CN118962103B - Tumor specific immune cell marker and application thereof - Google Patents

Tumor specific immune cell marker and application thereof

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CN118962103B
CN118962103B CN202411007718.3A CN202411007718A CN118962103B CN 118962103 B CN118962103 B CN 118962103B CN 202411007718 A CN202411007718 A CN 202411007718A CN 118962103 B CN118962103 B CN 118962103B
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cells
tumor
rep
marker
immune cell
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CN118962103A (en
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金华君
尹锋
何周
马星明
黄晨
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Shanghai Juncai Biotech Co Ltd
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Shanghai Junsai Biotechnology Co ltd
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Abstract

本发明涉及肿瘤特异性免疫细胞标志物及其用途。具体提供一种免疫细胞标志物组合，包括：(1)免疫细胞活化标志物，和(2)免疫细胞抑制标志物，任选地还包括(3)胞内标志物，和/或(4)组织驻留记忆标志物。本发明的标志物组合能够更加准确地鉴定和富集肿瘤特异性免疫细胞，富集的细胞群具有更好的肿瘤反应性和肿瘤杀伤功能。

The present invention relates to tumor-specific immune cell markers and uses thereof. 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

Tumor specific immune cell marker and application thereof
The application is a divisional application of China application 202310242995.1 filed on 14 th year 2023.
Technical Field
The invention relates to the technical field of biology, in particular to a tumor specific immune cell marker and application thereof.
Background
Tumor-infiltrating lymphocyte (Tumor Infiltrating Lymphocyte, TIL) therapy has become an important therapy for solid tumors in tumor immune cell therapy technology in recent years, and has achieved very remarkable clinical effects on various solid tumors such as melanoma, cervical cancer, head and neck tumor and non-small cell lung cancer. T cells are the major component of TIL, which are a heterogeneous population of T cells, including tumor-specific T cells and bystander (bystander) T cells, i.e., T cells that specifically recognize tumor antigens and T cells that recognize epitopes unrelated to tumors. Bystander TILs typically include T cells that recognize viral antigens, such as EBV, HCMV, and influenza virus epitopes. In the context of TIL as an adoptive T cell therapy (ACT) for the treatment of tumors, how to elevate the content of tumor-specific T cells in TIL, reduce the T cell fraction of bystanders, and obtain a population of TIL cells that can survive in vivo for a long period of time becomes a critical factor in elevating the therapeutic effect.
A number of different indicators have been proposed in published reports to distinguish tumor-specific T cell populations from bystander T cell populations in TIL. Thomas Duhen et al Nat Commun.2018Jul 13;9 (1): 2724 describes the high enrichment of the presence of tumor antigen specific T cells in a CD39+CD103+ cell population among a variety of primary and metastatic tumor CD8+ TIL cells. Kim E Kortekaas et al.cancer Immunol Res.2020Oct 8 (10): 1311-1321. It is described that tumor-specific CD4+ T cells are present centrally in the CD39+ TIL subpopulation. WO2021226085A1 discloses a method for amplifying TIL for clinical therapeutic use, comprising a step of sorting for positive TIL expression for specific indicators, including PD-1, CD39, CD38, CD103, CD101, LAG3, TIM3 and/or TIGIT. There is still a need for more accurate and efficient criteria for identifying and/or sorting TILs to determine the proportion of tumor-specific and tumor-reactive T cells in the TIL cells prepared and possibly for further sorting and expansion of this fraction of cells to enhance the clinical efficacy of TILs.
Disclosure of Invention
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,
Optionally further comprising (3) an intracellular marker, and/or (4) a tissue resident memory marker.
In one or more embodiments, the immune cell activation marker is an immune cell surface marker.
In one or more embodiments, the immune cell activation marker comprises one or more selected from the group consisting of CD25, CD38, CD69, CD137, CD107a, CD226, CD150, and Ly 108.
In one or more embodiments, the immune cell suppression marker comprises one or more selected from the group consisting of CD39, PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160, and CD 161.
In one or more embodiments, the intracellular markers include intracellular cytokines and/or immune cell intracellular activation markers. Preferably, the intracellular cytokine comprises any one or more selected from the group consisting of intracellular IFN-gamma, TNF-a, CXCL10, CXCL13, IL-2, IL-4, IL-6, IL-8 and IL-10, and preferably, the intracellular activation marker comprises any one or more selected from the group consisting of intracellular CD137, intracellular CD69 and intracellular CD107 a.
In one or more embodiments, the tissue resident memory marker comprises any one or more selected from the group consisting of CD69, CD103, and CD49 a.
In one or more embodiments, the immune cell is a T cell, NK cell, NKT cell, or TIL.
The invention also provides an agent for detecting a combination of markers as described in any of the embodiments herein, said agent being a binding molecule that specifically recognizes each marker.
In one or more embodiments, the binding molecule is an antibody or antigen-binding fragment thereof.
In one or more embodiments, the binding molecules are conjugated with a detectable label, such as biotin or a fluorescent group.
The invention also provides a composition comprising a marker combination or agent as described in any of the embodiments herein.
The invention also provides a kit for identifying or preparing a tumor-specific immune cell, comprising a marker combination, reagent, or composition as described in any of the embodiments herein.
In one or more embodiments, the immune cell is a T cell, NK cell, NKT cell, or TIL.
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.
In one or more embodiments, the kit is suitable for use or method as described in any of the embodiments herein.
The invention also provides a method of identifying tumor-specific immune cells comprising detecting expression of an immune cell marker combination described herein of an immune cell, wherein expression positive is a tumor-specific immune cell.
In one or more embodiments, the immune cell is a T cell, NK cell, NKT cell, or TIL.
The invention also provides a method of screening for tumor-specific immune cells comprising screening for cells in an immune cell population that express positive combinations of the immune cell markers described herein.
In one or more embodiments, the immune cell is a T cell, NK cell, NKT cell, or TIL.
Use of a marker combination or agent as described in any of the embodiments herein in the manufacture of a product for identifying or preparing tumor-specific immune cells.
In one or more embodiments, the immune cell is a T cell, NK cell, NKT cell, or TIL.
In one or more embodiments, the product is a kit or device.
The present invention provides methods of preparing tumor-specific immune cells comprising screening isolated tumor-infiltrating lymphocytes for cells that express positive combinations of the immune cell markers described herein.
In one or more embodiments, the immune cell is a T cell, NK cell, or NKT cell.
In one or more embodiments, the method further comprises the step of obtaining isolated tumor-infiltrating lymphocytes from a tumor sample, comprising in particular:
(1.1) obtaining seed cells from a tumor sample, e.g., culturing a tumor sample using a seed cell culture medium, and
(1.2) Culturing seed cells to obtain isolated tumor-infiltrating lymphocytes.
In one or more embodiments, the isolated tumor-infiltrating lymphocytes are derived from a sample selected from the group consisting of ascites, surgical resection primary focus samples, simultaneous and allogeneic surgical resection metastasis samples, puncture samples, and body fluids in a subject in need thereof. The body fluid includes blood, interstitial fluid, lymph fluid and/or body cavity fluid.
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.
In one or more embodiments, the isolated tumor-infiltrating lymphocytes are derived from a tissue mass after cutting of tumor tissue, the tissue mass after cutting of tumor tissue having a diameter of from about 1mm to about 10mm.
In one or more embodiments, the method further comprises further culturing the obtained tumor-specific immune cells.
The application of the tumor specific immune cells prepared by the method for preparing the tumor specific immune cells in preparing the cancer therapeutic medicine is provided.
The marker combination can more accurately identify and enrich tumor specific immune cells, and the enriched cell population has better tumor reactivity and tumor killing function.
Drawings
FIG. 1 killing of primary melanoma target cells by T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18TIL cell populations;
FIG. 2 killing rates of T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18TIL cell populations on primary cervical cancer target cells;
FIG. 3 killing rates of T03-REP-7, T03-REP-8, T03-REP-9, and T03-REP-20TIL cell populations against primary gastric cancer target cells;
FIG. 4 killing of primary ovarian cancer target cells by T04-REP-10, T04-REP-11, T04-REP-12, and T04-REP-19TIL cell populations;
FIG. 5 killing rates of T05-REP-13, T05-REP-14, T05-REP-15, and T05-REP-18TIL cell populations on primary non-small cell lung cancer target cells;
FIG. 6 killing rates of T06-REP-16, T06-REP-17 and T06-REP-18TIL cell populations on primary colon cancer target cells.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, e.g., lackie, DICTIONARY OF CELLAND MOLECULAR BIOLOGY (dictionary of cell MOLECULAR biology), elsevier press (4 th edition 2007), green et al, MOLECULAR CLONING, ALABORATORY MANUAL (MOLECULAR cloning, laboratory Manual), cold spring harbor laboratory press (Cold spring harbor, new York 2012).
The inventors have intensively studied and found a marker of tumor-specific immune cells. By using the 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.
Tumor infiltrating lymphocytes are typically obtained by culturing in tumor samples. Herein, a tumor sample is any sample containing tumor cells, including, but not limited to, ascites, surgical resection primary focus samples, simultaneous and asynchronous surgical resection metastasis samples, puncture samples, and body fluids of a subject in need thereof.
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 comprise (3) an intracellular marker, and/or (4) a tissue resident memory marker. Compositions formed from these markers are also within the scope of the invention.
Herein, immune cells include T cells, NK cells, NKT cells or TIL. T cells derived from TIL are preferred.
The immunocyte markers herein are applicable to any tumor. Exemplary 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.
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 Ly 108. Preferably, the immune cell activation marker comprises one, two or more selected from CD226, ly108, CD69, CD107a, CD226, CD 150.
Herein, tumor-specific immune cell inhibition markers include one or more selected from the group consisting of CD39, PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160 and CD161. Preferably, the immune cell suppression marker comprises one, two or more selected from CD39, LAG3, CD161, TIGIT, TIM3, CD 160. In some embodiments, the immune cell suppression marker comprises CD39 and/or CD161.
Intracellular markers described herein include intracellular cytokines and/or intracellular activation markers. The intracellular cytokine comprises any one or more selected from the group consisting of IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10 and CXCL13, preferably the intracellular cytokine comprises any one or more selected from the group consisting of IFN-gamma, CXCL10, CXCL13 and TNF-alpha. The intracellular activation marker comprises any one or more selected from the group consisting of intracellular CD137, intracellular CD69 and intracellular CD107a, preferably the intracellular activation marker comprises intracellular CD137. In some embodiments, the intracellular markers comprise any one or more selected from IFN-gamma, CXCL10, CXCL13, TNF-alpha, intracellular CD137. Herein, "intracellular + marker" designates such a marker located within an immune cell, which differs in structure from a marker without an "intracellular" prefix (typically located at the surface of the cell membrane). For example, "intracellular CD137" designates a CD137 molecule located intracellular, while "CD137" refers to a CD137 molecule in the form of a membrane surface.
The tissue resident memory marker comprises any one or more selected from CD69, CD103 and CD49a, preferably comprising CD49a and/or CD103.
Herein, each component of the tumor-specific immune cell markers, i.e., immune cell activation markers, immune cell inhibition markers, intracellular markers, and tissue resident memory markers, may be selected from any one or more of the above immune cell activation markers, any one or more of the above immune cell inhibition markers, any one or more of the above intracellular markers, and any one or more of the above tissue resident memory markers, respectively. Thus, the markers of the tumor-specific immune cells herein can be any combination of the markers in the above components.
In some embodiments, the tumor-specific immune cell markers include (1) an immune cell activation marker, and (2) an immune cell suppression marker. Optionally, the markers further comprise (3) an intracellular marker. The immune cell activation marker comprises CD226. In addition, the immune cell activation marker may further include one, two or more selected from the group consisting of CD25, CD38, CD69, CD137, CD107a, CD150 and Ly 108. The immunocytostatic marker comprises one, two or more selected from CD39, LAG3 and CD 161. In addition, the immunocytosis marker may further comprise one, two or more selected from the group consisting of PD-1, TIM3, CTLA-4, TIGIT, CD101, CD 160. The intracellular marker comprises intracellular CD137. In addition, the intracellular markers may further comprise any one or more selected from IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69 and intracellular CD107a, and preferably may further comprise any one or more selected from IFN-gamma, CXCL10, CXCL13 and TNF-alpha. In one or more embodiments, the tumor is melanoma.
In some embodiments, the tumor-specific immune cell markers include (1) an immune cell activation marker, and (2) an immune cell suppression marker. Optionally, the marker of the tumor-specific immune cell further comprises (3) an intracellular marker. The immune cell activation marker comprises Ly108. In addition, the immune cell activation marker may further include one, two or more selected from CD25, CD38, CD69, CD137, CD107a, CD226, CD 150. The immune cell inhibition marker comprises one, two or more selected from CD39, TIGIT and CD 161. In addition, the immunocytostatic marker may further comprise one, two or more selected from the group consisting of PD-1, TIM3, LAG3, CTLA-4, CD101 and CD 160. The intracellular markers include IFN-gamma. In addition, the intracellular markers may further comprise any one or more selected from the group consisting of TNF- α, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69, intracellular CD137 and intracellular CD107a, and preferably may further comprise any one or more selected from the group consisting of CXCL10, CXCL13, TNF- α and intracellular CD 137. In one or more embodiments, the tumor is cervical cancer.
In some embodiments, the tumor-specific immune cell markers include (1) an immune cell activation marker, and (2) an immune cell suppression marker. Optionally, the marker of the tumor-specific immune cell further comprises (3) an intracellular marker. The immune cell activation marker comprises CD69. In addition, the immune cell activation marker may further include one, two or more selected from the group consisting of CD25, CD38, CD137, CD107a, CD226, CD150 and Ly 108. The immune cell inhibition marker comprises one, two or more selected from CD39, TIM3 and CD 161. In addition, the immunocytosis marker may further comprise one, two or more selected from the group consisting of PD-1, LAG3, CTLA-4, TIGIT, CD101, CD 160. The immunocyte intracellular marker comprises CXCL13. In addition, the immune cell intracellular marker may further comprise any one or more selected from IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10, intracellular CD69, intracellular CD137 and intracellular CD107a, and preferably may further comprise any one or more selected from IFN-gamma, CXCL10, TNF-alpha and intracellular CD 137. In one or more embodiments, the tumor is gastric cancer.
In some embodiments, the tumor-specific immune cell markers include (1) an immune cell activation marker, and (2) an immune cell suppression marker. Optionally, the marker of the tumor-specific immune cell further comprises (3) an intracellular marker. The immune cell activation marker is CD107a. In addition, the immune cell activation marker may further include one, two or more selected from the group consisting of CD25, CD38, CD69, CD137, CD226, CD150 and Ly 108. The immune cell inhibition marker comprises one, two or more selected from CD39, LAG3 and CD 161. In addition, the immunocytosis marker may further comprise one, two or more selected from the group consisting of PD-1, TIM3, CTLA-4, TIGIT, CD101, CD 160. The immunocyte intracellular marker comprises CXCL10. In addition, the immune cell intracellular marker may further comprise any one or more selected from IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69, intracellular CD137 and intracellular CD107a, and preferably may further comprise any one or more selected from IFN-gamma, CXCL13, TNF-alpha and intracellular CD 137. In one or more embodiments, the tumor is ovarian cancer.
In some embodiments, the tumor-specific immune cell markers include (1) an immune cell activation marker, and (2) an immune cell suppression marker. Optionally, the markers of the tumor-specific immune cells further comprise (3) an intracellular marker and/or (4) a tissue resident memory marker. The immune cell activation marker comprises one, two or more selected from CD226, ly108 and CD107 a. Preferably, the immune cell activation marker is selected from (1) CD226, (2) Ly108, or (3) Ly108 and CD107a in combination. In addition, the immune cell activation marker may further include one, two or more selected from CD25, CD38, CD69, CD137, CD 150. The immunocytostatic marker comprises CD39. In addition, the immunocytostatic marker may further comprise one, two or more selected from the group consisting of PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160 and CD 161. The immune cell intracellular marker comprises one or more selected from CXCL10, IFN-gamma and intracellular CD 137. Preferably, the immune cell intracellular marker is selected from (1) CXCL10, (2) IFN-gamma. In addition, the immunocyte intracellular marker may further comprise any one or more selected from TNF-alpha, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69 and intracellular CD107a, and preferably further comprise CXCL13 and/or TNF-alpha. The tissue resident memory marker of the immune cell comprises CD103. In addition, the tissue resident memory marker may also include CD69 and/or CD49a. In one or more embodiments, the tumor is lung cancer, e.g., non-small cell lung cancer.
In some embodiments, the tumor-specific immune cell markers include (1) an immune cell activation marker, and (2) an immune cell suppression marker. Optionally, the markers of the tumor-specific immune cells further comprise (3) an intracellular marker and/or (4) a tissue resident memory marker. The immune cell activation marker comprises one, two or more selected from CD226, ly108 and CD150. Preferably, the immune cell activation marker is selected from (1) CD226 and Ly108, or (2) CD226 and CD150. In addition, the immune cell activation marker may further include one, two or more selected from CD25, CD38, CD69, CD137, CD107 a. The immune cell inhibition marker comprises one, two or more selected from CD39, CD160 and CD 161. Preferably, the immune cell suppression marker is selected from (1) a combination of CD39 and CD160, or (2) a combination of CD39 and CD 161. In addition, the immunocytosis marker may further comprise one, two or more selected from PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD 101. The immune cell intracellular marker comprises one or two selected from IFN-gamma and TNF-alpha. Preferably, the immune cell intracellular marker is selected from (1) IFN-gamma, or (2) IFN-gamma and TNF-alpha. In addition, the immunocyte intracellular marker may further comprise any one or more selected from IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69 and intracellular CD107a, and preferably may further comprise a member selected from CXCL10 and/or CXCL13. The tissue resident memory markers of the immune cells include 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 also include CD69. In one or more embodiments, the tumor is a bowel cancer, such as colon cancer.
The invention also provides methods for identifying and screening tumor-specific immune cells by detecting whether the immune cells express the above markers, wherein positive expression is tumor-specific immune cells.
Any method that can be used to detect the above-described markers expressed (intracellular or membrane surface) or secreted by cells can be used in the present invention. Preferably, such methods accomplish the detection by incubating the cells with binding molecules (e.g., specific small molecules, nucleic acids, antibodies, or antigen binding fragments thereof) that specifically recognize each marker and identifying the binding molecules. To facilitate detection, the binding molecules are conjugated with a detectable label, such as biotin or a fluorescent group. Such binding molecules and suitable detectable labels are within the skill of the art. Illustratively, the detecting is accomplished by flow cytometry.
Reagents used in the method of detecting a marker (abbreviated as detection reagents) are also within the scope of the present invention. Such as binding molecules that specifically recognize the respective markers.
The invention also includes kits having the markers described herein and/or detection reagents thereof for identifying or preparing tumor-specific immune cells. The kit may also include immunoreactive reagents such as blocking solutions, washing solutions, enzyme-labeled reagents. The kit is suitable for use or method described herein.
The markers of the invention are particularly useful in methods of screening for and obtaining tumor-specific immune cells from a population of immune cells comprising the step of screening (e.g., by flow cytometry) isolated tumor-infiltrating lymphocytes for cells that express positive combinations of the immune cell markers described herein.
The method further comprises the step of obtaining isolated tumor-infiltrating lymphocytes from the tumor sample, and specifically comprises (1.1) obtaining seed cells from the tumor sample, e.g., culturing the tumor sample using a seed cell culture medium, and (1.2) culturing the seed cells to obtain isolated tumor-infiltrating lymphocytes.
The seed cell medium may be any medium used in the art to culture TIL seed cells. For example RPMI1640 medium containing 10% human AB serum, 2mM L-glutamine, 55uM BME, 6000IU/mL IL-2, glutamax and antibiotics (e.g. gentamicin).
In some embodiments, step (1.1) comprises (a) washing a tumor tissue sample (e.g., using physiological saline containing 100U/mL penicillin, 100. Mu.g/mL streptomycin, and 50. Mu.g/mL gentamicin) and cutting it into small pieces of 1mm-10mm diameter, (b) culturing tumor tissue pieces using seed cell culture for 3-20 days based on 30-42℃and 1-10% CO 2. Exemplary procedure (1.1) is described in example 2 of WO2021239083A1 and includes the steps of 1) washing fresh isolated tumor tissue samples in 10cm dishes with 30mL of physiological saline (containing 100U/mL penicillin, 100. Mu.g/mL streptomycin and 50. Mu.g/mL gentamicin) and transferring to a new 10cm dish with 30mL of the above physiological saline for a total of 3 washes, 2) removing adipose tissue and necrotic tissue with a sterile surgical knife, cutting the tumor tissue into small pieces of 3mm diameter, placing 12 pieces of randomly selected tumor tissue pieces in each G-REX10 culture tank (purchased from Wilsonwolf), adding TIL seed medium, 3) adding seed cell medium to different G-REX10 culture tanks, culturing the tumor tissue pieces with 5% CO2 at 37℃per 40mL, counting total number of TIL seed cells and measuring the phenotype of cells by a flow cytometer after 12 days.
The culturing described in step (1.2) may use any medium known in the art for culturing TIL. For example AIM-V medium containing 1000IU/mL IL-2 and 30ng/mL CD3 antibody (e.g. OKT 3).
In one or more embodiments, the method further comprises further culturing the obtained tumor-specific immune cells (e.g., TILs). The culturing may be performed using any medium known in the art suitable for immune cells (e.g., TIL, particularly tumor-specific TIL).
The tumor specific immune cells prepared by the method can be used for scientific research or for preparing therapeutic drugs for corresponding cancers. Accordingly, the present invention also provides a pharmaceutical composition comprising tumor-specific immune cells produced by the methods described herein and a pharmaceutically acceptable adjuvant.
In the present invention, a "pharmaceutically acceptable adjuvant" 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 human. Herein, pharmaceutically acceptable excipients are non-toxic to the recipient of the composition at the dosages and concentrations employed. Various types of carriers or excipients commonly used in the art of therapy for delivering immune cells may be included. Exemplary excipients may be liquids or solids including, but not limited to, pH modifiers, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavoring agents, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers. In some embodiments, the pharmaceutically acceptable excipients may include one or more inactive ingredients including, but not limited to, stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients for use with the pharmaceutically active compound. See, e.g., REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition, a.r. genrmo, 1990,Mack Publishing Company. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.
The pharmaceutical composition of the invention may be selected for parenteral delivery, for inhalation or delivery through the digestive tract (such as orally), for example for intravenous infusion delivery. The preparation of the composition is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising immune cells, particularly immune cells (e.g., T cells), in sustained or controlled release delivery formulations.
Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. Compositions for parenteral administration may be stored in lyophilized form or in solution (e.g., lyophilized formulations). Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution tape or vial having a stopper pierceable by a hypodermic injection needle.
Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, freezers, or as dehydrated or lyophilized powders. The pharmaceutical formulation (e.g., a lyophilized formulation) may be stored in a ready-to-use form or in a form that is further formulated prior to administration. For example, a pharmaceutical composition suitable for delivery as described herein may be a cryopreserved formulation, which can withstand long distance transport without damaging the cells. In addition to the cells themselves, cryopreservation formulations typically include components such as cell cryopreservation solution, human Serum Albumin (HSA), and the like. Prior to administration (e.g., intravenous infusion), the cryopreserved pharmaceutical composition is stored (e.g., in liquid nitrogen). The frozen preparation can be directly infused into a patient or formulated as an infusion composition after thawing. The composition and concentration of conventional frozen stock solutions are known to those skilled in the art. For example, the frozen stock solution or infusion composition may further comprise dimethylsulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, magnesium chloride, or the like, the concentration of which may be determined by one of skill in the art (e.g., an experienced physician) depending on the condition of the cell, disease, patient, or the like.
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, characterized in that the processor, when executing the program, performs the step of screening (e.g. by flow cytometry) isolated tumor-infiltrating lymphocytes for cells expressing positive in an immune cell marker combination as described herein. For example, the device is recorded with or contains a marker or a reagent for tumor-specific immune cells described herein, and whether or not cells in a sample contain the marker is detected or judged, thereby identifying and screening cells positive for the marker.
The invention also provides the use of a marker combination or agent as described in any of the embodiments herein for the preparation of a product for identifying or preparing tumor-specific immune cells. The product comprises a kit or device as described herein.
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 the manner in which the value is measured or determined, e.g., the limits of the measurement system. For example, "about" may mean within 1 or more standard deviations. Or "about" may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Or in particular for biological systems or processes, the term may denote within a certain order of magnitude, preferably within a factor of 5, more preferably within a factor of 2. In describing particular values in the present application and claims, unless otherwise indicated, the term "about" is assumed to mean within an acceptable error range for the particular value.
As used herein, "and/or" includes any and all combinations of one or more of the associated listed items.
All percentages and ratios/ratios are by weight unless explicitly indicated otherwise.
All percentages and ratios are calculated based on the total composition unless otherwise specified.
Each maximum numerical limitation given throughout this disclosure includes each lower numerical limitation as if such lower numerical limitation were explicitly written herein. Each minimum numerical limitation given throughout this disclosure includes each higher numerical limitation as if such higher numerical limitations were expressly written herein. Each numerical range given throughout this disclosure includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
The values recited herein should not be construed as being strictly limited to the exact numerical values recited. Rather, unless specifically stated otherwise, the values are each used to refer to the stated value and a functionally equivalent range surrounding that value. For example, a value disclosed as "50 μl" is intended to mean "about 50 μl".
Each document cited herein, including any cross-referenced and related patents or applications, is incorporated by reference herein unless specifically excluded or otherwise limited. 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 references, proposes, suggests or discloses any such invention. Furthermore, 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 the term herein shall govern.
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. The invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. Furthermore, the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the 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 that are within the scope of this disclosure.
Examples
Example 1, tumor-specific TIL immune cell marker combinations with immune cell marker combinations referred to in the examples of tumor tissue samples are shown in table 1 below:
TABLE 1 immunocyte marker combinations
The tumor tissue samples used in the examples are shown in table 2 below:
TABLE 2 tumor tissue samples
Sample numbering Cancer species
T01 Melanoma (HEI)
T02 Cervical cancer
T03 Stomach cancer
T04 Ovarian cancer
T05 Non-small cell lung cancer
T06 Colon cancer
The relevant coupled fluorophore flow antibody sources used in the examples are shown in table 3 below:
TABLE 3 flow-through fluorescent antibodies
Example 2 cultivation and sorting of melanoma-derived TIL
Fresh melanoma tissue T01 was mechanically cut into pieces of 3X 3mm size, and the pieces were mixed as uniformly as possible and then divided into 2 parts, and 1 part was cultured according to the method described in section Robert Suriano et al.Ex Vivo Derived Primary Melanoma Cells:Implications for Immunotherapeutic Vaccines J Cancer 2013;4(5):371-382.Materials and Methods to obtain primary tumor cells of T01 tissue. The remaining 1 part was cultured to obtain seed cells according to the method described in example 2 of WO2021239083A1, and the medium for culturing seed cells was a CM1 medium containing 6000IU/mL IL-2, glutamax and antibiotics prepared according to the recipe and method described in example 5 of CN110099998A description. The obtained seed cells were subjected to expansion culture to obtain REP cells by preparing an expansion medium according to the method described in example 5 of WO2021239083A1, and further dividing the obtained seed cells into 4 equal parts, each of which was then subjected to incubation and flow cytometry (BD FACSAria TM III, bdbiosciences) successively with the antibodies of the coupling fluorophor of each marker in the immune cell marker combinations numbered 1, 2, 3 and 18 (excluding intracellular markers) in table 1 of example 1, respectively, and after multiple sorting, immune cell marker combinations 1, 2, 3 and 18 positive cell populations were obtained, designated as T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18, respectively.
10 6 Cells were taken from the T01-REP-3 cell population, fixed with PBS containing 2v/v% paraformaldehyde, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cell pellet was washed by resuspension with PBS, repeated 2 times, PBS containing 0.7v/v% Tween-20 was added, and incubated at room temperature for 15 minutes, and membrane permeabilization was performed on the cells. Centrifugation at 800g for 5 min, removal of supernatant, washing of cell pellet with PBS for 2 times, resuspension, addition of appropriately diluted anti-CD 137 antibody conjugated with fluorophores, incubation at room temperature for 30 min, washing of cells with PBS for 2 times, detection by up-flow cytometry.
The results showed 93.2% of cells positive for intracellular CD137 in the T01-REP-3 cell population. Indicating that the vast majority of the T01-REP-3 cell population are intracellular CD137 positive cells.
Example 3 phenotypic detection of sorted melanoma derived TILs
Four populations of T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 cells obtained in example 2 were each examined by flow cytometry for 1) lymphocyte phenotype: CD45, CD3, CD4, CD8, 2) depletion index: PD-1; 3) activation index CD25, 4) memory T cell index: T CM(CD45RO+CCR7+);TEM (CD45RO+CCR7-). The secretion levels of the cytokines IFN-gamma from each of the four populations of cells were measured using the HTRF IFN-gamma assay kit (Cisbio Human IFN GAMMA KIT, cat# 62 HIFNGPET) according to the protocol described in the specification.
The results are shown in Table 4, where more than 99% of T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 are CD45+ and CD3+ cells. The ratio of the positive cell to the positive cell of the depletion marker and the positive cell of the activation marker of the T01-REP-1, the T01-REP-2 and the T01-REP-3 is higher than the corresponding index of the T01-REP-18, and the secretion level of the IFN-gamma of the cytokines of the T01-REP-1, the T01-REP-2 and the T01-REP-3 and the proportion of the memory T cells (especially T CM) are also commonly and obviously higher than the T01-REP-18.
TABLE 4T 01 tissue derived TIL phenotype
Example 4 tumor cell killing function assay of sorted melanoma-derived TIL
The T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18 cell populations obtained in example 2 were tested for their in vitro killing activity against their cognate melanoma primary cells using a real-time label-free cell function Analyzer (RTCA) from Eisen, comprising the following steps:
(1) Zeroing, namely adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;
(2) Target cell plating, namely, paving the T01 melanoma tissue primary cells obtained by culturing in the embodiment 1 in 10 4 cells/50 mu L per hole in a plate containing a detection electrode, standing for a plurality of minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) After the target cells are cultured for 18-24 h, observing cell indexes, when the cell indexes are 1, respectively adding effector cells T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18, and the effective target ratio is 4:1, and independently setting a group of control groups which are paved with only the target cells and are not added with the effector cells, starting the step (3), after the co-culture is performed for more than 48-72h, observing the killing level of the target cells, and calculating the killing rate of the target cells. The target cell killing rate calculation formula (the target cell killing rate calculation formula in the following examples is the same as this):
Wherein A is the cell index of the control group and B is the cell index of each group to which effector cells are added.
As shown in FIG. 1, the killing rate of T01-REP-1, T01-REP-2 and T01-REP-3 on melanoma tumor primary target cells is obviously higher than that of T01-REP-18, which indicates that the TILs of T01-REP-1, T01-REP-2 and T01-REP-3 have obviously stronger killing effect on homologous melanoma primary tumor cells compared with the TILs of T01-REP-18.
Example 5 cultivation and sorting of cervical cancer-derived TIL
Fresh cervical cancer tissue T02 is mechanically cut into fragments with the size of 3X 3mm, the fragments are uniformly mixed as much as possible and then divided into 2 parts, and 1 part of tissue is cultured according to the method described in 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.1999Jul;73(7):5402-10Materials and Methods part to obtain primary tumor cells of the T02 tissue. The remaining 1 part was cultured to obtain seed cells according to the method described in example 2 of WO2021239083A1, and the medium for culturing seed cells was a CM1 medium containing 6000IU/mL IL-2, glutamax and antibiotics prepared according to the recipe and method described in example 5 of CN110099998A description. The obtained seed cells were subjected to expansion culture to obtain REP cells by preparing an expansion medium according to the method described in example 5 of WO2021239083A1, and further dividing the obtained seed cells into 4 equal parts, each of which was then subjected to incubation and flow cytometry (BD FACSAria TM III, bdbiosciences) respectively, one by one, with the antibodies of the coupling fluorophores of the respective markers in the immune cell marker combinations (excluding intracellular markers) numbered 4, 5, 6 and 18 in table 1 of example 1, respectively, and after multiple sorting, immune cell marker combinations 4, 5, 6 and 18 positive cell populations were obtained, designated as T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18, respectively.
10 6 Cells were taken from the T02-REP-4 cell population, fixed with PBS containing 2v/v% paraformaldehyde, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cell pellet was washed by resuspension with PBS, repeated 2 times, PBS containing 0.7v/v% Tween-20 was added, and incubated at room temperature for 15 minutes, and membrane permeabilization was performed on the cells. Centrifugation at 800g for 5min, removal of supernatant, washing of cell pellet with PBS for 2 times, resuspension, addition of appropriately diluted anti-IFN-gamma antibody conjugated with fluorophores, incubation at room temperature for 30 min, washing of cells with PBS for 2 times, detection by up-flow cytometry.
The results showed that IFN-. Gamma.positive cells accounted for 97.5% in the T02-REP-4 cell population. Indicating that the vast majority of the T02-REP-4 cell population is IFN-gamma positive.
Example 6 phenotypic detection of sorted cervical cancer derived TIL
The four populations of T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 obtained in example 5 were examined by flow cytometry for each of 1) lymphocyte phenotypes CD45, CD3, CD4, CD8, 2) depletion index PD-1, 3) activation index CD25, 4) memory T cell index T CM(CD45RO+CCR7+);TEM (CD45RO+CCR7-). The secretion levels of the cytokines IFN-gamma from each of the four populations of cells were measured using the HTRF IFN-gamma assay kit (Cisbio Human IFN GAMMA KIT, cat# 62 HIFNGPET) according to the protocol described in the specification.
The results are shown in Table 5, where more than 90% of T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 were CD45+ and CD3+ cells. The ratio of the positive cell to the positive cell of the depletion marker and the positive cell of the activation marker of the T02-REP-4, the T02-REP-5 and the T02-REP-6 is higher than that of the T02-REP-18, the secretion level of the cytokine IFN-gamma of the T02-REP-4, the secretion level of the cytokine-gamma of the T02-REP-5 and the secretion level of the cytokine-gamma of the T02-REP-6 and the ratio of the memory T cells, particularly the T CM, are also generally and obviously higher than that of the T01-REP-18.
TABLE 5T 02 tissue derived TIL phenotype
Example 7 detection of tumor cell killing function of sorted cervical cancer-derived TIL
The in vitro killing activity of T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18 cell populations obtained in example 5 on homologous cervical cancer primary cells was examined using a real-time label-free cell function Analyzer (RTCA) from Eisen, and the specific procedures were as follows:
(1) Zeroing, namely adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;
(2) Target cell plating, namely, paving the T02 cervical cancer tissue primary cells obtained by culturing in the embodiment 5 in a plate containing detection electrodes according to 10 4 cells/50 mu L per hole, standing for a plurality of minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) After the target cells are cultured for 18-24 h, observing cell indexes, when the cell indexes are 1, respectively adding effector cells T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18, and the effective target ratio is 4:1, and independently setting a group of control groups which are paved with only the target cells and are not added with the effector cells, starting the step (3), after the co-culture is performed for more than 48-72h, observing the killing level of the target cells, and calculating the killing rate of the target cells.
As shown in the figure 2, the killing rate of T02-REP-4, T02-REP-5 and T02-REP-6 on primary target cells of cervical cancer tumors is obviously higher than that of T02-REP-18, which indicates that the TIL of T02-REP-4, T02-REP-5 and T02-REP-6 has obviously stronger killing effect on homologous primary target cells of cervical cancer compared with the TIL of T02-REP-18.
Example 8 cultivation and sorting of gastric cancer-derived TIL
Fresh gastric cancer tissue T03 is mechanically cut into fragments with the size of 3X 3mm, the fragments are uniformly mixed as much as possible and then divided into 2 parts, and 1 part of tissue is 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 part to obtain primary tumor cells of the T03 tissue. The remaining 1 part was cultured to obtain seed cells according to the method described in example 2 of WO2021239083A1, and the medium for culturing seed cells was a CM1 medium containing 6000IU/mL IL-2, glutamax and antibiotics prepared according to the recipe and method described in example 5 of CN110099998A description. The obtained seed cells were subjected to expansion culture to obtain REP cells by preparing an expansion medium according to the method described in example 5 of WO2021239083A1, and further dividing the obtained seed cells into 4 equal parts, and each part was then subjected to incubation and flow cytometry (BD FACSAria TM III, bdbiosciences) successively with the antibodies of the dew fluorescent groups of the markers in the immune cell marker combinations (excluding intracellular markers) numbered 7, 8, 9 and 20 in table 1 of example 1, respectively, and after multiple sorting, immune cell marker combinations 7, 8, 9 and 20 positive cell populations were obtained, designated as T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20, respectively.
10 6 Cells were taken from the T03-REP-9 cell population, fixed with PBS containing 2v/v% paraformaldehyde, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cell pellet was washed by resuspension with PBS, repeated 2 times, PBS containing 0.7v/v% Tween-20 was added, and incubated at room temperature for 15 minutes, and membrane permeabilization was performed on the cells. Centrifugation at 800g for 5min, removal of supernatant, washing of cell pellet with PBS for 2 times, resuspension, addition of appropriately diluted anti-CXCL 13 antibody conjugated with fluorophores, incubation at room temperature for 30 min, washing of cells with PBS for 2 times, detection by up-flow cytometry.
The results showed that CXCL 13-positive cells account for 91.3% of the T03-REP-9 cell population. Indicating that the vast majority of the T02-REP-4 cell population is CXCL 13-positive cells.
Example 9 phenotypic detection of sorted gastric cancer derived TIL
Four populations of cells, T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20, obtained in example 8 were examined by flow cytometry for each of 1) phenotype indicators CD45, CD3, CD4, CD8, 2) depletion indicator PD-1, 3) activation indicator CD25, 4) memory T cell indicator T CM(CD45RO+CCR7+);TEM (CD45RO+CCR7-). The secretion levels of the cytokines IFN-gamma from each of the four populations of cells were measured using the HTRF IFN-gamma assay kit (Cisbio Human IFN GAMMA KIT, cat# 62 HIFNGPET) according to the protocol described in the specification.
The results are shown in Table 6, in which more than 90% of T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20 were CD45+ and CD3+ cells. The proportion of the positive cells of the depletion markers of T03-REP-7, T03-REP-8 and T03-REP-9 is generally higher than that of T03-REP-20, and the secretion level of the cytokines IFN-gamma and the proportion of memory T cells (T CM、TEM) of T03-REP-7, T03-REP-8 and T03-REP-9 are also generally and obviously higher than that of T03-REP-20.
TABLE 6T 03 tissue derived TIL phenotype
Example 10 detection of tumor cell killing function of sorted gastric cancer-derived TIL
The T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20 cell populations obtained in example 5 were tested for their in vitro killing activity against their cognate gastric cancer primary cells using a real-time label-free cell function Analyzer (RTCA) from Eisen, comprising the following steps:
(1) Zeroing, namely adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;
(2) Target cell plating, namely, paving the T03 gastric cancer tissue primary cells obtained by culturing in the embodiment 5 into a plate containing a detection electrode according to 104 cells/50 mu L per hole, standing for a plurality of minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) After the target cells are cultured for 18-24 h, observing cell indexes, when the cell indexes are 1, respectively adding effector cells T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20, and setting 50 mu L of each hole, wherein the effective target ratio is 4:1, and independently setting a group of control groups which are paved with only the target cells and are not added with the effector cells, starting the step (3), after the co-culture is performed for more than 48-72h, observing the killing level of the target cells, and calculating the killing rate of the target cells.
As shown in FIG. 3, the killing rate of T03-REP-7, T03-REP-8 and T03-REP-9 on gastric cancer tumor primary target cells is obviously higher than that of T03-REP-20, which indicates that the TIL of T03-REP-7, T03-REP-8 and T03-REP-9 has obviously stronger killing effect on homologous gastric cancer primary tumor cells compared with that of T03-REP-20.
EXAMPLE 11 cultivation and sorting of ovarian cancer derived TIL
Fresh ovarian cancer tissue T04 is mechanically cut into fragments with the size of 3X 3mm, the fragments are uniformly mixed as much as possible and then divided into 2 parts, and 1 part is 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 part to obtain primary tumor cells of the T04 tissue. The remaining 1 part was cultured to obtain seed cells according to the method described in example 2 of WO2021239083A1, and the medium for culturing seed cells was a CM1 medium containing 6000IU/mL IL-2, glutamax and antibiotics prepared according to the recipe and method described in example 5 of CN110099998A description. The obtained seed cells were subjected to expansion culture to obtain REP cells by preparing an expansion medium according to the method described in example 5 of WO2021239083A1, and further dividing the obtained seed cells into 4 equal parts, each of which was then subjected to incubation and flow cytometry (BD FACSAria TM III, bdbiosciences) successively with the antibodies of the coupling fluorophores of the respective markers in the immune cell marker combinations numbered 10, 11, 12 and 19 (excluding the intracellular markers) in example 1, respectively, and after multiple sorting, immune cell marker combinations 10, 11, 12 and 19 positive cell populations were obtained, designated as T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19, respectively.
10 6 Cells were taken from the T04-REP-12 cell population, fixed with PBS containing 2v/v% paraformaldehyde, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cell pellet was washed by resuspension with PBS, repeated 2 times, PBS containing 0.7v/v% Tween-20 was added, and incubated at room temperature for 15 minutes, and membrane permeabilization was performed on the cells. Centrifugation at 800g for 5 min, removal of supernatant, washing of cell pellet with PBS for 2 times, resuspension, addition of appropriately diluted anti-CXCL 10 antibody conjugated with fluorophores, incubation at room temperature for 30 min, washing of cells with PBS for 2 times, detection by up-flow cytometry.
The results showed that CXCL 10-positive cells account for 89.9% of the T04-REP-12 cell population. Indicating that the vast majority of the T04-REP-12 cell population is CXCL 10-positive cells.
Example 12 phenotypic detection of sorted ovarian cancer derived TIL
Four populations of cells, T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19, obtained in example 11 were examined by flow cytometry for each of 1) phenotype indicators CD45, CD3, CD4, CD8, 2) depletion indicator TIM3, 3) activation indicator CD25, 4) memory T cell indicator T CM(CD45RO+CCR7+);TEM (CD45RO+CCR7-). The secretion levels of the cytokines IFN-gamma from each of the four populations of cells were measured using the HTRF IFN-gamma assay kit (Cisbio Human IFN GAMMA KIT, cat# 62 HIFNGPET) according to the protocol described in the specification.
The results are shown in Table 7, where approximately or more than 95% of T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19 are CD45+ and CD3+ cells. The proportion of the positive cells of the depletion markers of T04-REP-10, T04-REP-11 and T04-REP-12 is generally higher than that of T04-REP-19, the secretion level of the cytokines of T04-REP-10, T04-REP-11 and T04-REP-12 and the proportion of memory T cells (T CM、TEM) are also generally and obviously higher than that of T04-REP-19.
TABLE 7T 04 tissue derived TIL phenotype
Example 13 detection of tumor cell killing function of sorted ovarian cancer-derived TIL
The T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19 cell populations obtained in example 11 were tested for their in vitro killing activity against homologous ovarian cancer primary cells using a real-time label-free cell function analyzer (RTCA) from Eisen, comprising the following steps:
(1) Zeroing, namely adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;
(2) Target cell plating, namely, paving the T04 ovarian cancer tissue primary cells obtained by culturing in the example 11 in a plate containing a detection electrode according to 10 4 cells/50 mu L per hole, standing for a plurality of minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) After the target cells are cultured for 18-24h, observing cell indexes, when the cell indexes are 1, respectively adding effector cells T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19, and setting 50 mu L of each hole, wherein the effective target ratio is 4:1, and independently setting a group of control groups which are paved with only the target cells and are not added with the effector cells, starting the step 3, after the co-culture is performed for more than 48-72h, observing the killing level of the target cells, and calculating the killing rate of the target cells.
As shown in FIG. 4, the killing rate of T04-REP-10, T04-REP-11 and T04-REP-12 on ovarian cancer primary target cells is obviously higher than that of T04-REP-19, which indicates that the TIL of T04-REP-10, T04-REP-11 and T04-REP-12 has obviously stronger killing effect on homologous ovarian cancer primary tumor cells compared with the TIL of T04-REP-19.
EXAMPLE 14 cultivation and sorting of non-Small cell lung cancer derived TIL
Fresh non-small cell lung cancer tissue T05 is mechanically cut into fragments with the size of 3 multiplied by 3mm, the fragments are uniformly mixed as much as possible and then divided into 2 parts, and 1 part is cultured according to the method described in D P.Kodack et al.Primary Patient-Derived Cancer Cells and Their Potential for Personalized Cancer Patient Care Cell Rep.2017Dec 12;21(11):3298–3309.EXPERIMENTAL PROCEDURES part to obtain primary tumor cells of the T05 tissue. The remaining 1 part was cultured to obtain seed cells according to the method described in example 2 of WO2021239083A1, and the medium for culturing seed cells was a CM1 medium containing 6000IU/mL IL-2, glutamax and antibiotics prepared according to the recipe and method described in example 5 of CN110099998A description. The obtained seed cells were subjected to expansion culture to obtain REP cells by preparing an expansion medium according to the method described in example 5 of WO2021239083A1, and further dividing the obtained seed cells into 4 equal parts, each of which was then subjected to incubation and flow cytometry (BD FACSAria TM III, bdbiosciences) successively with the antibodies of the coupling fluorophores of the respective markers in the immune cell marker combinations numbered 13, 14, 15 and 18 (excluding the intracellular markers) in table 1 of example 1, respectively, and after multiple sorting, immune cell marker combinations 13, 14, 15 and 18 positive cell populations were obtained, designated as T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18, respectively.
10 6 Cells from each of the T05-REP-13, T05-REP-14 and T05-REP-15 cell populations were fixed with PBS containing 2v/v% paraformaldehyde, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cell pellet was washed by resuspension with PBS, repeated 2 times, PBS containing 0.7v/v% Tween-20 was added, and the cells were incubated at room temperature for 15 minutes, and membrane permeation treatment was performed on the cells. Centrifugation at 800g for 5 min, removal of supernatant, washing of cell pellet with PBS for 2 times, resuspension, adding appropriate dilution of anti-CXCL 10 antibody, anti-IFN-gamma antibody and anti-IFN-gamma antibody of conjugated fluorescent groups to T05-REP-13, T05-REP-14 and T05-REP-15 cells, respectively, incubating at room temperature for 30 min, washing cells with PBS for 2 times, and detecting by an up-flow cytometer.
The results showed 90.7% CXCL 10-positive cells in T05-REP-13 cells and 87.6% and 88.1% IFN-gamma positive cells in T05-REP-14 and T05-REP-15 cells, respectively. The above results demonstrate that the vast majority of the T05-REP-13 cell population is CXCL10 positive cells, while the vast majority of the T05-REP-14 and T05-REP-15 cell populations are IFN-gamma positive cells.
Example 15 phenotypic detection of sorted non-small cell lung carcinoma derived TIL
Four populations of cells, T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18, obtained in example 14 were examined by flow cytometry for each of 1) phenotype indicators CD45, CD3, CD4, CD8, 2) depletion indicator PD-1, 3) activation indicator CD25, 4) memory T cell indicator T CM(CD45RO+CCR7+);TEM (CD45RO+CCR7-). The secretion levels of the cytokines IFN-gamma from each of the four populations of cells were measured using the HTRF IFN-gamma assay kit (Cisbio Human IFN GAMMA KIT, cat# 62 HIFNGPET) according to the protocol described in the specification.
The results are shown in Table 8, where more than 99% of the T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18 were CD45+ and the CD3+ cell fraction was higher than 80%. The proportion of the positive cells of the depletion markers of T05-REP-13, T05-REP-14 and T05-REP-15 is generally higher than that of T05-REP-18, and the secretion level and the proportion of memory T cells (T CM、TEM) of the cytokines IFN-gamma of T05-REP-13, T05-REP-14 and T05-REP-15 are also generally and obviously higher than that of T05-REP-18.
TABLE 8T 05 tissue derived TIL phenotype
Example 16 detection of tumor cell killing function of sorted non-Small cell Lung cancer-derived TIL
The T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18 cell populations obtained in example 14 were tested for their in vitro killing activity against their cognate non-small cell lung cancer primary cells using a real-time label-free cell function Analyzer (RTCA) from Eisen, comprising the following steps:
(1) Zeroing, namely adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;
(2) Target cell plating, namely, paving the T05 non-small cell lung cancer tissue primary cells obtained by culturing in the example 14 in a plate containing a detection electrode according to 10 4 cells/50 mu L per hole, standing for a plurality of minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) After the target cells are cultured for 18-24 h, observing cell indexes, when the cell indexes are 1, respectively adding the effector cells T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18, and the effective target ratio is 4:1, and independently setting a group of control groups which are paved with only the target cells and are not added with the effector cells, starting the step (3), after the co-culture is performed for more than 48-72h, observing the killing level of the target cells, and calculating the killing rate of the target cells. .
As shown in FIG. 5, the killing rate of T05-REP-13, T05-REP-14 and T05-REP-15 on primary target cells of non-small cell lung cancer is obviously higher than that of T05-REP-18, which indicates that the TIL of T05-REP-13, T05-REP-14 and T05-REP-15 has obviously stronger killing effect on homologous primary tumor cells of non-small cell lung cancer compared with the TIL of T05-REP-18.
Example 17 cultivation and sorting of colon cancer derived TIL
Fresh colon cancer tissue T06 is mechanically cut into fragments with the size of 3 multiplied by 3mm, the fragments are evenly mixed as much as possible and then divided into 2 parts, and 1 part is 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.2016Sep;37(9):12833-12842.Materials and methods part to obtain primary tumor cells of the T06 tissue. The remaining 1 part was cultured to obtain seed cells according to the method described in example 2 of WO2021239083A1, and the medium for culturing seed cells was a CM1 medium containing 6000IU/mL IL-2, glutamax and antibiotics prepared according to the recipe and method described in example 5 of CN110099998A description. Preparing an expansion culture medium for the obtained seed cells according to the method described in example 5 of WO2021239083A1, carrying out expansion culture on the obtained seed cells to obtain REP cells, dividing the REP cells into 3 equal parts, and carrying out incubation and flow cytometry (BD FACSaria TM III, bdbiosciences) on each part by using antibodies of coupling fluorescent groups of the markers in immune cell marker combinations (except for intracellular markers) numbered 16, 17 and 18 in table 1 of example 1, respectively, so as to obtain immune cell marker combinations 16, 17 and 18 positive cell groups which are named as T06-REP-16, T06-REP-17 and T06-REP-18 respectively.
2E6 cells were taken from the T06-REP-16 cell population, fixed with PBS containing 2v/v% paraformaldehyde, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cell pellet was washed by resuspension with PBS, repeated 2 times, PBS containing 0.7v/v% Tween-20 was added, and incubated at room temperature for 15 minutes, and membrane permeabilization was performed on the cells. Centrifugation at 800g for 5 min, removal of supernatant, washing of cell pellet with PBS for 2 times, resuspension, addition of appropriate dilution of anti-IFN-gamma and anti-TNF-alpha conjugated fluorophores, incubation at room temperature for 30 min, washing of cells with PBS for 2 times, detection by up-flow cytometry.
2E6 cells were taken from the T06-REP-17 cell population, fixed with PBS containing 2v/v% paraformaldehyde, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cell pellet was washed by resuspension with PBS, repeated 2 times, PBS containing 0.7v/v% Tween-20 was added, and incubated at room temperature for 15 minutes, and membrane permeation treatment was performed on the cells. Centrifugation at 800g for 5min, removal of supernatant, washing of cell pellet with PBS for 2 times, resuspension, addition of appropriately diluted anti-IFN-gamma antibody conjugated with fluorophores, incubation at room temperature for 30 min, washing of cells with PBS for 2 times, detection by up-flow cytometry.
The results showed that 84.6% of IFN-gamma and TNF-alpha double positive cells were present in the T06-REP-16 cell population, and 96.9% of IFN-gamma positive cells were present in the T06-REP-17 cell population. The majority of T06-REP-16 cells were IFN-gamma and TNF-alpha double positive cells, and the majority of T06-REP-17 cells were IFN-gamma positive cells.
Example 18 phenotypic detection of sorted colon cancer derived TIL
Three populations of cells, T06-REP-16, T06-REP-17 and T06-REP-18, obtained in example 2 were each examined by flow cytometry for 1) phenotype indicators CD45, CD3, CD4, CD8, 2) depletion indicator PD-1, 3) activation indicator CD25, 4) memory T cell indicator T CM(CD45RO+CCR7+);TEM (CD45RO+CCR7-). The secretion levels of the cytokines IFN-gamma from each of the four populations of cells were measured using the HTRF IFN-gamma assay kit (Cisbio Human IFN GAMMA KIT, cat# 62 HIFNGPET) according to the protocol described in the specification.
The results are shown in Table 9, where more than 99% of T06-REP-16, T06-REP-17 and T06-REP-18 are CD45+ and CD3+ cells. The proportion of the positive cells of the depletion markers of T06-REP-16 and T06-REP-17 is generally higher than that of T06-REP-18, and the secretion level and the proportion of memory T cells (T CM、TEM) of the cytokines IFN-gamma of T06-REP-16 and T06-REP-17 are also generally and obviously higher than that of T06-REP-18.
TABLE 9T 06 tissue derived TIL phenotype
Example 19 detection of tumor cell killing function of sorted colon cancer derived TIL
The T06-REP-16, T06-REP-17 and T06-REP-18 cell populations obtained in example 17 were tested for their in vitro killing activity against homologous colon cancer primary cells using a real-time label-free cell function Analyzer (RTCA) from the Eisen company, comprising the following steps:
(1) Zeroing, namely adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;
(2) Target cell plating, namely, paving the T06 colon cancer tissue primary cells obtained by culturing in the example 17 in a plate containing a detection electrode according to 10 4 cells/50 mu L per hole, standing for a plurality of minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) After the target cells are cultured for 18-24 h, observing cell indexes, when the cell indexes are 1, respectively adding 50 mu L of effector cells T06-REP-16, T06-REP-17 and T06-REP-18 in each hole, and calculating the killing rate of the target cells, wherein the effective target ratio is 4:1, and a group of control groups which are paved with only the target cells and are not added with the effector cells are independently arranged, starting the step 3, and after the co-culture is performed for more than 48-72h, observing the killing level of the target cells, and calculating the killing rate of the target cells. .
The results are shown in FIG. 6, wherein the killing rate of T06-REP-16 and T06-REP-17 on colon cancer primary target cells is obviously higher than that of T06-REP-18, which shows that the TIL of T06-REP-16 and T06-REP-17 has obviously stronger killing effect on homologous colon cancer primary tumor cells compared with the TIL of T06-REP-18.
Changes and modifications may be made to the invention to adapt it to various uses and conditions, and such embodiments are intended to fall within the scope of the claims herein. References to a list of elements in any definition of a variable herein include the definition of the variable as any single element or combination (or sub-combination) of elements listed. References to an embodiment herein include that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Claims (24)

1. An immune cell marker combination for enriching immune cells for increased tumor killing capacity, comprising (1) an immune cell activation marker, (2) an immune cell suppression marker, the marker combination optionally further comprising (3) a tissue resident memory marker comprising (a) CD226, (b) Ly108, or (c) Ly108 and CD107a combination, the immune cell suppression marker comprising CD39, the tissue resident memory marker comprising CD103, the immune cell marker combination comprising CXCL10 and/or intracellular IFN- γ, the tumor being non-small cell lung cancer.
2. The immune cell marker combination of claim 1, wherein the immune cell marker composition further comprises any one or more selected from TNF-a, CXCL13, IL-2, IL-4, IL-6, IL-8, IL-10, intracellular CD137, intracellular CD69, and intracellular CD107 a.
3. The immune cell marker combination of claim 1 wherein:
the immune cell activation marker further comprises one or more selected from the group consisting of CD25, CD38, CD69, CD137 and CD150, and/or
The immunocytostatic marker further comprises one or more selected from PD-1, TIM3, LAG3, CTLA-4, TIGIT, CD101, CD160 and CD161, and/or
The tissue resident memory marker comprises any one or more selected from CD69 and CD49 a.
4. A reagent for detecting a combination of immune cell markers according to any one of claims 1-3, which reagent is a binding molecule specifically recognizing each marker.
5. The reagent of claim 4, wherein:
the binding molecule is an antibody or antigen-binding fragment thereof, and/or
The binding molecules are conjugated with a detectable label.
6. The reagent of claim 5, wherein the detectable label is a biotin or a fluorophore.
7. A composition for enriching immune cells with increased tumor killing capacity comprising the immune cell marker combination of any one of claims 1-3 or the agent of any one of claims 4-6, wherein the tumor is non-small cell lung cancer.
8. A kit for identifying or preparing an immune cell having increased tumor killing capability, comprising the immune cell marker combination of any one of claims 1-3, the agent of any one of claims 4-6, or the composition of claim 7, wherein the tumor is non-small cell lung cancer.
9. The kit of claim 8, further comprising an immunoreactive reagent.
10. A method of identifying immune cells having increased tumor killing capacity comprising detecting expression of the immune cell marker combination of any one of claims 1-3 of the immune cells, wherein expression positive is an immune cell having increased tumor killing capacity, the tumor being non-small cell lung cancer.
11. The method of claim 10, wherein the immune cells are T cells, NK cells, or NKT cells.
12. The method of claim 10, wherein the immune cell is TIL.
13. A method of screening for immune cells having increased tumor killing capacity comprising screening for cells in an immune cell population that express positive for a combination of immune cell markers according to any one of claims 1-3, wherein the tumor is non-small cell lung cancer.
14. The method of claim 13, wherein the immune cells are T cells, NK cells, or NKT cells.
15. The method of claim 13, wherein the immune cell is TIL.
16. Use of the immune cell marker combination of any one of claims 1-3, the agent of any one of claims 4-6 and/or the composition of claim 7 in the manufacture of a product for identifying or preparing an immune cell having increased tumor killing capacity, said tumor being non-small cell lung cancer.
17. The use according to claim 16, wherein:
the immune cells are T cells, NK cells or NKT cells, and/or
The product is a kit or device.
18. The use according to claim 16, wherein the immune cell is TIL.
19. A method for preparing an immune cell having an increased tumor killing capacity, comprising screening isolated tumor infiltrating lymphocytes for cells positive for expression of the immune cell marker combination of any one of claims 1 to 3, wherein the tumor is non-small cell lung cancer.
20. The method of claim 19, wherein:
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, surgical excision of primary foci, simultaneous and heterologous surgical excision of metastasis, puncture and body fluids in a subject in need thereof, and/or
The isolated tumor-infiltrating lymphocytes are derived from non-small cell lung cancer.
21. The method of claim 19 or 20, wherein:
the method further comprises the step of obtaining isolated tumor-infiltrating lymphocytes from the tumor sample, and/or
The method further comprises the step of further culturing the obtained immune cells with increased tumor killing capacity.
22. The method of claim 21, wherein the step of obtaining isolated tumor-infiltrating lymphocytes from the 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.
23. The method of claim 22, wherein step (1.1) is culturing the tumor sample with a seed cell culture medium to obtain seed cells.
24. Use of immune cells with increased tumor killing capacity made by the method of any one of claims 19-23 in the manufacture of a medicament for the treatment of cancer, said cancer being non-small cell lung cancer.
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