The application is a divisional application of China application 202310242995.1 filed on 14 th year 2023.
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 (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 excision of primary foci samples, simultaneous and asynchronous surgical excision of metastasis samples, puncture samples and body fluids in a subject in need thereof.
The tumor-specific immune cell markers of the present invention include: (1) An immune cell activation marker, and (2) an immune cell inhibition marker. 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. Illustratively, the tumor comprises: 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 cytokines include any one or more selected from the group consisting of intracellular IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL-8, IL-10, CXCL10 and CXCL 13; preferably, the intracellular cytokine comprises any one or more selected from IFN-gamma, CXCL10, CXCL13, TNF-alpha. The intracellular activation marker comprises any one or more selected from the group consisting of intracellular CD137, intracellular CD69, and intracellular CD107 a; 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 marker of tumor-specific immune cells comprises: (1) An immune cell activation marker, and (2) an immune cell inhibition 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 CD107 a; preferably, any one or more selected from IFN-gamma, CXCL10, CXCL13 and TNF-alpha can be further included. In one or more embodiments, the tumor is melanoma.
In some embodiments, the marker of tumor-specific immune cells comprises: (1) An immune cell activation marker, and (2) an immune cell inhibition 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 CD107 a; preferably, any one or more selected from CXCL10, CXCL13, TNF- α, intracellular CD137 may be further included. In one or more embodiments, the tumor is cervical cancer.
In some embodiments, the marker of tumor-specific immune cells comprises: (1) An immune cell activation marker, and (2) an immune cell inhibition 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 CD107 a; preferably, any one or more selected from IFN-gamma, CXCL10, TNF-alpha, intracellular CD137 may be further included. In one or more embodiments, the tumor is gastric cancer.
In some embodiments, the marker of tumor-specific immune cells comprises: (1) An immune cell activation marker, and (2) an immune cell inhibition 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 CD107 a; preferably, any one or more selected from IFN-gamma, CXCL13, TNF-alpha, intracellular CD137 may be further included. In one or more embodiments, the tumor is ovarian cancer.
In some embodiments, the marker of tumor-specific immune cells comprises: (1) An immune cell activation marker, and (2) an immune cell inhibition 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 the group consisting of: (1) CD226, (2) Ly108, or (3) Ly108 in combination with CD107 a. 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 the group consisting of: (1) CXCL10, (2) IFN-gamma. In addition, the immune cell 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 and intracellular CD107 a; CXCL13 and/or TNF- α may also be preferably included. 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 marker of tumor-specific immune cells comprises: (1) An immune cell activation marker, and (2) an immune cell inhibition 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 the group consisting of: (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 the group consisting of: (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 the group consisting of: (1) IFN-gamma, or (2) IFN-gamma and TNF-alpha. In addition, the immune cell intracellular markers may further comprise any one or more selected from the group consisting of IL-2, IL-4, IL-6, IL-8, IL-10, CXCL13, intracellular CD69 and intracellular CD107 a; preferably, it may also 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 the group consisting of: (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 a method of screening and obtaining tumor-specific immune cells from a population of immune cells comprising the steps of: cells expressing positive in combination with the immune cell markers described herein are selected (e.g., by flow cytometry) from isolated tumor-infiltrating lymphocytes.
The method further comprises the step of obtaining isolated tumor-infiltrating lymphocytes from the tumor sample; the method specifically comprises the following steps: (1.1) obtaining seed cells from a tumor sample, e.g., culturing the tumor sample using a seed cell culture medium to obtain seed cells, and (1.2) culturing the seed cells to obtain isolated tumor-infiltrating lymphocytes.
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 in diameter, (b) culturing the tumor tissue pieces using seed cell culture for 3-20 days based on 30-42℃and 1-10% CO2. Exemplary step (1.1) is as described in example 2 of WO2021239083A1, comprising the steps of: 1) The obtained freshly isolated tumor tissue samples were washed in a 10cm dish to which 30mL of physiological saline (containing 100U/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL gentamicin) had been added under sterile conditions in a secondary biosafety cabinet, and transferred to a new 10cm dish to which 30mL of the above physiological saline had been added, and washed repeatedly 3 times in total; 2) Removing adipose tissue and necrotic tissue with a sterile scalpel, cutting tumor tissue into small pieces with diameter of 3mm, placing 12 tumor tissue pieces selected randomly in each G-REX10 culture tank (purchased from Wilsonwolf), and adding TIL seed culture medium; 3) Seed cell culture media are respectively added into different G-REX10 culture tanks, 40mL of each tank is used for culturing 5% CO2 at 37 ℃ of tumor tissue blocks, the total number and the activity rate of cells are counted after TIL seed cells are harvested on 12 th day, and the phenotype of the cells is detected by a flow cytometer.
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 liquid or solid, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, odorants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers. In some embodiments, pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients for use with the pharmaceutically effective compounds. 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 an apparatus for identifying or preparing tumour infiltrating lymphocytes, the apparatus comprising a memory, a processor and a computer program stored on the memory and executable on the processor, characterised in that the processor when executing the program implements the steps of: cells expressing positive in combination with the immune cell markers described herein are selected (e.g., by flow cytometry) from isolated tumor-infiltrating lymphocytes. 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 and tumor tissue samples
The immune cell marker combinations referred to in the examples 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 FACSAriaTM 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.
106 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 5min, 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 30min, 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 cells, T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18, obtained in example 2 were each examined by flow cytometry: 1) Lymphocyte phenotype: CD45, CD3, CD4, CD8; 2) Depletion index: PD-1; 3) An activation index CD25; 4) Memory T cell index: tCM(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 cells of the depletion markers to the positive cells of the T01-REP-1, the T01-REP-2 and the T01-REP-3 to the positive cells of the activation markers is higher than the corresponding indexes of the T01-REP-18; the secretion levels of the T01-REP-1, T01-REP-2 and T01-REP-3 cytokines IFN-gamma and the proportion of memory T cells (in particular TCM) are also generally significantly higher than 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: 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: placing the T01 melanoma tissue primary cells obtained in the culture of the example 1 in a plate containing a detection electrode according to 104 cells/50 mu L per hole for a plurality of minutes, placing the cells in an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) Adding effector cells: after the target cells are cultured for 18-24 h, observing the cell index, and when the cell index is 1, respectively adding effector cells T01-REP-1, T01-REP-2, T01-REP-3 and T01-REP-18, wherein the effective target ratio is 4:1, and the total volume of 50 mu L per hole; and (3) a group of control groups which are paved with target cells only and are not added with effector cells are independently arranged, the step (3) is started, after the co-culture is performed for more than 48-72 hours, the killing level of the target cells is observed, and the killing rate of the target cells is calculated. 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 FACSAriaTM 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.
106 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 30min, 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
Four populations of cells, T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18, obtained in example 5 were each examined by flow cytometry: 1) Lymphocyte phenotypes CD45, CD3, CD4, CD8; 2) A depletion index PD-1; 3) An activation index CD25; 4) Memory T cell index: tCM(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 cells of the depletion markers to the positive cells of the T02-REP-4, the T02-REP-5 and the T02-REP-6 to the positive cells of the activation markers is higher than that of the T02-REP-18; the secretion levels of the T02-REP-4, T02-REP-5, T02-REP-6 cytokines IFN-gamma and the proportion of memory T cells, in particular TCM, are also generally significantly higher than 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: 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: paving 104 cells/50 mu L of primary cells of the T02 cervical cancer tissue obtained by culturing in the example 5 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) Adding effector cells: after the target cells are cultured for 18-24 h, observing the cell index, and when the cell index is 1, respectively adding effector cells T02-REP-4, T02-REP-5, T02-REP-6 and T02-REP-18, wherein the effective target ratio is 4:1, and the total volume of 50 mu L per hole; and (3) a group of control groups which are paved with target cells only and are not added with effector cells are independently arranged, the step (3) is started, after the co-culture is performed for more than 48-72 hours, the killing level of the target cells is observed, and the killing rate of the target cells is calculated.
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 FACSAriaTM 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.
106 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 each examined by flow cytometry: 1) Phenotype indicators CD45, CD3, CD4, CD8; 2) A depletion index PD-1; 3) An activation index CD25; 4) Memory T cell index: tCM(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; T03-REP-7, T03-REP-8, T03-REP-9 cytokine IFN-gamma secretion levels and memory T cells (TCM、TEM) were also generally significantly higher than 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: 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: spreading the T03 gastric cancer tissue primary cells obtained by culturing in the embodiment 5 in 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) Adding effector cells: after the target cells are cultured for 18-24 h, observing the cell index, and when the cell index is 1, respectively adding effector cells T03-REP-7, T03-REP-8, T03-REP-9 and T03-REP-20, wherein the effective target ratio is 4:1, and the total volume of 50 mu L per hole; and (3) a group of control groups which are paved with target cells only and are not added with effector cells are independently arranged, the step (3) is started, after the co-culture is performed for more than 48-72 hours, the killing level of the target cells is observed, and the killing rate of the target cells is calculated.
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 FACSAriaTM 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.
106 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 each examined by flow cytometry: 1) Phenotype indicators CD45, CD3, CD4, CD8; 2) A depletion index TIM3; 3) An activation index CD25; 4) Memory T cell index: tCM(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 depletion marker positive cells of T04-REP-10, T04-REP-11 and T04-REP-12 is generally higher than that of T04-REP-19; T04-REP-10, T04-REP-11, T04-REP-12 cytokine secretion levels and memory T cell (TCM、TEM) ratios are also generally significantly higher than 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: 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: paving 104 cells/50 mu L of T04 ovarian cancer tissue primary cells obtained by culturing in example 11 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 step 2 to culture the cells;
(3) Adding effector cells: after the target cells are cultured for 18-24 hours, observing the cell index, and when the cell index is 1, respectively adding effector cells T04-REP-10, T04-REP-11, T04-REP-12 and T04-REP-19, wherein the effective target ratio is 4:1, and the total volume of 50 mu L per hole; and (3) independently setting a group of control groups which are paved with target cells only and are not added with effector cells, starting the step (3), and after the co-culture is performed for more than 48-72 hours, 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 FACSAriaTM 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.
106 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 30min, washing cells with PBS for 2 times, and detecting by an up-flow cytometer.
The results showed that among T05-REP-13 cells, CXCL 10-positive cells account for 90.7%; among T05-REP-14 and T05-REP-15 cells, IFN-gamma positive cells accounted for 87.6% and 88.1%, 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 each examined by flow cytometry: 1) Phenotype indicators CD45, CD3, CD4, CD8; 2) A depletion index PD-1; 3) An activation index CD25; 4) Memory T cell index: tCM(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 depletion marker positive cells of the T05-REP-13, the T05-REP-14 and the T05-REP-15 is generally higher than that of the T05-REP-18; the secretion levels of the T05-REP-13, T05-REP-14, T05-REP-15 cytokines IFN-gamma and the memory T cell (TCM、TEM) ratio are also generally significantly higher than 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: 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: paving 104 cells/50 mu L of T05 non-small cell lung cancer tissue primary cells obtained by culturing in example 14 in a plate containing a detection electrode, standing for several minutes, putting the cells into an instrument after the cells are stabilized, and starting step 2 to culture the cells;
(3) Adding effector cells: after the target cells are cultured for 18-24 h, observing the cell index, and when the cell index is 1, respectively adding effector cells T05-REP-13, T05-REP-14, T05-REP-15 and T05-REP-18, wherein the effective target ratio is 4:1, and the total volume of 50 mu L per hole; and (3) a group of control groups which are paved with target cells only and are not added with effector cells are independently arranged, the step (3) is started, after the co-culture is performed for more than 48-72 hours, the killing level of the target cells is observed, and the killing rate of the target cells is calculated. .
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 FACSariaTM 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 30min, 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 30min, washing of cells with PBS for 2 times, detection by up-flow cytometry.
The results showed that IFN-gamma and TNF-alpha double positive cells account for 84.6% in the T06-REP-16 cell population; IFN-gamma positive cells account for 96.9% of the T06-REP-17 cell population. Indicating that the vast majority of the T06-REP-16 cell populations are medium IFN-gamma and TNF-alpha biscationic cells; the vast majority of the T06-REP-17 cells in the population are IFN-gamma positive.
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: 1) Phenotype indicators CD45, CD3, CD4, CD8; 2) A depletion index PD-1; 3) An activation index CD25; 4) Memory T cell index: tCM(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 depletion marker positive cells of the T06-REP-16 and the T06-REP-17 is generally higher than that of the T06-REP-18; the secretion levels of the cytokines IFN-gamma and the memory T cells (TCM、TEM) of T06-REP-16 and T06-REP-17 are also generally and obviously higher than those 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: 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: paving 104 cells/50 mu L of T06 colon cancer tissue primary cells obtained by culturing in example 17 in a plate containing a detection electrode, standing for several minutes, putting the cells into an instrument after the cells are stable, and starting step 2 to culture the cells;
(3) Adding effector cells: after the target cells are cultured for 18-24 h, observing the cell index, and when the cell index is 1, respectively adding effector cells T06-REP-16, T06-REP-17 and T06-REP-18, wherein the effective target ratio is 4:1, and the total concentration of 50 mu L of each hole is 50 mu L; and (3) independently setting a group of control groups which are paved with target cells only and are not added with effector cells, starting the step (3), and after the co-culture is performed for more than 48-72 hours, 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.