphocytes, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the following steps are performed: screening (e.g., by flow cytometry) isolated tumor-infiltrating lymphocytes for cells that positively express the combination of immune cell markers described herein. For example, the device may record or contain markers or reagents for tumor-specific immune cells described herein, and by detecting or determining whether cells in a sample contain the markers, cells positive for the markers may be identified and screened.

[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 with respect to 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 this 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] Every maximum numerical limitation given throughout this disclosure includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this disclosure includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this disclosure includes every narrower numerical range that falls within that broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0084] The values described herein should not be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specifically stated, each of the values is intended to refer to the recited value and a functionally equivalent range surrounding 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 with respect to any invention disclosed or claimed herein, or that it, alone or in any combination with any other reference, refers to, proposes, 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] To facilitate understanding of the present invention, the present invention will be described in more detail 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 provide a more thorough and comprehensive understanding of the disclosure of the present invention. In addition, the terms used in the description of the present invention are only for the purpose of 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 such changes and modifications within the scope of the present disclosure.

[0087] Example

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

[0089] Table 1. Immune cell marker combinations

[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 type 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, Culture and Sorting of Melanoma-Derived TILs

[0099] Fresh melanoma tissue T01 was mechanically cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and then divided into two portions. One portion 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 from T01 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 medium containing 6000 IU / 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. Each part was incubated 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 (excluding intracellular markers) and analyzed by flow cytometry (BD FACSAria TMIII, bdbiosciences) sorting, and after multiple sorting, immune cell marker combination 1, 2, 3 and 18 positive cell populations were obtained and 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 2% v / v paraformaldehyde and centrifuged at 800 g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended and washed twice with PBS. The cells were then permeabilized by adding PBS containing 0.7% v / v Tween-20 and incubating at room temperature for 15 minutes. The cells were centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended. Fluorophore-conjugated anti-CD137 antibody was added with appropriate dilution and incubated at room temperature for 30 minutes. The cells were then washed twice with PBS and analyzed by flow cytometry.

[0101] The results showed that 93.2% of the T01-REP-3 cell population were intracellular CD137-positive cells, 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 populations 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 The HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) was used to detect the secretion level of the cytokine IFN-γ in each of the four cell populations 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 cells 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 the expression of 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 TILs

[0108] The in vitro cytotoxicity of the T01-REP-1, T01-REP-2, T01-REP-3, and T01-REP-18 cell populations obtained in Example 2 against their homologous primary melanoma cells was detected using a real-time label-free cell function analyzer (RTCA) from Eisen. The specific steps are as follows:

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

[0110] (2) Target cell plating: The primary T01 melanoma tissue cells obtained by culture in Example 1 were plated at 10 per well. 4 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 to culture the cells.

[0111] (3) Adding effector cells: After culturing the target cells for 18-24 hours, observe the cell index. When the cell index is 1, add effector cells T01-REP-1, T01-REP-2, T01-REP-3, and T01-REP-18, 50 μL per well, with an effector-target ratio of 4:1. In addition, set up a control group with only target cells and no effector cells, and start step (3). After co-culturing for more than 48-72 hours, observe the target cell killing level and calculate the target cell killing rate. The target cell killing rate calculation formula is as follows (the target cell killing rate calculation formula in the following examples is the same):

[0112]

[0113] A is the cell index of the control group, and B is the cell index of the groups with added 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 melanoma tumor target 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, Culturing and Sorting of Cervical Cancer-Derived TILs

[0116] Fresh cervical cancer tissue T02 was mechanically cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and then divided into two 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 cells in 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 6000 IU / 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 to obtain REP cells, which were further divided into 4 equal parts. Each part was incubated with antibodies coupled to fluorescent groups of each marker in the immune cell marker combination numbered 4, 5, 6 and 18 (excluding intracellular markers) in Table 1 of Example 1, and flow cytometry (BD FACS Aria) was performed. TM III, bdbiosciences) sorting, and after multiple sorting, immune cell marker combination 4, 5, 6 and 18 positive cell populations were obtained and 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 2% v / v paraformaldehyde and centrifuged at 800 g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended and washed twice with PBS. The cells were then permeabilized by adding PBS containing 0.7% v / v Tween-20 and incubating at room temperature for 15 minutes. The cells were centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended. Fluorophore-conjugated anti-IFN-γ antibody was added with appropriate dilution and incubated at room temperature for 30 minutes. The cells were then washed twice with PBS and analyzed by flow cytometry.

[0118] The results showed that 97.5% of the T02-REP-4 cell population were IFN-γ positive, 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 populations 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 The HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) was used to detect the secretion level of the cytokine IFN-γ in each of the four cell populations 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+ cells. The proportion 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 the expression of memory T cells, especially T cells, were significantly increased. 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 TILs

[0125] The in vitro cytotoxicity of the T02-REP-4, T02-REP-5, T02-REP-6, and T02-REP-18 cell populations obtained in Example 5 against their homologous primary cervical cancer cells was detected using a real-time label-free cell function analyzer (RTCA) from Eisen. The specific steps are as follows:

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

[0127] (2) Target cell plating: The T02 cervical cancer tissue primary cells obtained by culture in Example 5 were plated at 10 per well. 4 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 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 containing only target cells but no effector cells is set up separately, and step (3) is started. After co-culture for more than 48-72 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 TIL of T02-REP-18, TIL of T02-REP-4, T02-REP-5, and T02-REP-6 had significantly stronger killing effects on homologous primary tumor cells of cervical cancer.

[0130] Example 8, Culture and Sorting of Gastric Cancer-Derived TILs

[0131] Fresh gastric cancer tissue T03 was mechanically cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and then divided into two portions. One portion 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 from T03 tissue. The remaining portion was cultured according to the method described in Example 2 of WO2021239083A1 to obtain seed cells. The culture medium for seed cells was CM1 medium containing 6000 IU / 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. Each part was incubated with antibodies expressing fluorescent groups of each marker in the immune cell marker combination numbered 7, 8, 9 and 20 in Table 1 of Example 1 (excluding intracellular markers) and analyzed by flow cytometry (BD FACS Aria) one by one. TM III, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 7, 8, 9 and 20 positive cell populations were obtained and 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 2% v / v paraformaldehyde and centrifuged at 800 g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended and washed twice with PBS. The cells were then permeabilized by adding PBS containing 0.7% v / v Tween-20 and incubating at room temperature for 15 minutes. The cells were centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended. A properly diluted fluorophore-conjugated anti-CXCL13 antibody was added and incubated at room temperature for 30 minutes. The cells were then washed twice with PBS and analyzed by flow cytometry.

[0133] The results showed that CXCL13-positive cells accounted for 91.3% of the T03-REP-9 cell population, 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 populations 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 The HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) was used to detect the secretion level of the cytokine IFN-γ in each of the four cell populations 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+ cells. 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 derived from T03 tissue

[0138]

[0139]

[0140] Example 10, Detection of Tumor Cell Killing Function of Sorted Gastric Cancer-derived TILs

[0141] The in vitro cytotoxicity 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 was detected using a real-time label-free cell function analyzer (RTCA) from Eisen. The specific steps are as follows:

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

[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 10<4 > cells / 50 μL per well. The cells were allowed to stand for several minutes until they stabilized, and then placed in the instrument to start step 2, culturing 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 48-72 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 gastric cancer target 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, Culture and Sorting of Ovarian Cancer-Derived TILs

[0147] Fresh ovarian cancer tissue T04 was mechanically cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and then divided into two 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 from 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 medium containing 6000 IU / 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. Each part was incubated 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 analyzed by flow cytometry (BD FACSAria TM III, bdbiosciences) sorting, and after multiple sorting, immune cell marker combination 10, 11, 12 and 19 positive cell populations were obtained and 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 2% v / v paraformaldehyde and centrifuged at 800 g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended and washed twice with PBS. The cells were then permeabilized by adding PBS containing 0.7% v / v Tween-20 and incubating at room temperature for 15 minutes. The cells were centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended. A properly diluted fluorophore-conjugated anti-CXCL10 antibody was added and incubated at room temperature for 30 minutes. The cells were then washed twice with PBS and analyzed 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 populations 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 The HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) was used to detect the secretion level of the cytokine IFN-γ in each of the four cell populations 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 cells 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 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 TILs

[0156] The in vitro cytotoxicity 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 was detected using a real-time label-free cell function analyzer (RTCA) from Eisen. The specific steps are as follows:

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

[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 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 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 and no effector cells, and start step 3. After co-culturing for more than 48-72 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, Culture and Sorting of TILs from Non-Small Cell Lung Cancer

[0162] Fresh non-small cell lung cancer tissue T05 was mechanically cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and then divided into two 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 from 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 medium containing 6000 IU / 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. Each part was incubated with antibodies coupled to fluorescent groups of each marker in the immune cell marker combination numbered 13, 14, 15 and 18 (excluding intracellular markers) in Table 1 of Example 1, and flow cytometry (BD FACS Aria) was performed. TMIII, bdbiosciences) sorting, and after multiple sorting, the immune cell marker combination 13, 14, 15 and 18 positive cell populations were obtained and named T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18, respectively.

[0163] Take 10 cells from each of T05-REP-13, T05-REP-14 and T05-REP-15 cell populations. 6 Cells were fixed with PBS containing 2 v / v% paraformaldehyde and centrifuged at 800 g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended and washed with PBS twice. The cells were permeabilized by adding PBS containing 0.7 v / v% Tween-20 and incubating at room temperature for 15 minutes. Cells were centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the cell pellet was washed twice with PBS and resuspended. Appropriately diluted fluorophore-conjugated anti-CXCL10 antibody, anti-IFN-γ antibody, and anti-IFN-γ antibody were added to T05-REP-13, T05-REP-14, and T05-REP-15 cells, respectively. After incubation at room temperature for 30 minutes, the cells were washed twice with PBS and analyzed by flow cytometry.

[0164] The results showed that 90.7% of T05-REP-13 cells were CXCL10-positive, while 87.6% and 88.1% of T05-REP-14 and T05-REP-15 cells were IFN-γ-positive, respectively. These results indicate that the vast majority of T05-REP-13 cell populations were CXCL10-positive, while the vast majority of T05-REP-14 and T05-REP-15 cell populations were IFN-γ-positive.

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

[0166] The four cell populations 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 The HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) was used to detect the secretion level of the cytokine IFN-γ in each of the four cell populations 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 cells 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 levels of cytokines 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 derived from T05 tissue

[0169]

[0170] Example 16, Detection of Tumor Cell Killing Function of Sorted Non-Small Cell Lung Cancer-Derived TILs

[0171] The in vitro cytotoxicity of the T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18 cell populations obtained in Example 14 against their homologous primary non-small cell lung cancer cells was detected using a real-time label-free cell function analyzer (RTCA) from Eisen. The specific steps are as follows:

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

[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 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 to culture the cells.

[0174] (3) Adding effector cells: After culturing the target cells for 18-24 hours, observe the cell index. When the cell index is 1, add effector cells T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18, 50 μL per well, with an effector-target ratio of 4:1. In addition, set up a control group with only target cells and no effector cells, and start step (3). After co-culturing for more than 48-72 hours, observe the target cell killing level and calculate the target cell killing rate.

[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 TIL of T05-REP-18, TIL of T05-REP-13, T05-REP-14, and T05-REP-15 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 TILs

[0177] Fresh colon cancer tissue T06 was mechanically cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and then divided into two portions. One portion 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 from T06 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 medium containing 6000 IU / 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 three equal parts. Each part was incubated 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 (excluding intracellular markers) and flow cytometry (BD FACSAria) respectively. TM III, bdbiosciences) sorting, and after multiple sorting, immune cell marker combination 16, 17, and 18 positive cell populations were obtained and named T06-REP-16, T06-REP-17, and T06-REP-18, respectively.

[0178] 2E6 cells were obtained from a T06-REP-16 cell population and fixed with PBS containing 2% v / v paraformaldehyde. The cells were centrifuged at 800 g for 5 minutes, the supernatant discarded, and the cell pellet was resuspended and washed twice with PBS. The cells were then permeabilized by adding PBS containing 0.7% v / v Tween-20 and incubated at room temperature for 15 minutes. The cells were centrifuged at 800 g for 5 minutes, the supernatant discarded, and the cell pellet was washed twice with PBS and resuspended. Appropriately diluted fluorophore-conjugated anti-IFN-γ and anti-TNF-α antibodies were added and incubated at room temperature for 30 minutes. The cells were then washed twice with PBS and analyzed by flow cytometry.

[0179] 2E6 cells were obtained from a T06-REP-17 cell population and fixed with PBS containing 2% v / v paraformaldehyde. The cells were centrifuged at 800 g for 5 minutes, the supernatant discarded, and the cell pellet was resuspended and washed twice with PBS. The cells were then permeabilized by adding PBS containing 0.7% v / v Tween-20 and incubating at room temperature for 15 minutes. The cells were centrifuged at 800 g for 5 minutes, the supernatant discarded, and the cell pellet was washed twice with PBS and resuspended. A properly diluted fluorophore-conjugated anti-IFN-γ antibody was added and incubated at room temperature for 30 minutes. The cells were then washed twice with PBS and analyzed 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, while 96.9% of the T06-REP-17 cell population were IFN-γ-positive. This suggests that the vast majority of the T06-REP-16 cell population were double-positive for IFN-γ and TNF-α, while 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 cell populations 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 The HTRF IFN-γ detection kit (CisbioHuman IFN gamma kit, catalog number: 62HIFNGPET) was used to detect the secretion level of the cytokine IFN-γ in each of the four cell populations 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+ cells. 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 cytokines 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 derived from T06 tissue

[0185]

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

[0187] The in vitro cytotoxicity 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 a real-time label-free cell function analyzer (RTCA) from Eisen. The specific steps are as follows:

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

[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 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 to culture the cells.

[0190] (3) Adding effector cells: After culturing the target cells for 18-24 hours, observe the cell index. When the cell index is 1, add effector cells T06-REP-16, T06-REP-17, and T06-REP-18, 50 μL per well, with an effector-target ratio of 4:1. Set up a separate control group with only target cells and no effector cells, and start step 3. After co-culturing for more than 48-72 hours, observe the target cell killing level and calculate the target cell killing rate.

[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 colon cancer target cells were significantly higher than those of T06-REP-18, indicating that compared with T06-REP-18 TIL, T06-REP-16 and T06-REP-17 TILs had significantly stronger killing effects on homologous primary colon cancer tumor cells.

[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 sub-combination) 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 were specifically and individually incorporated herein by reference.

Claims

1. An immune cell marker combination for enriching immune cells with enhanced tumor-killing ability, comprising: (1) an immune cell activation marker, and (2) an immune cell inhibition marker, wherein the immune cell activation marker includes CD69, and the immune cell inhibition marker includes one or more selected from CD39, TIM3, and CD161, and the tumor is gastric cancer.

2. The immune cell marker combination according to claim 1, wherein The immune cell marker combination further includes a tissue-resident memory marker, and the tissue-resident memory marker includes any one or more selected from CD69, CD103 and CD49a.

3. The immune cell marker combination according to claim 1, wherein The immune cell marker combination also includes: any one or more of intracellular IFN-γ, TNF-a, CXCL10, CXCL13, IL-2, IL-4, IL-6, IL-8, IL-10, intracellular CD137, intracellular CD69 and intracellular CD107a.

4. The immune cell marker combination according to claim 1, wherein: 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 inhibition markers also include one or more selected from PD-1, LAG3, CTLA-4, TIGIT, CD101 and CD160. 5 . A reagent for detecting the immune cell marker combination according to any one of claims 1 to 4 , wherein the reagent is a binding molecule that specifically recognizes each marker.

6. The reagent according to claim 5, wherein: The binding molecule is an antibody or an antigen-binding fragment thereof, and / or The binding molecule is conjugated to a detectable label.

7. The reagent according to claim 6, characterized in that The detectable label is biotin or a fluorescent group.

8. A composition for enriching immune cells with improved tumor-killing ability, comprising the immune cell marker combination according to any one of claims 1 to 4 or the reagent according to any one of claims 5 to 7, wherein the tumor is gastric cancer.

9. A kit for identifying or preparing immune cells with enhanced tumor-killing ability, comprising the immune cell marker combination according to any one of claims 1-4, the reagent according to any one of claims 5-7, or the composition according to claim 8, wherein the tumor is gastric cancer.

10. The kit according to claim 9, wherein The kit also includes an immunoreactive reagent.

11. A method for identifying immune cells with improved tumor-killing ability, comprising detecting the expression of the immune cell marker combination of any one of claims 1-4 in immune cells, wherein the immune cells expressing positive are immune cells with improved tumor-killing ability, and the tumor is gastric cancer.

12. The method according to claim 11, wherein The immune cells are T cells or NK cells.

13. The method according to claim 11, wherein The immune cells are NKT cells.

14. The method according to claim 11, wherein The immune cells are TILs.

15. A method for screening immune cells with enhanced tumor-killing ability, comprising screening an immune cell population for cells that positively express the immune cell marker combination according to any one of claims 1 to 4, wherein the tumor is gastric cancer.

16. The method according to claim 15, wherein The immune cells are T cells or NK cells.

17. The method according to claim 15, wherein The immune cells are NKT cells.

18. The method according to claim 15, wherein The immune cells are TILs.

19. Use of the immune cell marker combination of any one of claims 1-4, the reagent of any one of claims 5-7 and / or the composition of claim 8 in preparing a product for identifying or preparing immune cells with improved tumor-killing ability, wherein the tumor is gastric cancer.

20. The use according to claim 19, characterized in that: The immune cells are T cells or NK cells, and / or The product is a kit or a device.

21. The use according to claim 19, characterized in that The immune cells are NKT cells.

22. The use according to claim 19, wherein The immune cells are TILs.

23. A method for preparing immune cells with enhanced tumor-killing ability, comprising: Screening isolated tumor-infiltrating lymphocytes for cells positively expressing the immune cell marker combination according to any one of claims 1 to 4, wherein the tumor is gastric cancer.

24. The method of claim 23, wherein: The immune cells are T cells or NK 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.

25. The method of claim 23, wherein: The immune cells are NKT cells.

26. The method according to any one of claims 23 to 25, wherein The method further comprises a step of obtaining isolated tumor-infiltrating lymphocytes from the tumor sample, and / or the method further comprises a step of further culturing the obtained immune cells with enhanced tumor-killing ability.

27. The method according to claim 26, wherein The steps of obtaining isolated tumor-infiltrating lymphocytes from a tumor sample include: (1.1) obtaining seed cells from the tumor sample, and (1.2) culturing the seed cells to obtain isolated tumor-infiltrating lymphocytes.

28. The method of claim 27, wherein: The tumor sample is cultured in a seed cell culture medium to obtain seed cells.

29. Use of immune cells with enhanced tumor-killing ability obtained by the method according to any one of claims 23 to 28 in the preparation of drugs for treating cancer, wherein the cancer is gastric cancer.

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