Tumor infiltrating lymphocyte amplification composition, kit and amplification method and application thereof

Abstract

The invention belongs to the technical field of immune cell culture, and particularly relates to a tumor infiltration lymphocyte amplification composition, a kit and an amplification method thereof. The invention provides the tumor infiltration lymphocyte amplification composition aiming at a trace amount of tumor tissue, namely a tumor puncture sample, the tumor infiltration lymphocyte amplification composition is used for amplifying the tumor infiltration lymphocyte, the problem that the tumor tissue sample size required by the tumor infiltration lymphocyte is large is solved, and the tumor infiltration lymphocyte amplification composition is suitable for a patient who cannot receive the tumor tissue obtained by surgical excision. Besides, by optimizing the antibody stimulant combination and the working concentration and establishing the technological process for amplifying the tumor infiltration lymphocytes based on the puncture sample, the dryness of the tumor infiltration lymphocytes obtained through amplification is maintained, the lethality is improved, and therefore the anti-tumor curative effect after the tumor infiltration lymphocytes are infused back into the body is guaranteed.

Description

Technical Field

[0001] This application belongs to the field of immune cell culture technology, specifically relating to a tumor-infiltrating lymphocyte amplification composition, kit, and amplification method thereof. Background Technology

[0002] Tumor-infiltrating lymphocytes (TILs) are a heterogeneous cell population composed of T cells (including CD8+ T cells, CD4+ T cells, and γδ T cells), B cells, NK cells, and innate lymphocytes (ILCs). After in vitro expansion and culture, CD3+ T cells become the dominant cell population (usually accounting for more than 95%). Among them, CD3+ T cells express TCR receptors on their surface and play a key role in mediating the immune killing of cancerous cells. Because different TCR receptors recognize different antigenic peptides presented by the major histocompatibility complex (MhC) molecule, T cells are endowed with antigen specificity. Therefore, once T cells specific to a certain antigen are stimulated by a specific antigen, these cells can promote programmed cell death of target cells through the release of cytotoxins and receptor-mediated mechanisms. Unlike chimeric antigen T-cell (CAR-T) / TCR (T cell receptor) T-cell therapy products, TILs typically exert their anti-tumor effects by recognizing tumor-associated antigens, such as neoantigens, presented by tumor cells. Therefore, TIL therapy can achieve its anti-tumor therapeutic goals by recognizing and targeting multiple tumor-associated antigens.

[0003] TIL cell therapy refers to the treatment method of separating, expanding, activating and then reinfusing lymphocytes from tumor tissue obtained by surgery into the patient to kill tumor cells. Traditional TIL preparation requires obtaining a sufficient amount of tumor tissue sample through surgical resection. The process is as follows: (1) Obtain a sufficient amount of tumor tissue, usually larger than 1 cm, through tumor tissue resection and other methods in the hospital. 3 (2) Transport tumor tissue to the GMP cell production workshop via cold chain; (3) Process tumor tissue in a GMP-grade environment, including tumor tissue mincing, digestion, lymphocyte separation and other steps to obtain TIL; (4) Place the separated TIL in a T cell selective expansion medium containing high concentrations of IL-2 and T cell activation antibody (usually anti-CD3 / CD28 antibody), and expand the culture to 10 after no less than 4 weeks. 10The above number of cells are harvested, washed and packaged as final product cells; (5) the final product cells are tested for quality and released after passing the test, and transported to the hospital by liquid nitrogen for patients to receive reinfusion.

[0004] Traditional TIL culture techniques have the following limitations: a large volume of tumor tissue is required (usually larger than 1 cm). 3 This makes it impossible for late-stage cancer patients who cannot undergo surgical sampling to prepare TILs; the time required to culture and expand T cells is long, and the T cells have poor stemness and low killing power, which limits the anti-tumor efficacy after TIL infusion. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a tumor-infiltrating lymphocyte amplification composition. The tumor-infiltrating lymphocytes amplified in vitro using this amplification composition have high stemness and strong killing power.

[0006] This application provides a tumor-infiltrating lymphocyte expansion composition, including a tumor-infiltrating lymphocyte expansion culture medium and an activation antibody mixture.

[0007] The tumor-infiltrating lymphocyte expansion culture medium includes a basal culture medium and components at the following concentrations: 2 v / v% to 10 v / v% SR serum substitute, 300 IU / mL to 9000 IU / mL IL-2, 0 ng / mL to 10 ng / mL IL-7 and 0 ng / mL to 30 ng / mL IL-15.

[0008] The activated antibody mixture comprises a combination of two or more of the following: 0 μg / mL to 5 μg / mL anti-CD3 antibody, 0 μg / mL to 5 μg / mL anti-CD28 antibody, and 0 μg / mL to 5 μg / mL anti-41BB antibody.

[0009] In one embodiment, the activating antibody mixture comprises one or more combinations of 0.11 μg / mL to 0.35 μg / mL anti-CD3 antibody, 0.11 μg / mL to 0.35 μg / mL anti-CD28 antibody, and 0.11 μg / mL to 0.35 μg / mL anti-41BB antibody.

[0010] In one embodiment, the concentration of the anti-CD3 antibody is 0.30 μg / mL to 0.35 μg / mL.

[0011] Optionally, the concentration of the anti-CD28 antibody is 0.30 μg / mL to 0.35 μg / mL.

[0012] Optionally, the concentration of the anti-41BB antibody is 0.30 μg / mL to 0.35 μg / mL.

[0013] Optionally, the basal culture medium includes one or more of X-VIVO15, RPMI-1640 and AIM-V media.

[0014] Another aspect of this application provides a tumor-infiltrating lymphocyte amplification kit, comprising the above-described tumor-infiltrating lymphocyte amplification composition.

[0015] This application also provides a method for in vitro expansion of tumor-infiltrating lymphocytes, comprising:

[0016] Take a tumor puncture sample, and culture the tumor puncture sample using the above-mentioned tumor-infiltrating lymphocyte amplification composition to prepare amplified tumor-infiltrating lymphocytes.

[0017] In one embodiment, the method includes: adding the activated antibody mixture to a culture container and incubating it to prepare a coated culture container; and

[0018] The tumor puncture sample was inoculated into the coated culture container, and the tumor-infiltrating lymphocyte amplification medium was added for culture to prepare amplified tumor-infiltrating lymphocytes.

[0019] In one embodiment, the volume of the tumor biopsy sample is 1.130 mm. 3 ~22.61 mm 3 .

[0020] In one embodiment, the culture container includes a culture well plate;

[0021] In one embodiment, the incubation conditions include: a temperature of 36 ℃ to 38 ℃ for 3 h to 6 h; or a temperature of 2 ℃ to 8 ℃ for 12 h to 16 h.

[0022] In one embodiment, the conditions for culturing the tumor-infiltrating lymphocytes with the added culture medium include: a temperature of 33°C to 38°C and a CO2 concentration of 4.8 v / v% to 5.2 v / v.

[0023] In one embodiment, the step of expanding the culture of tumor-infiltrating lymphocytes is also included;

[0024] The expansion culture of tumor-infiltrating lymphocytes includes: filtering the expanded tumor-infiltrating lymphocytes through a 40 μm~70 μm filter, centrifuging and resuspending them, and then culturing them at 1×10⁻⁶ ppm. 5 / mL~5×10 5Expanded inoculation culture was carried out at a cell density of / mL.

[0025] This application also provides the tumor-infiltrating lymphocytes amplified by the above-described method for in vitro amplification of tumor-infiltrating lymphocytes.

[0026] This application also provides the use of the aforementioned tumor-infiltrating lymphocytes in the preparation of drugs for treating tumors.

[0027] This application also provides a medicament for treating tumors, wherein the active ingredient of the medicament composition contains the aforementioned tumor-infiltrating lymphocytes, as well as pharmaceutically acceptable excipients.

[0028] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Figure showing the effect of different activating antibodies on the TIL amplification rate;

[0031] Figure 2 Figure showing the effect of different antibody combinations on the TIL amplification rate;

[0032] Figure 3 To validate the optimal working concentrations for different antibody pairwise combinations;

[0033] Figure 4 To validate the optimal working concentrations for different antibody pairwise combinations;

[0034] Figure 5 TIL amplification and validation were performed on different batches of puncture samples;

[0035] Figure 6 To validate the amplification of TILs in puncture samples using different usage methods (coated / free);

[0036] Figure 7 Phenotypic detection for TIL amplification using anti-CD3 / CD28 / 41BB antibody combination;

[0037] Figure 8 To detect the in vitro killing function of TILs amplified by anti-CD3 / CD28 / 41BB antibody combination;

[0038] Figure 9 In vivo efficacy experiment for amplifying TILs using a combination of anti-CD3 / CD28 / 41BB antibodies. Detailed Implementation

[0039] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0040] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0041] the term

[0042] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0043] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0044] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0045] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0046] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0047] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0048] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0049] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0050] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0051] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0052] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0053] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0054] Tumor-infiltrating lymphocytes (TILs), innate lymphocytes (ILCs), major histocompatibility complex (MhC), chimeric antigen receptor T-cells (CAR-T), T-cell receptor (TCR), central memory T-cells (TCM), effector memory T-cells (TEM), IL-2, interleukin-2, IL-7, and interleukin-15.

[0055] In the process of T cells killing tumor cells, central memory T cells (TCMs) or stem-like T cells play a crucial role in sustained anti-tumor effects. Maintaining T cell stemness during the initial culture phase enhances their sustained anti-tumor function. Numerous studies have shown that adding IL-7 and IL-15 to T cell culture media can significantly induce the generation of memory phenotype cells while maintaining T cell stemness. Furthermore, 41BB (CD137) is the second stimulatory signaling molecule on T cells, and its expression is significantly upregulated in activated or antibody-stimulated T cells. Therefore, 41BB is considered a molecular marker for tumor antigen-specific T cells. Activating T cells using 41BB ligands or 41BB antibodies can selectively expand antigen-specific T cells in TILs while upregulating the proportion of memory cells. Therefore, adding 41BB antibodies to the traditional anti-CD3 / CD28 antibody-stimulated T cell proliferation regimen can further increase the proportion of tumor antigen-specific memory cells.

[0056] This application provides a tumor-infiltrating lymphocyte expansion composition, including a tumor-infiltrating lymphocyte expansion culture medium and an activation antibody mixture;

[0057] The tumor-infiltrating lymphocyte expansion medium includes a basal medium and components in the basal medium at the following concentrations: 2 v / v%~10 v / v% SR serum substitute, 300 IU / mL~9000 IU / mL IL-2, 0 ng / mL~10 ng / mL IL-7 and 0 ng / mL~30 ng / mL IL-15.

[0058] It is understood that SR serum substitutes include, but are not limited to, one or more of AB serum and autologous serum. The activating antibody mixture includes 0 μg / mL to 5 μg / mL anti-CD3 antibody, 0 μg / mL to 5 μg / mL anti-CD28 antibody, and 0 μg / mL to 5 μg / mL anti-41BB antibody.

[0059] In a specific example, the concentration of anti-CD3 antibody was 0.30 μg / mL to 0.35 μg / mL.

[0060] In a specific example, the concentration of anti-CD28 antibody was 0.30 μg / mL to 0.35 μg / mL.

[0061] In a specific example, the concentration of anti-41BB antibody was 0.30 μg / mL to 0.35 μg / mL.

[0062] For example, in one specific embodiment, the concentration of the SR serum substitute is 2 v / v; in another specific embodiment, the concentration of the SR serum substitute is 10 v / v; it is understood that in other specific embodiments, the concentration of the SR serum substitute is 3 v / v%, 4 v / v%, 5 v / v%, 6 v / v%, 7 v / v%, 8 v / v%, or 9 v / v.

[0063] For example, in one specific embodiment, the concentration of IL-2 is 300 IU / mL, and in another specific embodiment, the concentration of IL-2 is 9000 IU / mL. It is understood that in other specific embodiments, the concentration of IL-2 may be 500 IU / mL, 1000 IU / mL, 1500 IU / mL, 2000 IU / mL, 2500 IU / mL, 3000 IU / mL, 3500 IU / mL, 4000 IU / mL, 4500 IU / mL, 5000 IU / mL, 5500 IU / mL, 6000 IU / mL, 6500 IU / mL, 7000 IU / mL, 7500 IU / mL, 8000 IU / mL, 8500 IU / mL, and 9000 IU / mL, as well as other values ​​within the range.

[0064] For example, in one specific embodiment, the concentration of IL-7 is 0 ng / mL, and in another specific embodiment, the concentration of IL-7 is 10 ng / mL. It is understood that in other specific embodiments, the concentration of IL-7 is 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, or 9 ng / mL, as well as other values ​​within the range.

[0065] For example, in one specific embodiment the concentration of IL-15 is 0 ng / mL, in another specific embodiment the concentration of IL-15 is 30 ng / mL, and it is understood that in other specific embodiments the concentration of IL-15 is 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL or 25 ng / mL, and other values ​​within the range.

[0066] In a specific example, the basal culture medium includes, but is not limited to, one or more of X-VIVO 15, RPMI-1640, and AIM-V media.

[0067] It is understandable that the basal culture medium is a commercially available medium, such as X-VIVO 15 medium, which contains the following components: L-glutamine, gentamicin, phenol red, pharmaceutical grade human albumin, recombinant human insulin, and human transferrin. Some media use recombinant transferrin instead of natural transferrin.

[0068] For example, RPMI-1640 medium contains various amino acids, vitamins, and inorganic salts required for cell growth.

[0069] For example, AIM-V medium contains L-glutamylamine, streptomycin sulfate, and gentamicin sulfate.

[0070] Another aspect of this application provides a tumor-infiltrating lymphocyte amplification kit, comprising a tumor-infiltrating lymphocyte amplification composition and an amplification buffer.

[0071] This application also provides a method for in vitro expansion of tumor-infiltrating lymphocytes, comprising:

[0072] A tumor biopsy sample was taken; the volume of the tumor biopsy sample was 1.130 mm. 3 ~ 22.61 mm 3 During collection, an 18 g puncture needle with a diameter of 1.2 mm and a length ranging from 1 mm to 20 mm was used. The tumor puncture sample was cultured using a tumor-infiltrating lymphocyte amplification composition to obtain amplified tumor-infiltrating lymphocytes.

[0073] In a specific example, the activation antibody mixture is added to a culture container and incubated to prepare a coated culture container.

[0074] Tumor puncture samples were inoculated into coated culture containers, and tumor-infiltrating lymphocyte amplification medium was added for culture to obtain amplified tumor-infiltrating lymphocytes.

[0075] In a specific example, the culture container includes a culture plate. For example, a 24-well plate or a 12-well plate, with the added volume being 500 μL for a 24-well plate and 1000 μL for a 12-well plate.

[0076] In a specific example, the incubation conditions include a temperature of 36 ℃ to 38 ℃ and a time of 3 h to 6 h. For example, temperatures of 36 ℃, 37 ℃, and 38 ℃, and times of 3 h, 4 h, 5 h, or 6 h.

[0077] Alternatively, incubation conditions may include a temperature of 2°C to 8°C and a time of 12 h to 16 h. For example, temperatures of 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, and 8°C, with times of 12 h, 13 h, 14 h, 15 h, or 16 h.

[0078] In a specific example, the conditions for culturing tumor-infiltrating lymphocytes with the added culture medium include: a temperature of 36°C–38°C, a CO2 concentration of 4.8 v / v %–5.2 v / v %, and half-volume medium replacement on days 1, 3, and 5 post-inoculation, supplemented with fresh TIL complete culture medium. For example, temperatures of 36°C, 37°C, or 38°C, and CO2 concentrations of 4.8 v / v %, 4.9 v / v %, 5.0 v / v %, 5.1 v / v %, or 5.2 v / v %.

[0079] Optionally, it also includes a step of expanding the culture of tumor-infiltrating lymphocytes.

[0080] Specifically, the expanded culture of tumor-infiltrating lymphocytes includes: filtering the expanded tumor-infiltrating lymphocytes through a 35μm ~ 75μm filter, centrifuging and resuspending them, and then... 5 / mL~5×10 5 Expanded seeding culture was carried out at a cell density of / mL. For example, the filter size was 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, or 75 μm.

[0081] Specifically, on day 7 of culture, the cultured cells were collected, filtered through a 40 μm filter, centrifuged, resuspended, and counted. A suitable container was selected with 1×10⁶ cells. 5 / mL~5×10 5 The cells were seeded and cultured at a density of 1 × 10⁶ / mL. During subsequent culture, equal volumes of culture medium were added and cell counts were performed every 2-3 days. An appropriate container was selected based on the size of the culture system. For example, a cell density of 1 × 10⁶ / mL was used. 5 / mL, 2×10 5 / mL, 3×10 5 / mL, 4×10 5 / mL or 5×10 5 / mL.

[0082] This application also provides tumor-infiltrating lymphocytes amplified by the above-described method for in vitro amplification of tumor-infiltrating lymphocytes.

[0083] This application also provides the use of tumor-infiltrating lymphocytes in the preparation of drugs for treating tumors.

[0084] This application also provides a pharmaceutical composition containing tumor-infiltrating lymphocytes and pharmaceutically acceptable excipients.

[0085] In this application, "pharmaceuticalally acceptable excipient" means pharmaceutically acceptable material, composition or mediator, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials.

[0086] As used herein, the phrase “pharmaceuticalally acceptable excipient” includes buffers compatible with drug administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, and the like. Each carrier must be “pharmaceuticalally acceptable” in the sense of being compatible with other components in the formulation and harmless to the patient.

[0087] Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn... Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0088] In this application, "excipients" include, but are not limited to, mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, fructooligosaccharides, dextran, glycine, starch, sucrose, dextrin (such as maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipids, kaolin, talc, calcium stearate, and magnesium stearate.

[0089] Optionally, the tumor includes, but is not limited to, one or more of the following: thyroid cancer, gallbladder cancer, lung cancer, breast cancer, and liver cancer.

[0090] Understandably, tumors can also be selected from solid tumors.

[0091] This application also provides a method for treating tumors, including taking a tumor sample from a subject, expanding tumor-infiltrating lymphocytes using an in vitro expansion method, and reinfusing the tumor-infiltrating lymphocytes into the patient.

[0092] It is understood that treatment refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease and / or adverse reactions caused by the disease. As used in this invention, treatment includes any treatment of a disease in mammals, particularly humans, including: (a) preventing the onset of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development or progression; and (c) alleviating the disease, i.e., causing disease remission and / or alleviating one or more disease symptoms. Treatment also means including the delivery of agents to provide a pharmacological effect, even in the absence of disease or symptom. For example, treatment includes the delivery of compositions that can elicit an immune response or confer immunity in the absence of disease, such as in the case of vaccines.

[0093] This application provides a composition for expanding tumor-infiltrating lymphocytes from minute amounts of tumor tissue, specifically tumor biopsy samples. This composition addresses the issue of requiring large sample volumes of tumor tissue for tumor-infiltrating lymphocyte expansion, making it suitable for patients who cannot undergo surgical resection to obtain tumor tissue. Furthermore, this application optimizes the combination and working concentration of antibody stimulants to establish a process for expanding tumor-infiltrating lymphocytes from biopsy samples. This maintains the stemness of the expanded tumor-infiltrating lymphocytes, enhances their killing power, and thus ensures the anti-tumor efficacy after reinfusion into the body.

[0094] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0095] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0096] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0097] Example 1

[0098] This embodiment provides a TIL amplification culture composition, comprising the following components:

[0099] X-VIVO 15 medium, 5v / v% SR serum substitute, 6000 IU / mL IL-2, 5ng / mL IL-7, 5ng / mL IL-15, 0.33μg / mL anti-CD3 antibody, 0.33μg / mL anti-CD28 antibody and 0.33μg / mL anti-41BB antibody.

[0100] The specific item numbers and manufacturers are shown in Table 1 below:

[0101] Table 1

[0102]

[0103] Example 2

[0104] This embodiment provides a TIL amplification culture composition, with the same catalog number as in Embodiment 1, comprising the following components:

[0105] X-VIVO 15 medium, 5 v / v% SR serum substitute, 6000 IU / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 0.35 μg / mL anti-CD3 antibody, 0.35 μg / mL anti-CD28 antibody and 0.35 μg / mL anti-41BB antibody.

[0106] Example 3

[0107] This embodiment provides a TIL amplification culture composition, with the same catalog number as in Embodiment 1, comprising the following components:

[0108] X-VIVO 15 medium, 5v / v% SR serum substitute, 6000 IU / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 0.11 μg / mL anti-CD3 antibody, 0.11 μg / mL anti-CD28 antibody and 0.11 μg / mL anti-41BB antibody.

[0109] Example 4

[0110] This embodiment provides a method for in vitro amplification and culture of TILs in puncture samples:

[0111] (1) Activation of antibody coating: Prepare an antibody coating solution corresponding to the working concentration according to the antibody concentration described in the TIL amplification culture composition of Example 1, and add it to a 24-well plate (500 μL) / 12-well plate (1000 μL) in a certain volume, and incubate at 37°C for 3-6 h, or at 4°C for 12-16 h. After incubation, wash away the coating solution, wash the plate twice with 2 times the volume, and then use it to inoculate puncture samples for culture.

[0112] (2) Inoculation of puncture samples: According to the experimental groups, each group was inoculated with one tumor puncture tissue, with a length of about 0.5 cm (volume of about 5.652 mm). 3 The culture medium was added to the TIL amplification complete medium (500 μL for 24-well plates and 1000 μL for 12-well plates) and cultured at 37°C in a 5% CO2 system.

[0113] (3) TIL culture medium replacement: Half-volume medium replacement was performed on the 1st, 3rd and 5th days after inoculation, and fresh TIL complete culture medium was added.

[0114] (4) TIL expansion culture: On day 7 of culture, the cultured cells were collected, filtered through a 40 μm filter, centrifuged, resuspended, and counted. A suitable container was selected for 1×10⁶ cells / day. 5 / mL~5×10 5Inoculate and culture cells at a density of / mL. During subsequent culture, replenish the culture medium with an equal volume every 2-3 days and count the cells. Select an appropriate container based on the size of the culture system.

[0115] (5) When the cells reach the experimental endpoint, collect the endpoint cells for TIL phenotype detection, in vitro killing experiment, or mouse experiment.

[0116] Comparative Example 1

[0117] This comparative example provides a TIL amplification culture composition that, compared to Example 1, uses only an anti-CD3 antibody, and is otherwise the same as Example 1.

[0118] X-VIVO 15 medium, 5v / v% SR serum substitute, 6000 IU / mL IL-2, 5ng / mL IL-7, 5ng / mL IL-15, 0.33μg / mL anti-CD3 antibody.

[0119] Comparative Example 2

[0120] This comparative example provides a TIL amplification culture composition that, compared to Example 1, uses only the anti-CD28 antibody, and is otherwise the same as Example 1.

[0121] X-VIVO 15 medium, 5v / v% SR serum substitute, 6000 IU / mL IL-2, 5ng / mL IL-7, 5ng / mL IL-15, 0.33μg / mL anti-CD28 antibody.

[0122] Comparative Example 3

[0123] This comparative example provides a TIL amplification culture composition. Compared with Example 1, this comparative example only uses 41BB antibody, and the rest is the same as Example 1.

[0124] X-VIVO 15 medium, 5v / v% SR serum substitute, 6000 IU / mL IL-2, 5ng / mL IL-7, 5ng / mL IL-15, 0.33μg / mL anti-41BB antibody.

[0125] Comparative Example 4

[0126] This comparative example provides a TIL amplification culture composition. Compared with Example 1, this comparative example uses a combination of anti-CD3 antibody and anti-CD28 antibody, and the rest is the same as Example 1.

[0127] This comparative study used X-VIVO 15 medium, 5v / v% SR serum substitute, 6000 IU / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 0.33 μg / mL anti-CD3 antibody, and 0.33 μg / mL anti-CD28 antibody.

[0128] Comparative Example 5

[0129] This comparative example provides a TIL amplification culture composition. Compared with Example 1, this comparative example uses a combination of anti-CD3 antibody and anti-41BB antibody, and the rest is the same as Example 1.

[0130] X-VIVO 15 medium, 5v / v% SR serum substitute, 6000 IU / mL IL-2, 5ng / mL IL-7, 5ng / mL IL-15, 0.33μg / mL anti-CD3 antibody and 0.33μg / mL anti-41BB antibody.

[0131] Comparative Example 6

[0132] This comparative example provides a TIL amplification culture composition. Compared with Example 1, this comparative example uses a combination of anti-CD28 antibody and anti-41BB antibody, and the rest is the same as Example 1.

[0133] X-VIVO 15 medium, 5 v / v% SR serum substitute, 6000 IU / mL IL-2, 5 ng / mL IL-7, 5 ng / mL IL-15, 0.33 μg / mL anti-CD28 antibody and 0.33 μg / mL anti-41BB antibody.

[0134] Comparative Example 7

[0135] This comparative example provides a method for in vitro amplification and culture of TILs in puncture samples. The difference between this comparative example and Example 4 is that the antibody activation solution is added in a free form.

[0136] (1) Inoculation of puncture samples: According to the experimental groups, each group was inoculated with one tumor puncture tissue, with a length of about 0.5 cm (volume of about 5.652 mm). 3 The TIL amplification culture composition was added (500 μL for 24-well plates and 1000 μL for 12-well plates), and cultured at 37 °C in a 5% CO2 system.

[0137] (2) TIL culture medium replacement: Half-volume medium replacement was performed on the 1st, 3rd and 5th days after inoculation, and fresh TIL complete culture medium was added.

[0138] (3) TIL expansion culture: On day 7 of culture, the cultured cells were collected, filtered through a 40 μm filter, centrifuged, resuspended, and counted. A suitable container was selected with a 1 × 10⁻⁶ inch diameter. 5 / mL~5×10 5 Inoculate and culture cells at a density of / mL. During subsequent culture, replenish the culture medium with an equal volume every 2-3 days and count the cells. Select an appropriate container based on the size of the culture system.

[0139] (5) When the cells reach the experimental endpoint, collect the endpoint cells for TIL phenotype detection, in vitro killing experiment, or mouse experiment.

[0140] I. Experimental steps for verifying relevant results:

[0141] 1. Flow cytometry detection of TIL phenotype

[0142] (1) Centrifuge the collected cells at 600 g for 5 min and remove the supernatant.

[0143] (2) Resuspend the cells in 1 mL of MACS buffer, centrifuge at 600 g for 5 min, and discard the supernatant.

[0144] (3) Repeat step (2).

[0145] (4) Resuspend cells in 100 μL of MACS buffer, add 1 μL each of flow cytometry antibodies CD3-APC-CY7, CD4-BV650, CD8-PE, CCR7-APC, CD45RA-BV786, CD39-AF700, and CD69-BV605, mix well, and incubate at 2℃~8℃ in the dark for 30 min.

[0146] (5) After incubation, add 1 mL of MACS buffer to resuspend the cells, centrifuge at 600 g for 5 min, and remove the supernatant.

[0147] (6) Repeat step (5).

[0148] (7) Resuspend cells in 200 μL MACS buffer and perform flow cytometry analysis.

[0149] The specific item numbers and usage quantities are shown in Table 2:

[0150] Table 2

[0151]

[0152] 2. In vitro sustained killing test

[0153] The liver cancer cell line SK-hep-1 (code name: SK-BN105) was selected as the target cell line to test the in vitro killing function of TILs obtained by stimulation with different antibody combinations. Target cells were digested and counted at a ratio of 1×10⁻⁶. 4 The target cells were seeded into 96-well plates at a specific density, and effector cells (TILs) of appropriate numbers were added according to the effector-to-target ratio for co-culture. The long-term killing effect was then assessed using an Incucyte instrument (3-7 days), with target cells added every 2-3 days to detect the sustained killing function of the TILs.

[0154] The culture medium catalog numbers and components required for target cell culture are shown in Table 3 below:

[0155] Table 3

[0156]

[0157] 3. In vivo tumor suppression experiment

[0158] Target cells (SK-BN105) in the logarithmic growth phase and in good growth condition were collected using trypsin digestion. After washing once with physiological saline, the cell density was adjusted to 2 × 10⁻⁶ cells / year. 7 / mL. 100 μL of cell suspension was subcutaneously injected into the right axillary region of NOG mice, i.e., 2 × 10⁹ cells per mouse. 6 The target cells were inoculated, and the inoculation diary was dated day 0.

[0159] Day 7 after inoculation of target cells (or when the average tumor volume is approximately 50 mm) 3 At that time, TILs stimulated with a combination of anti-CD3 / CD28 antibodies (1×10⁻⁶) were injected via the tail vein. 7 / ani), TILs stimulated by anti-CD3 / CD28 / 41BB antibody combination (1×10 7 The test substance was administered to each mouse (400 μL / mouse) and a solvent (0.9% sodium chloride and 12.5% ​​human serum albumin). Day 0 of treatment was marked as the day of injection. Tumor size and mouse weight were measured 2-3 times per week.

[0160] II. Experimental Results:

[0161] 1. The effect of different activating antibodies on the amplification rate of TILs

[0162] The results of Example 1 and Comparative Examples 1-3 are verified as follows: Figure 1 As shown: by Figure 1Results A show that the anti-CD3 antibody at coating concentrations of 1 μg / mL and 0.33 μg / mL had comparable effects on stimulating TIL proliferation, and was higher than the 0.33 μg / mL group. This suggests that the use of anti-CD3 antibody alone can significantly promote the proliferation of TILs from puncture samples, and the optimal concentration is 0.33 μg / mL.

[0163] Depend on Figure 1 Results B show that, compared to the anti-CD3 antibody stimulation group, the anti-CD28 antibody coating had a very limited effect on stimulating TIL proliferation, with the number of harvested cells at 28 days of culture being less than 10. 5 ~10 7 The results suggest that using anti-CD28 antibody alone cannot significantly promote TIL amplification. On the other hand, comparing the coating concentrations of different anti-CD28 antibodies, it can be seen that the 0.33 μg / mL group is more conducive to TIL amplification than the 1 μg / mL and 0.11 μg / mL groups, suggesting that the optimal concentration of anti-CD28 antibody is 0.33 μg / mL.

[0164] Depend on Figure 1 The C-results show that, compared with the anti-CD3 antibody and anti-CD28 antibody stimulation groups, coating with anti-41BB antibody has a certain effect on stimulating TIL proliferation, but the number of cells harvested on day 28 of culture is less than 10. 7 ~ 10 9 The anti-41BB antibody was weaker than the anti-CD3 antibody stimulation group, suggesting that the anti-41BB antibody needs to be used in combination with other antibodies to enhance the proliferative effect on TILs.

[0165] Furthermore, a comparison of different anti-41BB antibody coating concentrations showed that, compared to 0.11 μg / mL, the 1 μg / mL and 0.33 μg / mL groups were more conducive to TIL amplification, suggesting that the optimal concentration of anti-41BB antibody is 0.33 μg / mL.

[0166] 2. Effects of different antibody combinations on TIL amplification rate

[0167] The results of Comparative Examples 4-6 are shown below: Since T cell proliferation requires not only a primary stimulation signal primarily composed of anti-CD3 antibodies, but also signals from co-stimulatory molecules (CD28, 41BB, etc.), this application further combines the above three antibodies to optimize the antibody stimulation composition and concentration, based on the optimal single antibody concentration for stimulating TIL proliferation obtained above.

[0168] Based on the above experimental results, this application proposes to use anti-CD3 antibody, anti-CD28 antibody, and anti-41BB antibody in combination.

[0169] Figure 2 The concentration of anti-CD3 / CD28 / 41BB antibodies used in the experiment was 0.33 μg / mL.

[0170] Depend on Figure 2 Results A show that, compared with the groups using anti-CD3 antibody or anti-CD28 antibody alone, the combination of anti-CD3 antibody and anti-CD28 antibody can enhance the amplification capacity of TIL, suggesting that the use of combined anti-CD3 / CD28 antibody is superior to the use of either one antibody alone.

[0171] Depend on Figure 2 The results in B show that, compared with the groups using anti-CD3 antibody or anti-41BB antibody alone, the combination of anti-CD3 antibody and anti-41BB antibody can enhance the amplification capacity of TIL, suggesting that the use of combined antibody CD3 / 41BB antibody is better than the use of one of the antibodies alone.

[0172] Depend on Figure 2 The results from the C-test show that, compared with the groups using anti-CD28 antibody or anti-41BB antibody alone, the combination of anti-CD28 antibody and anti-41BB antibody can enhance the amplification capacity of TIL, suggesting that the use of combined anti-CD28 / 41BB antibody is superior to the use of either one antibody alone.

[0173] Figure 2 The experimental data came from the same batch of puncture samples (i.e., from the same tumor patients). By comparing the effects of different antibody combinations on TIL proliferation, it can be seen that the anti-CD3 / CD28 antibody combination has the best effect on TIL proliferation, followed by the anti-CD3 / 41BB antibody combination and the anti-CD28 / 41BB antibody combination.

[0174] 3. Validation of the optimal working concentrations for different antibody pair combinations (pair combinations)

[0175] The results above show that the optimal working concentration for each anti-CD3 / CD28 / 41BB antibody, used individually, is 0.33 μg / mL. However, the optimal working concentration for combining these three antibodies in pairs requires further validation. Therefore, antibody concentrations of 1 μg / mL, 0.33 μg / mL, and 0.11 μg / mL (these concentrations refer to the concentration of each antibody added in each pair of combinations) were set up for validation.

[0176] Depend on Figure 3 Results A show that when the working concentrations of the anti-CD3 / CD28 antibody combination were 1 μg / mL and 0.33 μg / mL, the proliferation ability of TILs was stronger. Furthermore, the TIL proliferation levels stimulated by the 1 μg / mL group and the 0.33 μg / mL group were quite similar, suggesting that the optimal working concentration of the anti-CD3 / CD28 antibody combination was 0.33 μg / mL.

[0177] Depend on Figure 3 Results B show that when the working concentrations of the anti-CD3 / 41BB antibody combination are 1 μg / mL and 0.33 μg / mL, the proliferation ability of TILs is stronger, and the TIL proliferation levels stimulated by the 1 μg / mL group and the 0.33 μg / mL group are relatively similar, suggesting that the optimal working concentration of the anti-CD3 / 41BB antibody combination is 0.33 μg / mL.

[0178] Depend on Figure 3 The results from the C-test show that when the working concentrations of the anti-CD28 / 41BB antibody combination are 1 μg / mL and 0.33 μg / mL, the TIL proliferation ability is stronger, and the 1 μg / mL group stimulates the TIL proliferation level more strongly, suggesting that the recommended concentration of the anti-CD3 / 41BB antibody combination is 1 μg / mL or 0.33 μg / mL.

[0179] 4. Optimization of anti-CD3 / CD28 / 41BB antibody combination conditions

[0180] The above experimental results show that the use of anti-CD3 / CD28 antibodies can stimulate TIL proliferation, and after 4 weeks of culture, more than 10 9 Order-of-magnitude final product cells. Although neither anti-41BB antibody alone, nor the anti-41BB / CD3 combination, nor the anti-41BB / CD28 antibody combination can achieve the level of TIL proliferation stimulated by anti-CD3 / CD28 antibody, since 41BB serves as an amplification signal for tumor antigen-specific T cells, this application further optimizes the working concentration of the anti-CD3 / CD28 / 41BB stimulation combination by adding anti-41BB antibody stimulation to the existing anti-CD3 / CD28 antibody stimulation conditions.

[0181] Depend on Figure 4 As shown in result A, compared with the anti-CD3 / CD28 antibody combination, the anti-CD3 / 41BB antibody combination, and the anti-CD28 / 41BB antibody combination, stimulation with the anti-CD3 / CD28 / 41BB antibody combination can further enhance the proliferation level of TILs.

[0182] Furthermore, this application optimizes the working concentration of the anti-CD3 / CD28 / 41BB antibody combination ( Figure 4 As shown in B in the table below, the groups are as follows: Compared with groups 1 and 3, the TIL amplification capacity and the final product cell number of groups 2, 4 and 5 are relatively close. Among them, group 4 has the highest cell number at the end of culture, indicating that the antibody concentration conditions of group 4 (0.33 μg / mL of anti-CD3 / CD28 / 41BB antibody each) are the optimal conditions.

[0183] Table 4

[0184]

[0185] 5. Validation of TIL amplification using anti-CD3 / CD28 / 41BB antibody combination.

[0186] Based on the above results, this application used three batches of tumor biopsy samples from different liver cancer patients to amplify TILs. Figure 5 The results showed that, compared with the anti-CD3 / CD28 antibody combination group, the anti-CD3 / CD28 / 41BB antibody combination group could further promote TIL proliferation, and the trend was consistent across the three batches of samples. These results indicate that using the anti-CD3 / CD28 / 41BB antibody stimulation combination, with each group at 0.33 μg / mL, can maximize the promotion of TIL amplification.

[0187] Furthermore, this application compared coating three different batches of TILs (from different patients) with the same concentration of anti-CD3 / CD28 / 41BB antibody combination (coating concentration of 0.33 μg / mL) or by directly adding it to the culture system. The cell expansion rate ( Figure 6 As can be seen, adding free antibodies cannot effectively activate T cells, and their proliferation level is limited. After 15 days of culture, the expansion folds were: Batch 1, 14.9-fold; Batch 2, 5.4-fold; Batch 3, 2.2-fold. In contrast, the antibody coating method of this application can effectively activate T cells and significantly promote their proliferation. After 15 days of culture, the cell numbers were: Batch 1, 2.1 × 10⁻⁶. 8 Batch 2, 3.79 × 10 7 Batch 3, 1.47 × 10 8 The amplification folds were: batch 1, 80.9 times; batch 2, 29.2 times; and batch 3, 29.3 times.

[0188] The data comparison above shows that using antibody coating is more effective than adding free antibodies in promoting the expansion of T cells from puncture samples in vitro.

[0189] 6. Phenotypic detection of TILs amplified by anti-CD3 / CD28 / 41BB antibody combination

[0190] Three batches of tumor biopsy samples from different liver cancer patients were used to amplify the TIL final product, and its phenotype was then tested.

[0191] Depend on Figure 7 The results show that, compared with the anti-CD3 / CD28 antibody combination group, the anti-CD3 / CD28 / 41BB antibody combination group is more effective against CD8. + T cell expansion is selective, specifically CD8. +The proportion of T cells showed an upward trend, and the trend was consistent across the three batches of samples. Furthermore, statistical analysis of the CD8 / CD4 T cell ratio revealed a significant upregulation in the anti-CD3 / CD28 / 41BB antibody combination group (P < 0.03). Figure 7 (As shown in A in the diagram).

[0192] Meanwhile, analysis of CD8+ T cell memory typing revealed that, compared to the anti-CD3 / CD28 antibody combination group, the anti-CD3 / CD28 / 41BB antibody combination group showed higher CD45RA levels. + CCR7 - Terminal differentiation CD8 + The proportion of T cells was significantly downregulated (P < 0.008), suggesting that the addition of anti-41BB antibody can inhibit CD8. + T cell differentiation ( Figure 7 (As shown in B). Analysis of stem cell-like cell phenotypes revealed that, compared to the anti-CD3 / CD28 antibody combination group, the proportion of CD39-CD69- stem cell-like CD8+ T cells in the anti-CD3 / CD28 / 41BB antibody combination group showed an upward trend and a statistically significant difference, suggesting that the addition of anti-41BB antibody can maintain the stemness of CD8+ T cells. Figure 7 (as shown in C).

[0193] The above results indicate that the combination of anti-CD3 / CD28 / 41BB antibodies can stimulate TIL amplification, increase the proportion of CD8+ T cells, and maintain the stemness of CD8+ T cells.

[0194] 7. Detection of the in vitro killing function of TIL amplified by anti-CD3 / CD28 / 41BB antibody combination

[0195] Three batches of tumor puncture samples from different liver cancer patients were used to amplify the final TIL product, and its tumor cell killing function was tested.

[0196] Depend on Figure 8 The results showed that the anti-CD3 / CD28 / 41BB antibody combination group exhibited stronger TIL-killing activity compared to the anti-CD3 / CD28 antibody combination group. These results indicate that stimulating TIL amplification with the anti-CD3 / CD28 / 41BB antibody combination can enhance its anti-tumor killing effect in vitro.

[0197] 8. In vivo antitumor efficacy of TIL amplified by anti-CD3 / CD28 / 41BB antibody combination

[0198] This application describes the construction of a subcutaneous xenograft model of the liver cancer cell line SK-hep-1 in immunodeficient (NOG) mice. The tumors were allowed to grow to 50 mm. 3 ~100mm3 Subsequently, TIL was reinfused to observe its anti-tumor efficacy. The specific technical route is as follows: Figure 9 As shown in A in the diagram.

[0199] Figure 9 Results B in the study showed that TIL reinfusion did not reduce the body weight of mice, indicating that TIL amplified using the above two antibody combinations had no significant toxic side effects in mice. Furthermore, compared to the anti-CD3 / CD28 antibody combination group, the anti-CD3 / CD28 / 41BB antibody combination group showed stronger anti-tumor activity in TIL (e.g., ...). Figure 9 As shown in C), this indicates that the combination of anti-CD3 / CD28 / 41BB antibodies can significantly enhance the anti-tumor efficacy of TIL in vivo.

[0200] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A composition for expanding tumor-infiltrating lymphocytes, characterized in that, Includes a culture medium for expanding tumor-infiltrating lymphocytes and a mixture of activating antibodies; The tumor-infiltrating lymphocyte expansion culture medium includes a basal culture medium and components at the following concentrations: 2 v / v%~10 v / v% SR serum substitute, 300 IU / mL~9000 IU / mL IL-2, 0 ng / mL~10 ng / mL IL-7 and 0 ng / mL~30 ng / mL IL-15; The activated antibody mixture comprises a combination of two or more of the following: 0 μg / mL to 5 μg / mL anti-CD3 antibody, 0 μg / mL to 5 μg / mL anti-CD28 antibody, and 0 μg / mL to 5 μg / mL anti-41BB antibody.

2. The tumor-infiltrating lymphocyte expansion composition according to claim 1, characterized in that, The activated antibody mixture comprises one or more combinations of 0.11 μg / mL to 0.35 μg / mL anti-CD3 antibody, 0.11 μg / mL to 0.35 μg / mL anti-CD28 antibody, and 0.11 μg / mL to 0.35 μg / mL anti-41BB antibody.

3. The tumor-infiltrating lymphocyte expansion composition according to claim 1, characterized in that, The concentration of the anti-CD3 antibody is 0.30 μg / mL to 0.35 μg / mL; Optionally, the concentration of the anti-CD28 antibody is 0.30 μg / mL to 0.35 μg / mL; Optionally, the concentration of the anti-41BB antibody is 0.30 μg / mL to 0.35 μg / mL; Optionally, the basal culture medium includes one or more of X-VIVO 15, RPMI-1640, and AIM-V media.

4. A kit for expanding tumor-infiltrating lymphocytes, characterized in that, The tumor-infiltrating lymphocyte expansion composition according to any one of claims 1 to 3 is included.

5. A method for in vitro expansion of tumor-infiltrating lymphocytes, characterized in that, include: Take a tumor puncture sample, and culture the tumor puncture sample using the tumor-infiltrating lymphocyte amplification composition according to any one of claims 1 to 3 to prepare amplified tumor-infiltrating lymphocytes.

6. The method according to claim 5, characterized in that, include: The activated antibody mixture was added to a culture container and incubated to prepare a coated culture container. as well as The tumor puncture sample was inoculated into the coated culture container, and the tumor-infiltrating lymphocyte amplification medium was added for culture to prepare amplified tumor-infiltrating lymphocytes.

7. The method according to claim 6, characterized in that, The method has one or more of the following conditions: (1) The volume of the tumor biopsy sample was 1.130 mm. 3 ~ 22.61 mm 3 ; (2) The culture container includes a culture well plate; (3) The incubation conditions include: a temperature of 36 ℃~38 ℃ and a time of 3 h~6 h; or a temperature of 2 ℃~8 ℃ and a time of 12 h~16 h; (4) The conditions for culturing the tumor-infiltrating lymphocytes in the culture medium include: a temperature of 33 ℃ to 38 ℃ and a CO2 concentration of 4.8 v / v % to 5.2 v / v %.

8. The method according to any one of claims 5 to 7, characterized in that, It also includes the step of expanding the culture of tumor-infiltrating lymphocytes; The expansion culture of tumor-infiltrating lymphocytes includes: filtering the expanded tumor-infiltrating lymphocytes through a 35 μm to 75 μm filter, centrifuging and resuspending them, and then culturing them at 1×10⁻⁶ ppm. 5 / mL~5×10 5 Expanded inoculation culture was carried out at a cell density of / mL.

9. Tumor-infiltrating lymphocytes amplified by the method for in vitro amplification of tumor-infiltrating lymphocytes according to any one of claims 5 to 8.

10. The use of the tumor-infiltrating lymphocytes according to claim 9 in the preparation of a drug for treating tumors.

11. A drug for treating tumors, characterized in that, The active ingredient of the pharmaceutical composition contains the tumor-infiltrating lymphocytes as described in claim 9, and pharmaceutically acceptable excipients.

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