Culture process for tumor-infiltrating lymphocyte

Abstract

Provided in the present application are a tumor-infiltrating lymphocyte and a culture method therefor, a culture medium, a related reagent combination, a pharmaceutical composition, and a pharmaceutical use. The culture method comprises: in an initial culture stage, adding a stimulating factor combination of IL-2, an anti-CD3 antibody, and an anti-4-1BB antibody, so as to achieve activation and expansion of the tumor-infiltrating lymphocyte, and the amount of IL-2 used can be controlled.

Description

A culture process of tumor infiltrating lymphocytes TECHNICAL FIELD

[0001] The present application relates to the field of cell culture, in particular to a culture process of tumor infiltrating lymphocytes. BACKGROUND

[0002] In the past few decades, immunotherapy, especially cell-based immunotherapy, has been the focus of cancer treatment. Clinical studies in recent years have shown that the use of tumor infiltrating lymphocytes (TIL) for cell transfer of advanced solid tumors shows good efficacy. TIL therapy is a kind of adoptive cell therapy (ACT), which uses the patient's own immune system to treat tumors, separates TIL from tumor sites by biopsy or surgery, stimulates and expands them in vitro with interleukin 2 (IL-2), and then returns them to the patient to enhance the immune attack on the tumor.

[0003] However, the TIL cell therapy faces many challenges when it is pushed from the laboratory to clinical application.

[0004] Firstly, TIL expansion involves the use of high doses of IL-2 for activating TIL in tumors, however, it has been reported that IL-2 is an important cytokine for the development and function of suppressive T cells (Tregs), which will compete with CD8+ T cells for IL-2 and inhibit the response of CD8+ T cells. High levels of IL-2 will up-regulate the inhibitory receptors of CD8+ T cells, reduce the production of cytokines and effector molecules, and cause CD8+ T cell dysfunction. Therefore, how to reduce the use of IL-2 during production and infusion is a problem to be solved.

[0005] In addition, the existing cytokine combination and culture conditions often need to be adjusted and optimized according to the specific circumstances of different patients, which not only increases the complexity of operation, but also increases the cost. One of the biggest challenges facing TIL therapy is the time required to harvest and expand T cell populations, which inevitably delays intervention on patients. At present, the typical manufacturing time is about 3 weeks to 3 months. Therefore, the field is very concerned about shortening the waiting time from the clinical decision to use TIL therapy to the product being available for infusion.

[0006] Finally, TIL cell therapy usually requires a large weight of tissue sample from the tumor of a patient to ensure that a sufficient number of TIL cells can be isolated for in vitro expansion. However, a larger sampling weight not only causes more trauma and pain to the patient, but also increases the time and risk of postoperative recovery. Therefore, there is a need for a more optimized process to reduce the tissue sampling weight, reduce the trauma and postoperative recovery time of the patient, and ensure that a sufficient number and quality of TIL cells can be obtained for treatment.

[0007] Therefore, there is an urgent need in the art to develop a culture method that reduces the use of IL-2, shortens the culture time of TILs, and can obtain a large number of TIL cells. SUMMARY

[0008] The purpose of the present application is to provide a culture method that reduces the use of IL-2, shortens the culture time of TILs, and can obtain a large number of TIL cells.

[0009] In a first aspect of the present application, a method for culturing tumor infiltrating lymphocytes (TILs) is provided, comprising the steps of:

[0010] A. providing a tumor tissue containing an initial TIL cell population;

[0011] B. placing the initial TIL cell population in a first culture medium for primary culture to obtain a TIL primary culture cell population; wherein the first culture medium comprises a combination of stimulating factors: IL-2, anti-CD3 antibody and anti-4-1BB antibody.

[0012] In another preferred embodiment, the method further comprises the step of:

[0013] C. performing quality detection verification on the TIL primary culture cell population.

[0014] In another preferred embodiment, the detection verification includes cell flow cytometric phenotype detection and expansion verification.

[0015] In another preferred embodiment, the tumor tissue is derived from a tumor patient.

[0016] In another preferred embodiment, the tumor is a solid tumor.

[0017] In another preferred embodiment, the tumor tissue is stored in a tissue preservative for 0-72 hours, and the storage temperature is 2-8°C.

[0018] In another preferred embodiment, the tumor tissue is subjected to washing treatment.

[0019] In another preferred embodiment, the mass of the tumor tissue is 0.01-5g, preferably 0.02g-3g, and more preferably 0.05g-1g.

[0020] In another preferred embodiment, the concentration of the IL-2 in the first culture medium is 500-3000 IU / ml, preferably 800-2500 IU / ml, more preferably 1000-2000 IU / ml.

[0021] In another preferred embodiment, the concentration of the anti-CD3 antibody in the first culture medium is 10-100 ng / ml, preferably 20-60 ng / ml, more preferably 25-50 ng / ml.

[0022] In another preferred embodiment, the concentration of the anti-4-1BB antibody in the first culture medium is 1-50 μg / ml, preferably 2-30 μg / ml, more preferably 3-10 μg / ml.

[0023] In another preferred embodiment, the antibody comprises an antibody active fragment or a derivative thereof.

[0024] In another preferred embodiment, the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a single-domain antibody, or a combination thereof.

[0025] In another preferred embodiment, the antibody comprises a humanized antibody, an animal-derived antibody, a chimeric antibody.

[0026] In another preferred embodiment, the anti-CD3 antibody is a monoclonal antibody.

[0027] In another preferred embodiment, the anti-4-1BB antibody is a humanized monoclonal antibody.

[0028] In another preferred embodiment, the components of the first culture medium further comprise a cell basal medium, a buffer, a serum substitute, L-glutamine or a substitute thereof, an antibiotic.

[0029] In another preferred embodiment, the cell basal medium comprises, but is not limited to, an RPMI 1640 medium, an SFM medium, and / or an X-VIVO medium, such as an Advanced RPMI 1640 medium.

[0030] In another preferred embodiment, the buffer is a HEPES buffer.

[0031] In another preferred embodiment, the first culture medium is free of serum.

[0032] In another preferred embodiment, the L-glutamine substitute is GlutaMax.

[0033] In another preferred embodiment, the antibiotic comprises, but is not limited to, gentamicin, levofloxacin, and / or a penicillin-streptomycin mixture.

[0034] In another preferred embodiment, the initial culture time in step B is 7-14 days.

[0035] In another preferred embodiment, the initial TIL cell population in 0.02-0.1 g tumor tissue, the number of TIL cells in the obtained TIL initial culture cell population is 1 x 10 7 ~ 1 x 10 9 , preferably 2 x 10 7 ~ 1 x 10 9 , more preferably 3 x 10 7 ~ 1 x 10 9 .

[0036] In another preferred embodiment, the proportion of viable cells in the TIL initial culture cell population is ≥ 95%, preferably ≥ 96%, more preferably ≥ 97%.

[0037] In another preferred embodiment, the number of CD3+ cells in the TIL initial culture cell population accounts for ≥ 80%, preferably ≥ 85%, more preferably ≥ 90% of the total number of cells.

[0038] In another preferred embodiment, the number of CD4+ cells in the TIL initial culture cell population accounts for ≥ 40%, preferably ≥ 45%, more preferably ≥ 50% of the number of CD3+ cells.

[0039] In another preferred embodiment, the number of CD352+ cells in the TIL initial culture cell population accounts for ≥ 95%, preferably ≥ 96%, more preferably ≥ 97% of the total number of viable cells.

[0040] In another preferred embodiment, the proportion of T CM cells in the TIL initial culture cell population accounts for ≥ 40%, preferably ≥ 45%, more preferably ≥ 50% of the number of CD8+ cells.

[0041] In another preferred embodiment, the proportion of T CM cells in the TIL initial culture cell population accounts for ≥ 60%, preferably ≥ 65%, more preferably ≥ 70% of the number of CD4+ cells.

[0042] In another preferred embodiment, the TIL initial culture cell population is expanded in a second culture medium, and the cell expansion factor reaches 1000 after 7 days of expansion.

[0043] In another preferred embodiment, the second culture medium comprises the following components: cell basal medium, buffer, serum substitute, L-glutamine or its substitute, antibiotic, IL-2, feeder cells.

[0044] In a second aspect of the present application, there is provided a TIL cell culture medium, comprising a combination of stimulating factors: IL-2, anti-CD3 antibody, anti-4-1BB antibody; wherein the concentration of IL-2 is 500-3000 IU / ml.

[0045] In another preferred embodiment, the concentration of IL-2 in the first culture medium is 800-2500 IU / ml, more preferably 1000-2000 IU / ml.

[0046] In another preferred embodiment, the concentration of anti-CD3 antibody in the first culture medium is 10-100 ng / ml, preferably 20-60 ng / ml, more preferably 25-50 ng / ml.

[0047] In another preferred embodiment, the concentration of anti-4-1BB antibody in the first culture medium is 1-50 μg / ml, preferably 2-30 μg / ml, more preferably 3-10 μg / ml.

[0048] In another preferred embodiment, the antibody comprises an antibody active fragment or a derivative thereof.

[0049] In another preferred embodiment, the anti-CD3 antibody is a monoclonal antibody.

[0050] In another preferred embodiment, the anti-4-1BB antibody is a humanized monoclonal antibody.

[0051] In another preferred embodiment, the components of the culture medium further comprise: a cell basal medium, a buffer, a serum replacement, L-glutamine or a replacement thereof, an antibiotic.

[0052] In another preferred embodiment, the cell basal medium comprises but is not limited to RPMI 1640 medium, SFM medium and / or X-VIVO medium, such as Advanced RPMI 1640 medium.

[0053] In another preferred embodiment, the buffer is HEPES buffer.

[0054] In another preferred embodiment, the complete medium is free of serum.

[0055] In another preferred embodiment, the L-glutamine replacement is GlutaMax.

[0056] In another preferred embodiment, the antibiotic comprises but is not limited to gentamicin, levofloxacin and / or penicillin-streptomycin mixture.

[0057] In another preferred embodiment, the culture medium is used for the culture of TIL cells in the initial culture stage.

[0058] In a third aspect of the present application, there is provided a reagent combination comprising components: (i) IL-2, (ii) an anti-CD3 antibody, and (iii) an anti-4-1BB antibody.

[0059] In another preferred embodiment, the reagent combination consists of components: (i) IL-2, (ii) an anti-CD3 antibody, and (iii) an anti-4-1BB antibody.

[0060] In another preferred embodiment, the concentration of IL-2 in the reagent combination is 500-3000 IU / ml, preferably 800-2500 IU / ml, more preferably 1000-2000 IU / ml.

[0061] In another preferred embodiment, the concentration of the anti-CD3 antibody in the reagent combination is 10-100 ng / ml, preferably 20-60 ng / ml, more preferably 25-50 ng / ml.

[0062] In another preferred embodiment, the concentration of the anti-4-1BB antibody in the reagent combination is 1-50 μg / ml, preferably 2-30 μg / ml, more preferably 3-10 μg / ml.

[0063] In a fourth aspect of the present application, there is provided use of the reagent combination as described in the third aspect of the present application, which is added to a TIL culture medium for culturing of TIL cells.

[0064] In another preferred embodiment, the concentration of IL-2 in the reagent combination is 500-3000 IU / ml, preferably 800-2500 IU / ml, more preferably 1000-2000 IU / ml.

[0065] In another preferred embodiment, the concentration of the anti-CD3 antibody in the reagent combination is 10-100 ng / ml, preferably 20-60 ng / ml, more preferably 25-50 ng / ml.

[0066] In another preferred embodiment, the concentration of the anti-4-1BB antibody in the reagent combination is 1-50 μg / ml, preferably 2-30 μg / ml, more preferably 3-10 μg / ml.

[0067] In a fifth aspect of the present application, there is provided a tumor infiltrating lymphocyte (TIL) cell, which is a cell cultured by the method as described in the first aspect of the present application.

[0068] In another preferred embodiment, the number of CD4+ cells in the TIL cell is ≥40%, preferably ≥45%, more preferably ≥50% of the number of CD3+ cells.

[0069] In another preferred embodiment, the TILs are further expanded in culture. CM The proportion of cells that are CD4+ cells is > 60%, preferably > 65%, more preferably > 70%.

[0070] In another preferred embodiment, the TIL cells are further expanded in culture.

[0071] In a sixth aspect of the present application, there is provided a pharmaceutical composition comprising:

[0072] (i) the TIL cells as described in the fifth aspect of the present application; and

[0073] (ii) a pharmaceutically acceptable carrier.

[0074] In a ninth aspect of the present application, there is provided the use of the TIL cells as described in the fifth aspect of the present application in the manufacture of a medicament for treating a tumor.

[0075] In another preferred embodiment, the tumor is a solid tumor, a hematological tumor, preferably a solid tumor.

[0076] In another preferred embodiment, the tumor comprises a melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a lung cancer, a bladder cancer, a head and neck cancer, a pancreatic cancer, a liver cancer, a gastric cancer, a colorectal cancer, a kidney cancer, a sarcoma, or a combination thereof.

[0077] In an eighth aspect of the present application, there is provided a method of treating a tumor, comprising administering to a subject in need thereof the TIL cells as described in the fifth aspect of the present application or the pharmaceutical composition as described in the sixth aspect of the present application.

[0078] In another preferred embodiment, the subject comprises a human, a non-human mammal.

[0079] It should be understood that, within the scope of the present application, all combinations of the above-described technical features of the present application and the technical features specifically described hereinafter (e.g., in the examples) can be interchanged, so as to constitute new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 shows the change in the number of TIL cells during the initial culture stage.

[0081] Figure 2 shows the cell phenotype of TIL cells after the initial culture.

[0082] Figure 3 shows the memory T cell phenotype of TIL cells after the initial culture.

[0083] Figure 4 shows the change in the number of TIL cells during the expansion culture stage.

[0084] Figure 5 shows the cell phenotype of TIL cells after expansion.

[0085] Figure 6 shows the memory T cell phenotype of TIL cells after expansion.

[0086] Figure 7 shows the lesion situation of melanoma patients at baseline, with the left panel showing target lesion 1 and the right panel showing target lesion 2.

[0087] Figure 8 shows the lesion situation of melanoma patients 84 days after reinfusion of TIL cells prepared by the method of the present application, with the left panel showing target lesion 1 and the right panel showing target lesion 2. DETAILED DESCRIPTION

[0088] After extensive and in-depth research, the present inventors have provided a TIL cell culture method, in which three stimulatory factors, IL-2, anti-CD3 antibody and anti-4-1BB antibody, are added in the primary culture stage, which can achieve efficient activation and expansion of TIL cells, and the amount of IL-2 used in the culture process is controlled at a very low level, which can avoid the side effects caused by excessive use of IL-2. In addition, the culture method of the present application shortens the total preparation time of TIL cells, and can achieve a large amount of expansion of TIL cells in a short time, greatly reducing the demand for tumor tissue, with at least 1.0 x 10 7 TIL primary culture cells, and the primary culture cells can be expanded by up to 1000 times after the expansion stage. The culture method of the present application is efficient, controllable and simplified, and can meet the requirements of TIL cell preparation in clinical or industrial production.

[0089] On this basis, the present application is completed.

[0090] DEFINITIONS

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0092] As used herein, the term "tumor infiltrating lymphocytes" or "TIL cells" refers to a class of immune cells present in the tumor microenvironment. TIL cells have significant advantages in the field of cancer treatment. First, they can be directly extracted from the patient's body, and after in vitro expansion and activation, they can be reinfused into the patient's body, thereby achieving individualized treatment. Second, since TIL cells are derived from the patient's own body, there is no risk of allogeneic rejection, and the safety is higher. In addition, TIL cell therapy has shown significant efficacy in clinical trials for some refractory tumors such as melanoma.

[0093] As used herein, the term "initial TIL cell population" or "initial cell population", which can also be referred to as "seed cells", refers to TIL cells before the start of TIL cell culture, which are usually located in tumor tissue mass of a patient. In the culture of TIL cells, it is necessary to first obtain tumor tissue from the patient, for example, by surgery, puncture, etc. The tumor is preferably a solid tumor.

[0094] As used herein, the term "priming", also referred to as "activation culture" or "pre-REP", refers to the first stage of culture of initial TIL cells in tumor tissue to obtain activated TIL cells with expansion potential. In the present application, the culture medium used in the priming stage is referred to as "priming complete medium" or "first culture medium", and the TIL cell population obtained after the completion of priming is referred to as "TIL priming cell population" or "first TIL cell population".

[0095] As used herein, the term "activating factor" refers to a combination of cytokines and antibodies added to the culture medium in the priming stage, which is used to activate the expansion ability of TIL cells. In the present application, a combination of IL-2, anti-CD3 antibody, and anti-4-1BB antibody is used as activating factors.

[0096] As used herein, the term "antibody" refers to an immunoglobulin, which is a four-chain structure of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds. In the present application, the term "antibody" also includes active fragments and derivatives of antibodies, such as single-chain antibodies, single-domain antibodies, Fab, etc., as long as they retain the activity of antibodies.

[0097] In the present application, the antibody can include, but is not limited to, polyclonal or monoclonal; and / or antibodies of humanized, animal (e.g., mouse, rabbit, etc.) origin, or chimeric antibodies (e.g., mouse-human chimeric antibodies). In addition, the antibodies of the present disclosure can include monospecific antibodies and multispecific antibodies, such as bispecific antibodies and trispecific antibodies, but are not limited thereto.

[0098] In some embodiments of the present application, the "anti-CD3 antibody" refers to an anti-CD3 monoclonal antibody, which is not limited in origin, for example, it can be self-developed or commercially available. In a preferred embodiment, the anti-CD3 monoclonal antibody is a commercially available antibody.

[0099] In some embodiments of the present application, the "anti-4-1BB antibody" refers to an anti-4-1BB monoclonal antibody, which is not limited in origin, for example, it can be self-developed or commercially available. In a preferred embodiment, the anti-4-1BB monoclonal antibody is a commercially available antibody. In a preferred embodiment, the anti-4-1BB monoclonal antibody is a humanized monoclonal antibody.

[0100] As used herein, the term "IL-2" or "interleukin 2" is a cytokine that plays an important role in regulating immune responses, promoting cell proliferation and differentiation, etc. The IL-2 in the present application includes all forms of IL-2, such as IL-2 derived from humans or other mammals, and includes IL-2 wild type and mutant, as long as the mutant has similar activity to the wild type. In the present application, IL-2 includes recombinant human IL-2.

[0101] As used herein, the terms "expansion", "rapid expansion culture", "REP" are used interchangeably to refer to the expansion culture of the cells after the priming culture, for verifying the rapid expansion ability of the TIL cells in a short time in the priming stage.

[0102] In the present application, the method of the expansion stage is not limited, and the conventional expansion method known in the art can be selected to rapidly expand the cells after the priming culture.

[0103] In a preferred embodiment, the culture medium used in the expansion stage is a complete culture medium, to which IL-2 and feeder cells are added.

[0104] As used herein, the term "CD3+ cells" refers to CD3 positive cells in TIL cells, which represent mature T cells and thymocytes infiltrated in tumors, including helper T cells, regulatory T cells and killer T cells, etc.

[0105] As used herein, the term "CD8+ cells" refers to CD8 positive cells in TIL cells, which represent cytotoxic T cells or cytotoxic CD8+ T lymphocytes (CTL), which are key components of the adaptive immune system and play an important role in the immune system against pathogens.

[0106] As used herein, the term "CD4+ cells" refers to CD4 positive T lymphocytes in TIL cells, which represent helper T cells, including Th-1, Th-2, Th-9, Th-17, regulatory T cells and follicular helper T cell subtypes, etc.

[0107] As used herein, the term "CD352+ cells" refers to CD352 positive cells in TIL cells, including natural killer cells, T cells and B cells, which can trigger the cytotoxicity of natural killer cells with high expression density of cell surface natural cytotoxicity receptors.

[0108] As used herein, the term "memory T cells" refers to a type of cells that have the ability of long-term survival and rapid response to the same antigen again after receiving antigen stimulation, which are formed from initial cells and can be divided into effector memory T cells (T EM ), central memory T cells (T CM ), stem cell memory T cells (TSCM ) etc.

[0109] TIL cell culture method of the present application

[0110] In the present application, a TIL cell culture protocol is provided, especially the method of the initial culture stage is optimized.

[0111] In the method of the present application, a very small amount of tumor tissue can meet the needs of subsequent culture, for example, 0.02-1g, preferably 0.05-1g, more preferably 0.1-1g of tumor tissue. The tumor tissue can be obtained from the patient by puncture or other methods. Before the culture starts, the tumor tissue can be treated by washing, soaking, etc. according to the situation.

[0112] In the present application, "complete medium" refers to a medium suitable for TIL cell culture, which includes a cell basal medium, and optionally added additives suitable or conducive to TIL cell culture. The additives include buffers, serum or serum substitutes, antibiotics, etc. In the present application, the composition of the additives is not limited as long as the additives are conducive to the growth or culture of TIL cells. In the present application, the medium or the components in the medium can be commercially available.

[0113] In a preferred embodiment, the complete medium comprises the following components: 2.5% V / V of Hepes buffer, 1%-10% V / V of serum substitute, 1% V / V of GlutaMax, 0.1% V / V of gentamicin, and RPMI 1640 medium.

[0114] In the present application, "initial culture complete medium", also referred to as "first medium", refers to a medium in which the complete medium is added with the combination of stimulating factors of the present application, including: IL-2, anti-CD3 antibody and anti-4-1BB antibody.

[0115] The concentration of IL-2 is lower than the amount used in the conventional method, which is 500-3000 IU / ml, preferably 800-2500 IU / ml, more preferably 1000-2000 IU / ml.

[0116] In a preferred embodiment, the concentration of the anti-CD3 antibody in the first medium is 10-100 ng / ml, preferably 20-60 ng / ml, more preferably 25-50 ng / ml.

[0117] In a preferred embodiment, the concentration of the anti-4-1BB antibody in the first medium is 1-50 μg / ml, preferably 2-30 μg / ml, more preferably 3-10 μg / ml.

[0118] In a preferred embodiment of the present application, the initial culture stage lasts for about 7-14 days. The culture can be performed in a cell culture vessel, preferably under culture conditions of 37°C, 5% CO2. The cell culture vessel is not limited, for example, it can be a gas-permeable culture bag or a culture flask.

[0119] In the initial culture stage, when ≥1×10 7 TIL cells, for example, ≥5×10 7 TIL cells, or ≥1×10 8 TIL cells are obtained, the initial culture stage ends.

[0120] For the first TIL cells harvested after the end of the initial culture, an appropriate amount of cells is taken for expansion culture. The expansion culture uses a second culture medium, the composition of which includes complete culture medium identical to that of the initial culture stage, and the addition of IL-2 at a conventional concentration of 1000 IU / mL, and the second culture medium does not add anti-CD3 antibody and anti-4-1BB antibody.

[0121] In a preferred embodiment, the expansion culture stage lasts for 7 days, and 2.0×10 5 cells taken from the first TIL cell population are expanded to harvest about 2.0×10 8 TIL cells after expansion culture, and the expansion culture stage has an expansion ratio of about 1000 times. In addition, the TIL cells after expansion culture are tested for viability, phenotype, etc., and the results show that the TIL cells obtained by the method of the present application have high viability and excellent cell phenotype.

[0122] Applications

[0123] The TIL cell culture method of the present application can obtain a large number of TIL cells in a short time, and reduces the amount of cytokines used, and is suitable for preparing the required TIL cells in clinical or industrial production for TIL cell therapy.

[0124] The core idea of TIL therapy is to separate TIL from tumor tissue, expand them in vitro, and then reinfuse them into the patient's body, which has the advantages of strong specificity and individualization, can treat based on the patient's own immune cells, and reduces the risk of rejection. The method of the present application shortens the cycle of TIL cell expansion in vitro, which can reduce the waiting time for treatment of patients with advanced cancer and improve survival rate. In addition, the TIL cell culture method of the present application has a high expansion rate, so it greatly reduces the demand for tumor tissue, and can realize the preparation of TIL cell products from tumor tissue puncture.

[0125] The main advantages of the present application include:

[0126] 1. The TIL cell culture method of the present application reduces the amount of IL-2 used, produces IL-2-dependent low TIL cells, and reduces side effects caused by excessive IL-2 concentration.

[0127] 2. The TIL cell culture method of the present application reduces the amount of tumor tissue required, and at least 1.0 x 10 7 primary culture cells can be produced from 0.02 g of tumor tissue, and tumor tissue puncture can be used to prepare TIL cells.

[0128] 3. The TIL cell culture method of the present application produces primary culture cells with strong amplification capacity, and an amplification fold of more than 1000 can be achieved after 7 days of expansion culture.

[0129] 4. In the TIL cell culture process of the present application, the use of a combination of three activators reduces process costs, reduces the detection of other factor residues, and more greatly reduces the physical discomfort response caused by the reinfusion of TIL preparations in patients.

[0130] 5. The TIL cell culture method of the present application can achieve rapid and large-scale expansion of TIL cells, greatly shortening the total preparation time, completing the primary culture stage in 7-14 days, reducing the waiting time for treatment of patients with advanced cancer, and improving survival rate.

[0131] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0132] Example 1: Primary culture of TIL cells

[0133] (I) Preparation of reagents

[0134] 1. Preparation of tissue preservation solution: 5% V / V human blood albumin, 5 mg / ml gentamicin, and 2.5 μg / ml amphotericin B were added to RPMI 1640 medium in sequence and mixed.

[0135] 2. Preparation of tissue washing solution: 5% V / V human blood albumin, 5 mg / ml gentamicin, and 2.5 μg / ml amphotericin B were added to 0.9% sodium chloride injection in sequence and mixed.

[0136] 3. Preparation of complete medium: Take 2.5% V / V of Hepes, 1%-10% V / V of serum replacement, 1% V / V of GlutaMax, and 0.1% V / V of gentamicin (concentration of 10 μg / ml) in turn and add them to RPMI 1640 medium, and mix them gently. Filter the prepared complete medium to remove bacteria.

[0137] 4. Sources of other reagents:

[0138] Anti-CD3 monoclonal antibody: Beijing Tongli Haiyuan Biotechnology Co., Ltd., item number: TL-101;

[0139] Anti-4-1BB humanized monoclonal antibody: Tai Zehuikang Biomedicine Co., Ltd., batch number: KR008F20210601;

[0140] Human interleukin-2 injection (IL-2): Beijing Sihuan Biopharmaceutical Co., Ltd., item number: S20040008.

[0141] (ii) Collection of tumor tissue

[0142] The tumor tissue obtained from a patient with a solid tumor is placed in a tissue preservation solution and transported at 2-8°C. It can be stored in the tissue preservation solution for 72 h.

[0143] (iii) Tumor tissue processing

[0144] 1. Discard the tissue preservation solution, and add an appropriate amount of tissue rinse solution to rinse the tumor tissue.

[0145] 2. Transfer the tissue to a 50 ml centrifuge tube, wash the tissue with tissue rinse solution and soak for 1-5 min.

[0146] 3. Transfer the tissue to a 10 cm 2 culture dish, remove the surface blood stains and non-usable tissue, and rinse the tumor tissue with tissue rinse solution.

[0147] 4. Transfer the tissue to a 10 cm 2 culture dish, add tissue rinse solution to wash and soak for 1-5 min, a total of 1-5 times.

[0148] 5. After washing, cut the sample tissue into 1-3 mm 3 size tissue pieces.

[0149] (iv) Primary culture

[0150] Take 0.02-0.1 g of tumor tissue and transfer it to a gas-permeable culture bag or culture bottle, and add 100 mL of complete medium.

[0151] In the experimental group-1, 30 ng / ml of anti-CD3 monoclonal antibody, 5 μg / ml of anti-4-1BB humanized monoclonal antibody, and human interleukin-2 injection (IL-2) with a concentration of 2000 IU / ml were added.

[0152] In the experimental group-2, 30 ng / ml of anti-CD3 monoclonal antibody, 5 μg / ml of anti-4-1BB humanized monoclonal antibody, and human interleukin-2 injection (IL-2) with a concentration of 1000 IU / ml were added.

[0153] In the control group, only human interleukin-2 injection (IL-2) was added with a concentration of 6000 IU / ml.

[0154] The gas-permeable culture bag or culture bottle was placed in a carbon dioxide incubator (37℃, 5% CO2) for culture, and the culture was performed for 7-14 days.

[0155] Example 2: Detection of primary cultured cells

[0156] The primary cultured cells were taken for cell counting and cell phenotype detection. The specific operation was as follows:

[0157] The experimental group TIL cells and the control group TIL cells of Example 1, which were primary cultured for 7 days, 9 days, 11 days, and 14 days, were taken, 10 mL pipettes were used to blow and suck to mix the cell suspensions in each well, a cell counter was used to count the cells, and the viable cell density, volume, cell viability, and average diameter were recorded. The TIL cell growth fold was calculated, according to the counting results, 1×10 6 The cells were labeled using fluorescent labeled antibodies, and a flow cytometer was used to monitor the flow cytometric phenotype of the TIL cells obtained by the primary culture method of Example 1.

[0158] The cell data of the TIL cells from lung cancer patients after primary culture is shown in Table 1 and FIG. 1:

[0159] Table 1: Number and viability of primary cultured cells

[0160] The flow cytometric results of the cells after primary culture are shown in Table 2, Table 3, FIG. 2, and FIG. 3:

[0161] Table 2: Phenotype of primary cultured cells

[0162] Table 3: Proportion of memory T cell subtypes in primary cultured cells

[0163] The above results show that the TIL cells obtained by the culture method of the present application (experimental-1 and experimental-2) have better performance in cell proliferation ability and cell viability than the control group;

[0164] In terms of cell phenotype, the experimental group has a higher CD3+ ratio, and the two experimental groups have a higher CD3+ ratio than the control group, indicating that the experimental group has a significant advantage in TIL cell expansion and activity.

[0165] In terms of memory T cell subtypes, the CD4+ T CM ratio in the experimental group increased significantly, and these cells are usually long-lived and can rapidly expand and differentiate when encountering the same antigen again, showing stronger stemness, indicating that the culture conditions of the experimental group can maintain or improve the stemness of memory T cells.

[0166] According to the comparison of three groups of culture experiments, it is found that when the concentration of human interleukin-2 injection (IL-2) is 1000 or 2000 IU / ml, and 30 ng / ml of CD3 monoclonal antibody and 5 μg / ml of anti-4-1BB humanized monoclonal antibody are added, TIL cells have obvious expansion, and the cell state is relatively better, which can meet the process requirement of ≥1.0×10 7 after 7 days of initial culture.

[0167] In two experimental groups, there is no significant difference in culture data and flow cytometry phenotype between the two IL-2 concentrations. In order to reduce the toxicity and dependence of IL-2, the optimal IL-2 concentration is 1000 IU / ml.

[0168] Example 3: Expansion of initial culture cells (REP)

[0169] The cells of the control group and the experimental group-2 obtained after 14 days of initial culture in Example 1 were subjected to expansion experiment to detect the performance of the initial culture cells in expansion, and the specific steps are as follows:

[0170] (1) The complete culture medium was placed in the carbon dioxide incubator for 30 min for rewarming.

[0171] (2) The experimental grouping is shown in Table 4:

[0172] Table 4 Expansion experiment grouping

[0173] (3) Thawing of feeder cells: After taking the frozen feeder cells from the liquid nitrogen tank, quickly put them into the preheated 37℃ water bath for recovery. When there is only a small amount of ice nucleus left in the feeder cell tube, take it out, add a small amount of complete culture medium to the centrifuge tube, and suck the thawed feeder cell suspension into the centrifuge tube, centrifuge at 400g for 5 min. Discard the supernatant, resuspend the feeder cells with a small amount of complete culture medium, take a sample for counting, and record the viable cell density, volume and cell viability.

[0174] (4) According to the grouping in Table 4, add 2.0×10 6 cells per well, mix well, and place in a carbon dioxide incubator for culture.

[0175] (5) Expansion-Day 2 treatment: 4 ml of culture supernatant was discarded using a pipette, and the remaining cell suspension in the two wells of the culture flask was mixed by blowing and sucking. The sample was counted, and the viable cell density, volume, cell viability, and average diameter were recorded. The TIL cell growth multiple was calculated. The initial culture complete medium was added to a final volume of 8 mL, and IL-2 was added according to the volume of the added complete medium in the two wells.

[0176] (6) Expansion-Day 4 treatment: 4 ml of culture supernatant was discarded using a pipette, and the remaining cell suspension in the two wells of the culture flask was mixed by blowing and sucking. The sample was counted, and the viable cell density, volume, cell viability, and average diameter were recorded. The TIL cell growth multiple was calculated. The initial culture complete medium was added to a final volume of 8 mL, and IL-2 was added according to the volume of the added complete medium in the two wells.

[0177] (7) Expansion-Day 7 collection: The cell suspension in the culture flask was mixed by blowing and sucking using a 10 mL pipette, and the sample was counted. The viable cell density, volume, cell viability, and average diameter were recorded, and the TIL cell growth multiple was calculated.

[0178] Example 4: Detection of expanded cells

[0179] The TIL cells on the 7th day of expansion in Example 3 were taken, and the cell suspensions in the two wells of the culture flask were mixed by blowing and sucking using a 10 mL pipette. Cell counting was performed using a cell counter, and the viable cell density, volume, cell viability, and average diameter were recorded. The TIL cell growth multiple was calculated, and according to the counting results, 1 × 10 6 The cells were labeled using fluorescently labeled antibodies, and the flow cytometric phenotype of the TIL cells obtained by the culture method of Example 1 was monitored using a flow cytometer.

[0180] 2.0 × 10 5 After 7 days of expansion, the number and viability of the cells were as shown in Table 5 and FIG. 4:

[0181] Table 5: Number and viability of expanded cells

[0182] According to the data calculated from Table 5, about 4 × 10 8 After 7 days of expansion, about 4.6 × 10 11 TIL cells were obtained.

[0183] The flow cytometric results of the expanded cells are shown in Tables 6, 7, and FIGS. 5, 6:

[0184] Table 6: Phenotype of expanded cells

[0185] Table 7 Proportion of subtypes of expanded memory T cells

[0186] The results show that the cells cultured by the primary culture method of the application have excellent expansion performance in the expansion stage, and can reach about 1000-fold expansion after only 7 days of expansion. In addition, in the phenotype of memory T cells, the proportion of CD8+T CM and CD4+T CM in the experimental-2 group is significantly higher than that in the control group, indicating that the TILs cultured by the method of the application have stronger stemness.

[0187] Example 5: Clinical treatment effect of TILs prepared by the method of the application

[0188] In this example, the effect of TIL cells prepared by the method of Example 1 and Example 3 of the application for clinical trials was verified, and the TIL cell reinfusion scheme is shown in the following table:

[0189] Table 8 TIL cell reinfusion scheme

[0190] The clinical effect of the patient after reinfusion is as follows:

[0191] The lesion condition of the patient at the baseline stage is shown in Figure 7: target lesion 1: 30.27 mm (right thigh metastasis), target lesion 2: 20.23 mm (right thigh metastasis), and non-target lesion: right thigh metastasis (present).

[0192] The lesion condition of the patient 84 days after reinfusion is shown in Figure 8: target lesion 1: 29.57 mm (right thigh metastasis), target lesion 2: 0 mm (right thigh metastasis), and non-target lesion: disappeared.

[0193] According to the imaging report results of this patient, the comprehensive efficacy evaluation is PR, and the sum of the diameters of the target lesions of the patient is reduced by 41%, and the non-target lesion disappears.

[0194] For another 7 patients with advanced melanoma, the same scheme was used for reinfusion treatment. Specifically, the TIL cells were autologous TIL cells derived from the patient, which were primary cultured by the method of Example 1, and expanded by the expansion method of experimental group-2 in Example 3, and then reinfused into the patient according to the TIL reinfusion scheme shown in Table 8, and the disease progression of the patient was monitored for a long time. The statistical results of the clinical trials of a total of 8 patients with melanoma are shown in Table 9.

[0195] Table 9 Clinical trial results of patients with advanced melanoma

[0196] Five of the subjects (62.5%) were male, with a median age of 59.5 years (47-67). Two subjects achieved complete remission (CR), and two subjects achieved partial remission (PR). The objective remission rate (ORR) was 50%, and the disease control rate (DCR) was 100%.

[0197] In addition, due to the significant reduction in the dosage of IL-2 used in the reinfusion regimen, the side effects caused by high-dose IL-2 were reduced. The clinical results showed that no serious clinical side effects occurred in 8 patients, and the most common adverse reactions (TEAEs) during treatment were mainly hematological toxicity and fever, and most of the TEAEs recovered within 2 weeks, and no grade 4 or above TEAEs occurred, which reflects a high level of safety.

[0198] The above results show that after the TIL cells initially cultured by the method described in Example 1 are reinfused into patients, they can effectively and durably kill tumor cells, and exhibit excellent tumor inhibition ability. In addition, the TIL cells of the present application exhibit a high level of safety in clinical trials.

[0199] Example 6: Initial culture of TIL cells from different cancer types

[0200] Using the same method as in Example 1, the TIL cell samples from melanoma (ME), lung cancer (LA), breast cancer (MB), cervical cancer (CC), head and neck cancer (HN), and ovarian cancer (OC) patients were initially cultured. During the initial culture, the cell phenotype was detected using the same method as in Example 2, where D represents days. The results are shown in Table 10 below:

[0201] Table 10: Initial culture results of different cancer types

[0202] The results show that, consistent with the results of Example 2, the TIL cells from melanoma, lung cancer, breast cancer, cervical cancer, head and neck cancer, and ovarian cancer have a high proportion of CD4+ cells to CD3+ cells after being initially cultured by the method of Example 1, with the lowest being more than 40%.

[0203] Discussion

[0204] I. Effectiveness of TIL therapy

[0205] TILs have shown good therapeutic effects in different cancer types as an effective adoptive immunotherapy, and the advantage of TIL therapy is mainly used in the treatment of solid tumors. Peter Sidaway reported that TIL infusion is an effective adoptive immunotherapy for HPV-associated cancers. Infusion of tumor-infiltrating lymphocytes (TILs) expanded ex vivo is an attractive anticancer treatment because it can achieve high levels of antitumor specificity. Inozume pointed out that TILs also have a good objective response rate in melanoma. Similarly, according to the literature of Rohaan, the progression-free survival of patients treated with TILs was significantly longer than that of patients treated with ipilimumab in patients with advanced melanoma. In the summary of lung cancer treatment methods by Lahiri et al., it is pointed out that TIL therapy is also a powerful means of treating lung cancer. Veatch et al. reported that TIL therapy has clinical benefits for ICB-resistant NSCLC patients and induces durable complete responses in some patients. Stanton et al. showed that tumor-infiltrating lymphocytes (TILs) play a crucial role in mediating chemotherapy response and improving clinical outcomes in all breast cancer subtypes. Triple-negative breast cancer (TN) is most likely to have tumors with >50% lymphocyte infiltration, referred to as lymphocyte-dominant breast cancer, and the largest survival benefit is obtained from a 10% increase in TILs.

[0206] II. Difficulties faced by TIL therapy

[0207] Since the cell product is specially manufactured for each patient, the cost of this treatment method is high for each manufacturer, and the growth cycle is long. The total production time of a TIL treatment may require up to 3 weeks of highly personalized, labor-intensive production, and the production personnel require highly trained and experienced technical personnel.

[0208] III. Comparison of existing TIL expansion protocols

[0209] The process of generating TILs usually starts with a pre-REP phase, in which TILs are dissociated or migrated from tumor fragments and undergo initial expansion. Then the TILs are further expanded in the REP phase in response to stimulators, such as IL-2 and / or feeder cells. The traditional TIL production procedure is specific for tumor recognition and usually takes 6-8 weeks. The rapid expansion of TIL cells in the prior art also usually takes 27 days. However, the method of the present application can obtain at least 1x10 7 primary culture cells in 7 days, and then expand the cells by more than 1000 times in 7 days, greatly shortening the culture time of TIL cells and achieving high-multiple expansion in a short time, which is beneficial to clinical application and industrial production.

[0210] IV. Influence of different TIL phenotypes

[0211] Adoptive cell therapy using tumor infiltrating lymphocytes (TIL) can mediate objective and durable tumor regression in patients with metastatic melanoma. Numerous studies have demonstrated that CD8+ tumor reactive TIL can acquire the ability to kill tumor cells directly, and in recent years, it has been found that tumor infiltrating CD4+ T cells can promote immune function and can kill tumor cells directly.

[0212] In addition, the proportion of memory T cells characterizes the stemness of TILs. The proportion of various memory T cell populations of CD8+ and CD4+ of the TILs of the present application is batched, and the present application continuously cultures TILs in batches. A large amount of flow data shows that the proportion of CD8+ TCM and CD4+ TCM of the TILs in the tumor of the present application after in vitro expansion is relatively high, and the stability between batches is good, which proves that the present application exhibits a high proportion of central memory T cell phenotype for different solid tumors (including melanoma, lung cancer, breast cancer, cervical cancer, head and neck tumors, etc.).

[0213] V. IL-2 dosage

[0214] Giving a large dose of IL-2 as a standard treatment to support the growth and activity of TIL infusion can cause serious clinical side effects, but based on the use of a large dose of IL-2 for culturing TIL cells in the past, resulting in the dependence of TIL cells on high-dose IL-2, most TILs still use high-dose IL-2 infusion after clinical reinfusion. The present application greatly reduces the amount of IL-2 used in the process of culturing TILs, which creates conditions for reducing the use of IL-2 after clinical TIL reinfusion. Clinical data show that the TIL produced by the present application can expand and function in the patient's body after reinfusion, and low-dose IL-2 (about 1 / 20 of the dose of IL-2 used in the traditional TIL treatment process) can be given.

[0215] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teachings of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A method of culturing tumor infiltrating lymphocytes (TILs), characterized by, The method comprises the steps of: A. providing a tumor tissue containing an initial TIL cell population; B. placing the initial TIL cell population in a first culture medium for primary culture to obtain a TIL primary culture cell population; wherein the first culture medium comprises a combination of stimulating factors: IL-2, anti-CD3 antibody and anti-4-1BB antibody.

2. The method of claim 1, wherein, In the first culture medium, the concentration of IL-2 is 500-3000 IU / ml.

3. The method of claim 1, wherein, In the first culture medium, the concentration of IL-2 is 800-2500 IU / ml.

4. The method of claim 1, wherein, In the first culture medium, the concentration of IL-2 is 1000-2000 IU / ml.

5. The method of claim 1, wherein, In the first culture medium, the concentration of anti-CD3 antibody is 10-100 ng / ml.

6. The method of claim 1, wherein, In the first culture medium, the concentration of anti-4-1BB antibody is 1-50 μg / ml.

7. The method of claim 1, wherein, The components of the first culture medium further include cell basal medium, buffer, serum substitute, L-glutamine or its substitute, and antibiotics.

8. The method of claim 1, wherein, In step B, the time for primary culture is 7-14 days.

9. The method of claim 1, wherein, 0.02-0.1 g of the initial TIL cell population in the tumor tissue, and the number of cells in the obtained TIL primary cell population is 1 x 10 7 ~1 x 10 9 ~1 x 10 10. A TIL cell culture medium, characterized in that, The culture medium comprises a combination of stimulating factors: IL-2, anti-CD3 antibody, and anti-4-1BB antibody; wherein the concentration of IL-2 is 500-3000 IU / ml, the concentration of anti-CD3 antibody is 10-100 ng / ml, and the concentration of anti-4-1BB antibody is 1-50 μg / ml.

11. The medium of claim 8, wherein, The components of the culture medium further include cell basal medium, buffer, serum substitute, L-glutamine or its substitute, and antibiotics.

12. A reagent combination, characterized in that The reagent combination consists of (i) IL-2, (ii) anti-CD3 antibody, and (iii) anti-4-1BB antibody; wherein the concentration of IL-2 in the reagent combination is 500-3000 IU / ml, the concentration of anti-CD3 antibody is 10-100 ng / ml, and the concentration of anti-4-1BB antibody is 1-50 μg / ml.

13. A tumor infiltrating lymphocyte (TIL), characterized in that, The TIL cell is a cell cultured by the method of claim 1.

14. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (i) the TIL cell of claim 13; and (ii) a pharmaceutically acceptable carrier.

15. The use of TIL cells according to claim 13, characterized in that, A medicament for treating tumors.

16. A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-15. The method comprises administering the TIL cell of claim 13 or the pharmaceutical composition of claim 14 to a subject in need. The method comprises administering the TIL cell of claim 13 or the pharmaceutical composition of claim 14 to a subject in need.

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