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, including: (1) an immune cell activation marker, and (2) an immune cell inhibition marker, optionally further including (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 No. 202310242995.1 filed on March 14, 2023. Technical Field

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

[0003] Tumor Infiltrating Lymphocyte (TIL) therapy has become an important type of therapy for solid tumors in tumor immunocyte therapy technology in recent years, and very significant clinical effects have been achieved for various solid tumors such as melanoma, cervical cancer, head and neck tumors, and non-small cell lung cancer. TIL mainly consists of T cells, which are a heterogeneous population of 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 using TIL as adoptive T cell therapy (ACT) to treat tumors, how to increase the content of tumor-specific T cells in TIL, reduce the proportion of bystander T cells, and obtain a population of TIL cells that can survive in vivo for a long time has become a crucial factor in improving the therapeutic effect.

[0004] Multiple different metrics have been mentioned in the published reports 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. documented that in multiple 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. documented that tumor-specific CD4+ T cells are concentrated in the CD39+ TIL subset. WO2021226085A1 discloses a method for expanding TIL for clinical treatment, which includes a sorting step for TIL expressing positive for specific metrics, and the specific metrics include PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3, and / or TIGIT. However, there is still a need for more accurate and effective criteria for identifying and / or sorting TIL to determine the proportion of tumor-specific and tumor-reactive T cells in the prepared TIL cells, and possibly further sorting and expanding this part of the cells subsequently to improve the clinical efficacy of TIL. Summary of the Invention

[0005] In a first aspect of the present invention, there is provided a combination of immune cell markers, comprising: (1) an immune cell activation marker, and (2) an immune cell inhibition marker,

[0006] Optionally, it further comprises (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 marker comprises one or more selected from CD25, CD38, CD69, CD137, CD107a, CD226, CD150, and Ly108.

[0009] In one or more embodiments, the immune cell inhibition marker comprises 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 activation markers of immune cells. 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 cells are T cells, NK cells, NKT cells, or TILs.

[0013] The present invention also provides a reagent for detecting the marker combination described in any one of the embodiments herein, and 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 with 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 one of the embodiments 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 one of the embodiments herein.

[0018] In one or more embodiments, the immune cells are T cells, NK cells, NKT cells, or TILs.

[0019] In one or more embodiments, the kit further includes immune reaction reagents. Preferably, the immune reaction reagents include a blocking solution, a washing solution, and an enzyme-labeled reagent.

[0020] In one or more embodiments, the kit is applicable to the use or method described in any one of the embodiments herein.

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

[0022] In one or more embodiments, the immune cells are T cells, NK cells, NKT cells, or TILs.

[0023] The present invention also provides a method for screening tumor-specific immune cells, including screening for cells that are positive for the combined expression of the immune cell markers described herein in a population of immune cells.

[0024] In one or more embodiments, the immune cells are T cells, NK cells, NKT cells, or TILs.

[0025] Use of the marker combination or reagent according to 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 cells are T cells, NK cells, NKT cells, or TILs.

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

[0028] The present invention provides a method for preparing tumor-specific immune cells, including: screening for cells that are positive for the combined expression of the immune cell markers described herein from isolated tumor-infiltrating lymphocytes.

[0029] In one or more embodiments, the immune cells are T cells, NK cells, or NKT cells.

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

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

[0032] (1.2) Culturing the 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 of a subject in need, surgically resected primary site samples, synchronous and metachronous surgically resected metastatic site samples, puncture samples, and body fluids. The body fluids include blood, tissue fluid, lymph fluid, and / or body cavity effusion.

[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 cholangiocarcinoma.

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

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

[0037] Use of the tumor-specific immune cells prepared by the method for preparing tumor-specific immune cells described herein 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. 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 against 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 against 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 against 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 against 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 against primary non-small cell lung cancer target cells;

[0044] Figure 6 : Killing rates of T06-REP-16, T06-REP-17, and T06-REP-18 TIL cell populations against primary colon cancer target cells. Detailed Description of the Invention

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one 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, N.Y. 2012).

[0046] Through in-depth research, the inventors discovered markers of tumor-specific immune cells. Using these markers, tumor-specific immune cells can be accurately and rapidly 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 obtained by culturing from tumor samples. In this article, a tumor sample is any sample containing tumor cells, including but not limited to: ascites of a subject in need, primary resection samples, synchronous and metachronous surgical resection of metastatic samples, puncture samples, and body fluids.

[0048] The markers of the tumor-specific immune cells 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 by these markers are also within the scope of the present invention.

[0049] In this article, immune cells include T cells, NK cells, NKT cells, or TIL. Preferably T cells derived from TIL.

[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 cholangiocarcinoma; 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. In this article, tumor-specific immune cell activation markers include one, two, or more selected from CD25, CD38, CD69, CD137, CD107a, CD226, CD150, and Ly108. Preferably, the immune cell activation markers include one, two, or more selected from CD226, Ly108, CD69, CD107a, CD226, CD150.

[0052] In this article, tumor-specific immune cell inhibitory markers include one or more selected from CD39, PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160, and CD161. Preferably, the immune cell inhibitory markers include one, two, or more selected from CD39, LAG3, CD161, TIGIT, TIM3, and CD160. In some embodiments, the immune cell inhibitory 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, and 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. In this article, "intracellular + marker" refers to the marker located within immune cells, whose structure is different from that of the marker without the "intracellular" prefix (usually located on the cell membrane surface). For example, "intracellular CD137" refers to the CD137 molecule located within the cell, while "CD137" refers to the CD137 molecule in the membrane surface form.

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

[0055] In this article, each component of the markers of the tumor-specific immune cells, namely immune cell activation markers, immune cell inhibitory markers, intracellular markers, and tissue-resident memory markers, can be respectively selected from any one or more of the above immune cell activation markers, any one or more of the above immune cell inhibitory markers, any one or more of the above intracellular markers, and any one or more of the above tissue-resident memory markers. Therefore, the markers of the tumor-specific immune cells in this article can be any combination of the markers in the above components.

[0056] In some embodiments, the markers of the 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 further 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 further include one, two, or more selected from PD-1, TIM3, CTLA-4, TIGIT, CD101, and 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 the tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of the tumor-specific immune cells further include (3) intracellular markers. The immune cell activation markers include Ly108. In addition, the immune cell activation markers may further 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 further 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 the tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of the tumor-specific immune cells further include (3) intracellular markers. The immune cell activation markers include CD69. In addition, the immune cell activation markers may further 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 further 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 may further include any one or more selected from IFN-γ, CXCL10, TNF-α, intracellular CD137. In one or more embodiments, the tumor is gastric cancer.

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

[0060] In some embodiments, the markers of the tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of the 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, 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 further include one, two or more selected from CD25, CD38, CD69, CD137, CD150. The immune cell inhibition marker includes CD39. In addition, the immune cell inhibition marker may further 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, intracellular CD137. Preferably, the immune cell intracellular markers are selected from: (1) CXCL10, (2) IFN-γ. In addition, the immune cell intracellular markers may further 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 may further include CXCL13 and / or TNF-α. The tissue-resident memory marker of the immune cells includes CD103. In addition, the tissue-resident memory marker of the immune cells may further 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 the tumor-specific immune cells include: (1) immune cell activation markers, and (2) immune cell inhibition markers. Optionally, the markers of the 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 further 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) the combination of CD39 and CD160, or (2) the combination of CD39 and CD161. In addition, the immune cell inhibition markers may further include one, two, or more selected from PD-1, TIM3, LAG3, CTLA-4, TIGIT, and CD101. The immune cell intracellular markers include one or two selected from IFN-γ and TNF-α. Preferably, the immune cell intracellular markers are selected from: (1) IFN-γ, or (2) IFN-γ and TNF-α. In addition, the immune cell intracellular markers may further 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, they may further include CXCL10 and / or CXCL13. The tissue-resident memory markers of the immune cells include CD49a and / or CD103. Preferably, the tissue-resident memory markers of the immune cells are selected from: (1) CD49a, or (2) CD49a and CD103. In addition, the tissue-resident memory markers may further include CD69. In one or more embodiments, the tumor is intestinal cancer, such as colon cancer.

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

[0063] Any method that can be used to detect the above-mentioned markers expressed (intracellularly or on the cell surface) or secreted by cells can be used in the present invention. Preferably, such a method is achieved by incubating the 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 the convenience of detection, the binding molecules are conjugated 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 for detecting the markers (abbreviated as detection reagents) are also within the scope of the present invention. For example, the above-mentioned binding molecules that specifically recognize each marker.

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

[0066] The markers of the present invention are particularly suitable for a method of screening and obtaining tumor-specific immune cells from a population of immune cells, including the steps of screening (such as by flow cytometry) cells that are positive for the combined expression of the immune cell markers 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 including: (1.1) obtaining seed cells from the tumor sample, such as culturing the tumor sample using a seed cell medium to obtain seed cells, and (1.2) culturing the seed cells to obtain isolated tumor-infiltrating lymphocytes.

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

[0069] In some embodiments, step (1.1) includes: (a) washing the tumor tissue sample (e.g., washing with normal 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 - 10 mm, (b) culturing the tumor tissue pieces at 30 - 42 °C and 1 - 10% CO2 using a seed cell culture medium for 3 - 20 days. Exemplary step (1.1) is as described in Example 2 of WO2021239083A1, and includes the following steps: 1) Place the freshly isolated tumor tissue sample obtained in a sterile environment in a class II biosafety cabinet into a 10 cm culture dish containing 30 mL of normal saline (containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 50 μg / mL gentamicin) for washing, and then transfer it to a new 10 cm dish containing 30 mL of the above normal saline for washing, repeating the washing 3 times in total; 2) Use a sterile surgical blade to remove adipose tissue and necrotic tissue, cut the tumor tissue into small pieces with a diameter of 3 mm, place 12 randomly selected tumor tissue pieces in each G-REX10 culture tank (purchased from Wilsonwolf), and add TIL seed medium; 3) Add the seed cell medium to different G-REX10 culture tanks respectively, 40 mL per tank, culture the tumor tissue pieces at 37 °C and 5% CO2, count the total number and viability of cells after harvesting TIL seed cells on the 12th day, and detect the phenotype of cells by flow cytometry.

[0070] The culture described in step (1.2) can use any medium in the art for culturing TIL. For example, AIM-V 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 includes further culturing the obtained tumor-specific immune cells (such as TIL). The culture can be carried out using any medium known in the art suitable for immune cells (such as TIL, especially tumor-specific TIL).

[0072] The tumor-specific immune cells prepared by the method described herein can be used for scientific research or for preparing therapeutic drugs for the corresponding cancer. 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, a "pharmaceutically acceptable excipient" is a pharmaceutically or food acceptable carrier, solvent, suspending agent or excipient for delivering the tumor-specific immune cells of the present invention to an animal or a human. As used herein, a pharmaceutically acceptable excipient is non-toxic to the recipient of the composition at the dosages and concentrations employed. It may include various types of carriers or excipients commonly used in the art for delivering immune cells in therapy. Exemplary excipients may be liquid or solid and include, but are not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifying agents. 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 for use in combination with a pharmaceutically active compound. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, A.R. 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 can be selected for parenteral delivery, for inhalation or for delivery through the digestive tract (such as orally), for example for intravenous infusion delivery. The preparation of the compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations that incorporate immune cells, particularly immune cells (such as T cells), in sustained or controlled release delivery formulations.

[0075] Pharmaceutical compositions for in vivo administration are generally provided in a sterile formulation. Sterilization is achieved by filtration through a sterile filtration membrane. Compositions for parenteral administration can be stored in lyophilized form or in solution (such as a frozen preparation). Parenteral compositions are usually placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper that can be pierced by a hypodermic needle.

[0076] Once formulated, the pharmaceutical composition is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal, frozen product, or as a 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 requires further formulation before administration. For example, a suitable formulation for delivering the pharmaceutical composition described herein can be a frozen preparation, which can tolerate long-distance transportation without damaging cells. In addition to the cells themselves, frozen preparations typically also include components such as cell cryopreservation medium and human serum albumin (HSA). Before administration (e.g., intravenous infusion), the frozen pharmaceutical composition needs to be stored at a low temperature (e.g., placed in liquid nitrogen). After thawing, the frozen pharmaceutical composition can be directly administered to the patient or formulated into an infusion composition for administration. Those skilled in the art are aware of the components and concentrations of conventional cryopreservation media. For example, the cryopreservation medium or infusion composition may also contain dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, or magnesium chloride, etc., and their concentrations can be determined by those skilled in the art (e.g., experienced physicians) according to the conditions of cells, diseases, patients, etc.

[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 on the memory and executable on the processor, wherein, when the processor executes the program, the following steps are implemented: screening (e.g., by flow cytometry) cells that are positive for the combination of immune cell markers described herein from isolated tumor-infiltrating lymphocytes. For example, the device records or contains markers or reagents of the tumor-specific immune cells described herein, and by detecting or determining whether the cells in the sample contain the markers, the cells positive for the markers are identified and screened.

[0078] The present invention also provides the use of the marker combination or reagent according to any one of the embodiments herein in the preparation of a product for identifying or preparing tumor-specific immune cells. The product includes the kits or devices described herein.

[0079] The term “about” or “approximately” means within an acceptable error range of a specific value determined by those of ordinary skill in the art, which will depend on how the value is measured or determined, e.g., the limitations 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. Or, particularly for biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5-fold, more preferably within 2-fold. When specific values are described in this application and the claims, unless otherwise stated, it is assumed that the term “about” means within the acceptable error range of the specific value.

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

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

[0082] Unless otherwise indicated, all percentages and ratios are calculated based on the total amount of the composition.

[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 falling within the broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0084] The values recited herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specifically stated, each such value is intended to refer to the recited value 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-reference 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, or reference, suggestion or disclosure of any such invention. Further, when any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to the term in this document shall govern.

[0086] For the sake of facilitating the understanding of the present invention, the present invention will be described more comprehensively hereinafter. However, the present invention can be implemented in many different ways and is not limited to the embodiments described herein. On the contrary, these embodiments are provided so that the disclosure of the present invention will be thorough and complete. 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. Without departing from the spirit and scope of the present disclosure, various other changes and modifications can be made. The scope of the appended claims includes all such changes and modifications within the scope of the present disclosure.

[0087] Examples

[0088] Example 1, Tumor-Specific TIL Immune Cell Marker Combinations and Tumor Tissue Samples

[0089] The immune cell marker combinations involved in the examples are shown in Table 1 below:

[0090] Table 1, Immune Cell Marker Combinations

[0091]

[0092] The tumor tissue samples used in the examples are shown in Table 2 below:

[0093] Table 2, Tumor Tissue Samples

[0094] Sample number Cancer type T01 Melanoma T02 Cervical cancer T03 Gastric cancer T04 Ovarian cancer T05 Non-small cell lung cancer T06 Colorectal cancer

[0095] The sources of the relevant conjugated fluorescent group flow antibodies used in the examples are shown in Table 3 below:

[0096] Table 3, Flow Fluorescent Antibodies

[0097]

[0098]

[0099] Example 2, Culture and Sorting of TILs Derived from Melanoma

[0100] Fresh melanoma tissue T01 was mechanically cut into pieces of 3×3×3 mm in size. After the pieces were evenly mixed as much as possible, they were divided into 2 parts. One part was cultured to obtain primary tumor cells of T01 tissue according to the method described in the "Materials and Methods" section of Robert Suriano et al. Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines J Cancer 2013; 4(5): 371-382. The remaining one part was cultured to obtain seed cells according to the method described in Example 2 of WO2021239083A1. The culture medium for culturing the 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 specification of CN110099998A. The obtained seed cells were prepared into an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded in culture to obtain REP cells. Then, the REP cells were further divided into 4 equal parts, and each part was successively incubated with the antibodies conjugated with fluorescent groups of each marker in the immune cell marker combinations numbered 1, 2, 3, and 18 in Table 1 of Example 1 (except for intracellular markers) one by one, and then analyzed by flow cytometry (BD FACSAria TMIII. Sorted by BD Biosciences, after multiple sorts, positive cell populations of immune cell markers 1, 2, 3, and 18 were obtained, named T01-REP-1, T01-REP-2, T01-REP-3, and T01-REP-18 respectively.

[0101] Take 10 6 cells from the T01-REP-3 cell population, fix the cells with PBS containing 2 v / v% paraformaldehyde, centrifuge at 800 g for 5 minutes, discard the supernatant, resuspend and wash the cell pellet with PBS, repeat 2 times, add PBS containing 0.7 v / v% Tween-20, incubate at room temperature for 15 minutes to permeabilize the cell membrane. Centrifuge at 800 g for 5 minutes, discard the supernatant, wash the cell pellet 2 times with PBS and then resuspend, add appropriately diluted anti-CD137 antibody conjugated with a fluorescent group, incubate at room temperature for 30 minutes, wash the cells 2 times with PBS, and detect on a flow cytometer.

[0102] The results showed that in the T01-REP-3 cell population, the proportion of cells positive for intracellular CD137 was 93.2%. It indicates that the vast majority of cells in the T01-REP-3 cell population are positive for intracellular CD137.

[0103] Example 3. Phenotypic detection of sorted melanoma-derived TIL

[0104] Use a flow cytometer to detect the following for the four cell populations of T01-REP-1, T01-REP-2, T01-REP-3, and T01-REP-18 obtained in Example 2: 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-). Use the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) to detect the secretion level of the cytokine IFN-γ for each of the four cell populations according to the method described in the instruction manual.

[0105] The results are shown in Table 4. More than 99% of T01-REP-1, T01-REP-2, T01-REP-3, and T01-REP-18 are CD45+ and CD3+ cells. The proportions of positive cells for exhaustion markers and activation markers in T01-REP-1, T01-REP-2, and T01-REP-3 are all higher than the corresponding indicators of T01-REP-18; the secretion levels of the cytokine IFN-γ and memory T cells (especially TCM ) The proportion is also generally significantly higher than that of T01-REP-18.

[0106] Table 4, T01 Tissue Source TIL Phenotype

[0107]

[0108] Example 4, Detection of Tumor Cell Killing Function of Sorted Melanoma-Derived TIL

[0109] Use the Real-Time Cell Analyzer (RTCA) of ACEA Biosciences 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:

[0110] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and perform zero adjustment;

[0111] (2) Target cell seeding: Seed the T01 melanoma tissue primary cells obtained by culturing in Example 1 at 10 4 cells / 50 μL in the plate containing detection electrodes. Let it stand for several minutes. After the cells are stable, place it in the instrument and start step 2 to culture the cells;

[0112] (3) Adding effector cells: After culturing the target cells for 18 h - 24 h, observe the cell index. When the cell index is 1, add the effector cells T01-REP-1, T01-REP-2, T01-REP-3, and T01-REP-18, 50 μL per well, and the effector-to-target ratio is 4:1. Additionally, set up a control group with only target cells seeded and no effector cells added. Start step (3). After co-culturing for more than 48 - 72 h, observe the killing level of the target cells and calculate the target cell killing rate. The formula for calculating the target cell killing rate is as follows (the formula for calculating the target cell killing rate in the following examples is the same):

[0113]

[0114] Where A is the cell index of the control group, and B is the cell index of each group with effector cells added.

[0115] The results are as Figure 1 shown. The killing rates of T01-REP-1, T01-REP-2, and T01-REP-3 against the melanoma tumor primary target cells are significantly higher than that of T01-REP-18, indicating that compared with the TIL of T01-REP-18, the TIL of T01-REP-1, T01-REP-2, and T01-REP-3 have significantly stronger killing effects on the homologous melanoma primary tumor cells.

[0116] Example 5, Cultivation and Sorting of TILs Derived from Cervical Cancer

[0117] The fresh cervical cancer tissue T02 was mechanically cut into pieces of 3×3×3 mm in size. After the pieces were evenly mixed as much as possible, they were divided into two parts. One part of the tissue was cultured to obtain primary tumor cells of the T02 tissue according to the method described in the "Materials and Methods" section of A D 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. The remaining one part was cultured to obtain seed cells according to the method described in Example 2 of WO2021239083A1. The culture medium for culturing the 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 specification of CN110099998A. The obtained seed cells were formulated into an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded in culture to obtain REP cells. Then, the REP cells were further divided into four equal parts, and each part was successively incubated with antibodies conjugated with fluorescent groups of each marker in the immunocyte marker combinations numbered 4, 5, 6, and 18 (excluding intracellular markers) in Table 1 of Example 1, and sorted by a flow cytometer (BD FACSAria TM III, bdbiosciences). After multiple sorting, immunocyte marker combination 4, 5, 6, and 18 positive cell populations were obtained, and were respectively named T02-REP-4, T02-REP-5, T02-REP-6, and T02-REP-18.

[0118] Take 10 from the T02-REP-4 cell population 6Cells were fixed with PBS containing 2 v / v% paraformaldehyde, centrifuged at 800 g for 5 minutes, the supernatant was discarded, the cell pellet was resuspended and washed with PBS, repeated 2 times, PBS containing 0.7 v / v% Tween-20 was added, incubated at room temperature for 15 minutes, and the cells were permeabilized. Centrifuged at 800 g for 5 minutes, the supernatant was discarded, the cell pellet was washed 2 times with PBS and then resuspended, appropriately diluted anti-IFN-γ antibody conjugated with a fluorescent group was added, incubated at room temperature for 30 minutes, and then the cells were washed 2 times with PBS and detected by flow cytometry.

[0119] The results showed that in the T02-REP-4 cell population, the proportion of IFN-γ-positive cells was 97.5%. It indicated that the vast majority of cells in the T02-REP-4 cell population were IFN-γ-positive cells.

[0120] Example 6, Phenotypic detection of sorted TILs from cervical cancer

[0121] The four cell populations of T02-REP-4, T02-REP-5, T02-REP-6, and T02-REP-18 obtained in Example 5 were detected by flow cytometry for their respective: 1) lymphocyte phenotypes 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 levels of the cytokine IFN-γ in the four cell populations were detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instructions.

[0122] The results are shown in Table 5. More than 90% of the cells in T02-REP-4, T02-REP-5, T02-REP-6, and T02-REP-18 were CD45+ and CD3+ cells. The proportions of exhausted marker-positive and activation marker-positive cells in T02-REP-4, T02-REP-5, and T02-REP-6 were all higher than those in T02-REP-18; the secretion levels of the cytokine IFN-γ and the proportion of memory T cells, especially T CM proportion in T02-REP-4, T02-REP-5, and T02-REP-6 were also generally significantly higher than those in T01-REP-18.

[0123] Table 5, Phenotype of TILs from T02 tissue

[0124]

[0125] Example 7, Detection of tumor cell killing function of sorted TILs from cervical cancer

[0126] Use the Real-Time Cell Analyzer (RTCA) of ACEA Biosciences, Inc. 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 primary cervical cancer cells. The specific steps are as follows:

[0127] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and perform zero adjustment;

[0128] (2) Seeding of target cells: Seed the primary T02 cervical cancer tissue cells obtained by culturing in Example 5 at a density of 10 4 cells / 50 μL in a plate containing detection electrodes. Let it stand for several minutes. After the cells are stable, place it in the instrument and start step 2 to culture the cells;

[0129] (3) Adding effector cells: After culturing the target cells for 18 h - 24 h, observe the cell index. When the cell index is 1, add the effector cells T02-REP-4, T02-REP-5, T02-REP-6, and T02-REP-18, 50 μL per well, with an effector-to-target ratio of 4:1. Additionally, set up a control group with only target cells seeded and no effector cells added. Start step (3). After co-culturing for more than 48 - 72 h, observe the killing level of the target cells and calculate the target cell killing rate.

[0130] The results are as Figure 2 shown. The killing rates of T02-REP-4, T02-REP-5, and T02-REP-6 against the primary target cervical cancer cells are significantly higher than that of T02-REP-18, indicating that compared with the TIL of T02-REP-18, the TIL of T02-REP-4, T02-REP-5, and T02-REP-6 have significantly stronger killing effects on the homologous primary cervical cancer cells.

[0131] Example 8. Culture and Sorting of TIL Derived from Gastric Cancer

[0132] Fresh gastric cancer tissue T03 was mechanically cut into pieces of 3×3×3 mm in size. After the pieces were evenly mixed as much as possible, they were divided into two parts. One part of the tissue was cultured to obtain primary tumor cells of T03 tissue according to the method described in the Methods section of 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. The remaining one part was cultured to obtain seed cells according to the method described in Example 2 of WO2021239083A1. The culture medium for culturing the 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 specification of CN110099998A. The obtained seed cells were prepared into an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded in culture to obtain REP cells, which were further divided into four equal parts. Each part was then incubated successively one by one with the antibodies of the fluorescent groups exposed by each marker in the immunocyte marker combinations numbered 7, 8, 9 and 20 in Table 1 of Example 1 (except for intracellular markers), and sorted by a flow cytometer (BD FACSAria TM III, bdbiosciences). After multiple sorting, positive cell populations of immunocyte marker combinations 7, 8, 9 and 20 were obtained, and were respectively named T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20.

[0133] Take 10 6 cells from the T03-REP-9 cell population, fix the cells with PBS containing 2 v / v% paraformaldehyde, centrifuge at 800 g for 5 minutes, discard the supernatant, resuspend and wash the cell pellet with PBS, repeat 2 times, add PBS containing 0.7 v / v% Tween-20, incubate at room temperature for 15 minutes, and perform membrane permeabilization treatment on the cells. Centrifuge at 800 g for 5 minutes, discard the supernatant, wash the cell pellet 2 times with PBS and then resuspend it, add appropriately diluted anti-CXCL13 antibody conjugated with a fluorescent group, incubate at room temperature for 30 minutes, wash the cells 2 times with PBS, and detect on a flow cytometer.

[0134] The results showed that in the T03-REP-9 cell population, the proportion of CXCL13-positive cells was 91.3%. It shows that the vast majority of cells in the T02-REP-4 cell population are CXCL13-positive cells.

[0135] Example 9, Phenotypic Detection of Sorted Gastric Cancer-derived TILs

[0136] The four cell populations of T03-REP-7, T03-REP-8, T03-REP-9, and T03-REP-20 obtained in Example 8 were detected by flow cytometry for their respective: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator PD-1; 3) activation indicator CD25; 4) memory T cell indicators: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion levels of the cytokine IFN-γ of the four cell populations were detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instructions.

[0137] As shown in Table 6, more than 90% of the cells in T03-REP-7, T03-REP-8, T03-REP-9, and T03-REP-20 were CD45+ and CD3+ cells. The proportion of positive cells for the exhaustion marker in T03-REP-7, T03-REP-8, and T03-REP-9 was generally higher than that in T03-REP-20; the secretion levels of the cytokine IFN-γ and the proportion of memory T cells (T CM 、T EM ) in T03-REP-7, T03-REP-8, and T03-REP-9 were also generally significantly higher than those in T03-REP-20.

[0138] Table 6, Phenotype of TILs from T03 Tissue

[0139]

[0140]

[0141] Example 10, Detection of Tumor Cell Killing Function of Sorted Gastric Cancer-derived TILs

[0142] The real-time label-free cell function analyzer (RTCA) of ACEA Biosciences was used to detect the in vitro killing activity of the cell populations of T03-REP-7, T03-REP-8, T03-REP-9, and T03-REP-20 obtained in Example 5 against their homologous gastric cancer primary cells. The specific steps are as follows:

[0143] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and perform zero adjustment;

[0144] (2) Seeding target cells: Seed the primary T03 gastric cancer tissue cells obtained from the culture in Example 5 at a density of 10^4 cells / 50 μL per well in a plate containing detection electrodes. Let it stand for several minutes. After the cells are stable, place it in the instrument and start Step 2 to culture the cells;

[0145] (3) Adding effector cells: After culturing the target cells for 18 h - 24 h, observe the cell index. When the cell index is 1, add effector cells T03-REP-7, T03-REP-8, T03-REP-9, and T03-REP-20, 50 μL per well, with an effector-to-target ratio of 4:1. Additionally, set up a control group with only target cells seeded and no effector cells added. Start Step (3). After co-culturing for more than 48 - 72 h, observe the killing level of the target cells and calculate the target cell killing rate.

[0146] The results are as Figure 3 shown. The killing rates of T03-REP-7, T03-REP-8, and T03-REP-9 against primary target cells of gastric cancer tumors are significantly higher than that of T03-REP-20, indicating that compared with the TIL of T03-REP-20, the TIL of T03-REP-7, T03-REP-8, and T03-REP-9 have significantly stronger killing effects on homologous primary gastric cancer tumor cells.

[0147] Example 11, Culture and Sorting of TIL Derived from Ovarian Cancer

[0148] Fresh ovarian cancer tissue T04 was mechanically cut into fragments of 3×3×3 mm in size. After the fragments were evenly mixed as much as possible, they were divided into two parts. One part was cultured to obtain primary tumor cells of T04 tissue according to the method described in the Protocol section of Lee J. Priby et al. Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens J Vis Exp. 2014;(84):51581. The remaining one part was cultured to obtain seed cells according to the method described in Example 2 of WO2021239083A1. The culture medium for culturing the 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 specification of CN110099998A. The obtained seed cells were prepared into an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded in culture to obtain REP cells. Then, the REP cells were further divided into four equal parts, and each part was incubated successively with antibodies conjugated with fluorescent groups of each marker in the immunocyte marker combinations numbered 10, 11, 12, and 19 in Table 1 of Example 1 (except for intracellular markers), and sorted by a flow cytometer (BD FACSAria TM III, bdbiosciences). After multiple sorting, positive cell populations of immunocyte marker combinations 10, 11, 12, and 19 were obtained, and were named T04-REP-10, T04-REP-11, T04-REP-12, and T04-REP-19, respectively.

[0149] Take 10 6 cells from the T04-REP-12 cell population, fix the cells with PBS containing 2 v / v% paraformaldehyde, centrifuge at 800 g for 5 minutes, discard the supernatant, resuspend and wash the cell pellet with PBS, repeat 2 times, add PBS containing 0.7 v / v% Tween-20, incubate at room temperature for 15 minutes, and perform membrane permeabilization treatment on the cells. Centrifuge at 800 g for 5 minutes, discard the supernatant, wash the cell pellet with PBS 2 times and then resuspend, add appropriately diluted anti-CXCL10 antibody conjugated with a fluorescent group, incubate at room temperature for 30 minutes, wash the cells with PBS 2 times, and detect on a flow cytometer.

[0150] The results showed that in the T04-REP-12 cell population, the proportion of CXCL10-positive cells was 89.9%. It indicated that the vast majority of cells in the T04-REP-12 cell population were CXCL10-positive cells.

[0151] Example 12, Phenotype Detection of TIL Derived from Ovarian Cancer by Sorting

[0152] The following were detected for the four cell populations of T04-REP-10, T04-REP-11, T04-REP-12, and T04-REP-19 obtained in Example 11 using a flow cytometer: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator TIM3; 3) activation indicator CD25; 4) memory T cell indicators: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). The secretion levels of the cytokine IFN-γ for each of the four cell populations were detected using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the method described in the instructions.

[0153] As shown in Table 7, nearly or more than 95% of the cells in T04-REP-10, T04-REP-11, T04-REP-12, and T04-REP-19 were CD45+ and CD3+ cells. The proportion of positive cells for the exhaustion marker 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 the proportion of memory T cells (T CM 、T EM ) in T04-REP-10, T04-REP-11, and T04-REP-12 were also generally significantly higher than those in T04-REP-19.

[0154] Table 7, T04 tissue-derived TIL phenotype

[0155]

[0156] Example 13, Detection of the tumor cell killing function of sorted ovarian cancer-derived TIL

[0157] The real-time label-free cell function analyzer (RTCA) from ACEA Biosciences was used to detect the in vitro killing activity of the cell populations of T04-REP-10, T04-REP-11, T04-REP-12, and T04-REP-19 obtained in Example 11 against their homologous ovarian cancer primary cells. The specific steps are as follows:

[0158] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and perform zero adjustment;

[0159] (2) Target cell plating: Plate the primary cells of T04 ovarian cancer tissue obtained in Example 11 at 10 4 cells / 50 μL per well in a plate containing detection electrodes. Let it stand for several minutes. After the cells are stable, place it in the instrument and start step 2 to culture the cells;

[0160] (3) Adding effector cells: After culturing the target cells for 18 h - 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-to-target ratio of 4:1. Additionally, set up a control group with only target cells plated and no effector cells added. Start step 3. After co-culturing for more than 48 - 72 h, observe the killing level of the target cells and calculate the target cell killing rate.

[0161] The results are as Figure 4 shown that the killing rates of T04-REP-10, T04-REP-11, and T04-REP-12 on primary ovarian cancer target cells are significantly higher than that of T04-REP-19, indicating that compared with the TIL of T04-REP-19, the TIL of T04-REP-10, T04-REP-11, and T04-REP-12 have significantly stronger killing effects on homologous primary ovarian cancer tumor cells.

[0162] Example 14, Culture and Sorting of TIL Derived from Non-Small Cell Lung Cancer

[0163] Mechanically cut fresh non-small cell lung cancer tissue T05 into fragments of 3×3×3 mm in size. After mixing the fragments as evenly as possible, divide them into 2 parts. One part is used to culture the primary tumor cells of T05 tissue according to the method described in the EXPERIMENTAL PROCEDURES section of D P.Kodack et al.Primary Patient-Derived Cancer Cellsand Their Potential for Personalized Cancer Patient Care Cell Rep.2017Dec 12;21(11):3298–3309. The remaining 1 part is used to culture the seed cells according to the method described in Example 2 of WO2021239083A1. The culture medium for culturing the seed cells is CM1 medium formulated with the formula and method described in Example 5 of the specification of CN110099998A, containing 6000 IU / mL IL-2, Glutamax, and antibiotics. The obtained seed cells are formulated into an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells are expanded in culture to obtain REP cells. Then, further divide them into 4 equal parts, and each part is successively incubated with the antibodies conjugated with fluorescent groups of each marker in the immune cell marker combinations numbered 13, 14, 15, and 18 in Table 1 of Example 1 (except for intracellular markers) one by one, and then analyzed by flow cytometry (BDFACSAria TMIII. Sorted by BD Biosciences. After multiple sorts, positive cell populations for immune cell markers 13, 14, 15, and 18 were obtained, named T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18, respectively.

[0164] Take 10 cells each from the cell populations of T05-REP-13, T05-REP-14, and T05-REP-15. 6 Fix the cells with PBS containing 2 v / v% paraformaldehyde, centrifuge at 800 g for 5 minutes, discard the supernatant, resuspend and wash the cell pellet with PBS, repeat 2 times, add PBS containing 0.7 v / v% Tween-20, incubate at room temperature for 15 minutes to permeabilize the cell membrane. Centrifuge at 800 g for 5 minutes, discard the supernatant, wash the cell pellet 2 times with PBS and then resuspend. Add appropriately diluted anti-CXCL10 antibody conjugated with a fluorescent group, anti-IFN-γ antibody, and anti-IFN-γ antibody to the cells of T05-REP-13, T05-REP-14, and T05-REP-15 respectively. After incubating at room temperature for 30 minutes, wash the cells 2 times with PBS and detect on a flow cytometer.

[0165] The results showed that in T05-REP-13 cells, the proportion of CXCL10-positive cells was 90.7%; in T05-REP-14 and T05-REP-15 cells, the proportions of IFN-γ-positive cells were 87.6% and 88.1% respectively. The above results indicate that the vast majority of cells in the T05-REP-13 cell population are CXCL10-positive cells, while the vast majority of cells in the T05-REP-14 and T05-REP-15 cell populations are IFN-γ-positive cells.

[0166] Example 15. Phenotype detection of TILs derived from non-small cell lung cancer by sorting

[0167] Use a flow cytometer to detect the following for the four cell populations of T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18 obtained in Example 14: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator PD-1; 3) activation indicator CD25; 4) memory T cell indicators: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). Use the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) to detect the secretion levels of the cytokine IFN-γ for each of the four cell populations according to the method described in the instruction manual.

[0168] The results are shown in Table 8. More than 99% of the cells in T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18 are CD45+, and the proportion of CD3+ cells is higher than 80% in all of them. The proportion of positive cells for exhaustion markers in T05-REP-13, T05-REP-14, and T05-REP-15 is generally higher than that in T05-REP-18; the secretion level of cytokine IFN-γ and the proportion of memory T cells (T CM , T EM ) in T05-REP-13, T05-REP-14, and T05-REP-15 are also generally significantly higher than those in T05-REP-18.

[0169] Table 8, Phenotype of TIL from T05 Tissue Source

[0170]

[0171] Example 16, Detection of Tumor Cell Killing Function of Sorted TIL from Non-Small Cell Lung Cancer

[0172] Use the real-time label-free cell function analyzer (RTCA) of ACEA Biosciences to detect the in vitro killing activity of the cell populations of T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18 obtained in Example 14 against their homologous non-small cell lung cancer primary cells. The specific steps are as follows:

[0173] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and perform zero adjustment;

[0174] (2) Target cell seeding: Seed the primary cells of T05 non-small cell lung cancer tissue obtained by culturing in Example 14 at a density of 10 4 cells / 50 μL in a plate containing detection electrodes. Let it stand for several minutes. After the cells are stable, place it in the instrument and start step 2 to culture the cells;

[0175] (3) Adding effector cells: After culturing the target cells for 18 h - 24 h, observe the cell index. When the cell index is 1, add the effector cells T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18, 50 μL per well, with an effector-to-target ratio of 4:1. Additionally, set up a control group with only target cells seeded and no effector cells added. Start step (3). After co-culturing for more than 48 - 72 h, observe the killing level of the target cells and calculate the target cell killing rate..

[0176] The results are as Figure 5It was shown that the killing rates of T05-REP-13, T05-REP-14, and T05-REP-15 against primary target cells of non-small cell lung cancer were significantly higher than that of T05-REP-18, indicating that compared with the TIL of T05-REP-18, the 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.

[0177] Example 17, Culture and Sorting of TIL Derived from Colon Cancer

[0178] Fresh colon cancer tissue T06 was mechanically cut into pieces of 3×3×3 mm in size. After the pieces were evenly mixed as much as possible, they were divided into two parts. One part was cultured to obtain primary tumor cells of T06 tissue according to the method described in the Materials and methods section of 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. The remaining part was cultured to obtain seed cells according to the method described in Example 2 of WO2021239083A1. The culture medium for culturing the 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 specification of CN110099998A. The obtained seed cells were prepared into an expansion medium according to the method described in Example 5 of WO2021239083A1, and the obtained seed cells were expanded in culture to obtain REP cells, which were further divided into three equal parts. Each part was then incubated successively with antibodies conjugated with fluorescent groups of each marker in the immunocyte marker combinations numbered 16, 17, and 18 in Table 1 of Example 1 (excluding intracellular markers), and sorted by a flow cytometer (BD FACSAria TM III, bdbiosciences). After multiple sorting, positive cell populations of immunocyte marker combinations 16, 17, and 18 were obtained, and were named T06-REP-16, T06-REP-17, and T06-REP-18, respectively.

[0179] Take 2E6 cells from the T06-REP-16 cell population, fix the cells with PBS containing 2 v / v% paraformaldehyde, centrifuge at 800 g for 5 minutes, discard the supernatant, resuspend and wash the cell pellet with PBS, repeat 2 times, add PBS containing 0.7 v / v% Tween-20, incubate at room temperature for 15 minutes to permeabilize the cell membrane. Centrifuge at 800 g for 5 minutes, discard the supernatant, wash the cell pellet 2 times with PBS and then resuspend, add appropriately diluted anti-IFN-γ antibody and anti-TNF-α antibody conjugated with fluorescent groups, incubate at room temperature for 30 minutes, then wash the cells 2 times with PBS, and detect by flow cytometry.

[0180] Take 2E6 cells from the T06-REP-17 cell population, fix the cells with PBS containing 2 v / v% paraformaldehyde, centrifuge at 800 g for 5 minutes, discard the supernatant, resuspend and wash the cell pellet with PBS, repeat 2 times, add PBS containing 0.7 v / v% Tween-20, incubate at room temperature for 15 minutes to permeabilize the cell membrane. Centrifuge at 800 g for 5 minutes, discard the supernatant, wash the cell pellet 2 times with PBS and then resuspend, add appropriately diluted anti-IFN-γ antibody conjugated with fluorescent groups, incubate at room temperature for 30 minutes, then wash the cells 2 times with PBS, and detect by flow cytometry.

[0181] The results showed that the proportion of IFN-γ and TNF-α double-positive cells in the T06-REP-16 cell population was 84.6%; the proportion of IFN-γ positive cells in the T06-REP-17 cell population was 96.9%. It shows that the vast majority of cells in the T06-REP-16 cell population are IFN-γ and TNF-α double-positive cells; the vast majority of cells in the T06-REP-17 cell population are IFN-γ positive cells.

[0182] Example 18, Phenotype Detection of Sorted Colon Cancer-derived TIL

[0183] Use flow cytometry to detect the following for the three cell populations of T06-REP-16, T06-REP-17, and T06-REP-18 obtained in Example 2: 1) phenotypic indicators CD45, CD3, CD4, CD8; 2) exhaustion indicator PD-1; 3) activation indicator CD25; 4) memory T cell indicators: T CM (CD45RO+CCR7+); T EM (CD45RO+CCR7-). Use the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) to detect the secretion level of the cytokine IFN-γ for each of the four cell populations according to the method described in the instruction manual.

[0184] As shown in Table 9, more than 99% of the cells in T06-REP-16, T06-REP-17, and T06-REP-18 are CD45+ and CD3+ cells. The proportion of cells positive for exhaustion markers in T06-REP-16 and T06-REP-17 is generally higher than that in T06-REP-18; the secretion levels of the cytokine IFN-γ and the proportion of memory T cells (T CM and T EM ) in T06-REP-16 and T06-REP-17 are also generally significantly higher than those in T06-REP-18.

[0185] Table 9, TIL phenotypes from T06 tissue

[0186]

[0187] Example 19, Detection of tumor cell killing function of sorted TILs from colon cancer

[0188] The real-time label-free cell function analyzer (RTCA) of ACEA Biosciences was used to detect the in vitro killing activities of the cell populations of T06-REP-16, T06-REP-17, and T06-REP-18 obtained in Example 17 against their homologous primary colon cancer cells. The specific steps are as follows:

[0189] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and perform zero adjustment;

[0190] (2) Seeding of target cells: Seed the primary T06 colon cancer tissue cells obtained by culturing in Example 17 at a density of 10 4 cells / 50 μL in a plate containing detection electrodes. After placing for several minutes and waiting for the cells to stabilize, then put it into the instrument and start step 2 to culture the cells;

[0191] (3) Adding effector cells: After culturing the target cells for 18 h - 24 h, observe the cell index. When the cell index is 1, add the effector cells T06-REP-16, T06-REP-17, and T06-REP-18, 50 μL per well, with an effector-to-target ratio of 4:1; in addition, set up a control group with only target cells seeded and no effector cells added. Start step 3. After co-culturing for more than 48 - 72 h, observe the killing level of the target cells and calculate the target cell killing rate..

[0192] The results are as Figure 6 shown. The killing rates of T06-REP-16 and T06-REP-17 against primary colon cancer target cells are significantly higher than that of T06-REP-18, indicating that compared with the TILs of T06-REP-18, the TILs of T06-REP-16 and T06-REP-17 have significantly stronger killing effects on homologous primary colon cancer tumor cells.

[0193] The present invention can be varied and modified to adapt to various uses and conditions, and such embodiments also fall within the scope of the claims herein. Any reference to a list of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or sub-combination) of the listed elements. Any reference to an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or part thereof. All patents and publications mentioned in this specification are incorporated herein by reference as if each individual patent or publication was 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) Immune cell activation markers, and (2) immune cell inhibition markers, wherein the immune cell activation markers include Ly108, and the immune cell inhibition markers include one, two or more selected from CD39, TIGIT, CD161, and the tumor is cervical cancer.

2. The immune cell marker combination according to claim 1, further comprising any one or more selected from intracellular IFN-γ, TNF-a, CXCL10, CXCL13, IL-2, IL-4, IL-6, IL-8 and IL-10.

3. The immune cell marker combination according to claim 1, further comprising any one or more selected from intracellular CD137, intracellular CD69 and intracellular CD107a.

4. The immune cell marker combination according to claim 1 or 2, further comprising tissue-resident memory markers.

5. The immune cell marker combination according to claim 4, wherein The tissue-resident memory markers include any one or more selected from CD69, CD103 and CD49a.

6. The immune cell marker combination according to claim 1 or 2, characterized in that: The immune cell activation markers further include one or more selected from CD25, CD38, CD69, CD137, CD107a, CD226 and CD150, and / or The immune cell inhibition markers further include one or more selected from PD-1, TIM3, LAG3, CTLA-4, CD101 and CD160.

7. A reagent for detecting the immune cell marker combination according to any one of claims 1-6, which is a binding molecule that specifically recognizes each marker.

8. The reagent according to claim 7, characterized in that: The binding molecule is an antibody or its antigen-binding fragment, and / or The binding molecule is conjugated with a detectable label.

9. The reagent according to claim 8, wherein, The detectable label is biotin or a fluorophore.

10. A composition for enriching immune cells with enhanced tumor-killing ability, comprising the immune cell marker combination according to any one of claims 1-6 or the reagent according to any one of claims 7-9, and the tumor is cervical cancer.

11. 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 1-6, the reagent according to any one of claims 7-9, or the composition according to claim 10.

12. The kit according to claim 11, wherein The kit further includes immune reaction reagents.

13. A method for identifying immune cells with enhanced tumor-killing ability, comprising detecting the expression of the immune cell marker combination according to any one of claims 1-6 in immune cells, wherein those with positive expression are immune cells with enhanced tumor-killing ability, and the tumor is cervical cancer.

14. The method according to claim 13, wherein The immune cells are T cells, NK cells or NKT cells.

15. The method according to claim 13, wherein The immune cells are TIL.

16. A method for screening immune cells with enhanced tumor killing ability, comprising screening cells that are positive for the expression of the combination of immune cell markers according to any one of claims 1-6 in a population of immune cells, wherein the tumor is cervical cancer.

17. The method according to claim 16, characterized in that, The immune cells are T cells, NK cells or NKT cells.

18. The method according to claim 16, wherein The immune cells are TIL.

19. Use of the combination of immune cell markers according to any one of claims 1-6, the reagent according to any one of claims 7-9 and / or the composition according to claim 10 in the preparation of a product for identifying or preparing immune cells with enhanced tumor killing ability, wherein the tumor is cervical cancer.

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

21. The use according to claim 19, wherein The immune cells are TIL.

22. A method for preparing immune cells with enhanced tumor killing ability, comprising: Screening cells that are positive for the expression of the combination of immune cell markers according to any one of claims 1-6 from isolated tumor-infiltrating lymphocytes, wherein the tumor is cervical cancer.

23. The method according to claim 22, characterized in that: 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 of a subject in need, a surgically resected primary tumor sample, synchronous and metachronous surgically resected metastatic tumor samples, a puncture sample and a body fluid, and / or The isolated tumor-infiltrating lymphocytes are derived from cervical cancer.

24. The method according to claim 22 or 23, characterized in that, The method further comprises the step of obtaining isolated tumor-infiltrating lymphocytes from a tumor sample, and / or the method further comprises the step of further culturing the obtained immune cells with enhanced tumor killing ability.

25. The method according to claim 24, wherein The step of obtaining isolated tumor-infiltrating lymphocytes from a tumor sample comprises: (1.1) obtaining seed cells from the tumor sample, and (1.2) culturing the seed cells to obtain isolated tumor-infiltrating lymphocytes.

26. The method according to claim 25, wherein Step (1.1) is culturing the tumor sample with a seed cell medium to obtain seed cells.

27. Use of the immune cells with enhanced tumor killing ability prepared by the method according to any one of claims 22-26 in the preparation of a cancer therapeutic drug, wherein the cancer is cervical cancer.

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