Culture medium for tumor infiltrating lymphocytes and in vitro expansion culture method

Abstract

A culture medium for tumor infiltrating lymphocytes and an in vitro expansion culture method. The culture medium comprises a basal medium and cell growth factors, the cell growth factors comprising a combination of at least two of IL-2, IL-7, IL-15, and TGF-β. The in vitro expansion culture method comprises the steps of culturing tumor infiltrating lymphocytes sequentially under a first culture condition and a second culture condition: a first culture medium that is the aforementioned culture medium is used for the first culture condition; and, relative to the first culture medium, a second culture medium that does not contain TGF-β is used for the second culture condition.

Description

Culture medium and in vitro expansion culture method of tumor infiltrating lymphocytes

[0001] Related applications

[0002] The present application claims priority to the Chinese patent application No.CN202411388989.8, filed on September 30, 2024, entitled "Culture medium and in vitro expansion culture method of tumor infiltrating lymphocytes", the contents of which are hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, in particular to a culture medium and in vitro expansion culture method of tumor infiltrating lymphocytes. BACKGROUND

[0004] Tumor infiltrating lymphocytes (TILs) are a heterogeneous cell population composed of CD8+T cells, CD4+T cells, B cells, NK cells, γδT cells and innate lymphocytes (ILCs). After in vitro expansion culture, CD3+T cells become the most important cell population (usually more than 95% of the proportion). Among them, CD3+T cells express TCR receptors on the surface, which plays a key role in mediating immune killing of cancer cells. Different TCR receptors recognize different antigen peptides presented by major histocompatibility complex (MHC) molecules, thereby conferring antigen specificity to T cells. Therefore, once antigen-specific T cells are stimulated by a specific antigen, these cells can promote the programmed death of target cells through the release of cytotoxins and receptor-mediated mechanisms. Unlike chimeric antigen T cells (CAR-T, Chimeric Antigen Receptor T-Cell) / TCR (T cell receptor)-T cell therapy products, TILs usually recognize tumor-associated antigens such as neoantigens presented by tumor cells, thereby exerting an anti-tumor effect. Therefore, TIL therapy can recognize and target multiple tumor-associated antigens to achieve the purpose of anti-tumor therapy.

[0005] In the process of T cell killing tumor cells, central memory T cells (TCM) play a key role in the sustained anti-tumor effect. TCM has the following characteristics: 1. homing: TCM homing to bone marrow or lymph nodes through high expression of CCR7; among them, TCM is more likely to accept tumor-related antigens presented by antigen-presenting cells in lymph nodes, and part of TCM activation / proliferation obtains a large number of effector T cells, which migrate to tumor tissues and exert anti-tumor effect. 2. memory: after being stimulated by antigens, part of T cells differentiate into TCM, retain memory to tumor antigens, and when this part of T cells is reinfused into the body and activated by antigens again, they can proliferate and activate more quickly to achieve the killing effect on tumor cells. 3. persistent expansion: TCM can proliferate and differentiate rapidly in vivo / in vitro. At the same time, TCM has a certain self-renewal ability, and can survive in the bone marrow / lymph node microenvironment for a long time after being reinfused into the body, and will proliferate and differentiate to exert anti-tumor effect once stimulated by tumor-related antigens again. The study of Christopher A.K. et al. (2005, PNAS, PMID: 15980149) pointed out that, compared with CD8 + TCM and CD8 + The anti-tumor effect of effector T cells (TEM, effector memory T cell) is more significant in the TCM group than in the TEM group after reinfusion into tumor-bearing mice, which shows that CD8 + TCM plays a key role in anti-tumor therapy.

[0006] At present, the CD8 + Most of the related technologies for inducing TCM generation are to add memory T cell generation related cytokines, such as IL-7 / IL-15, to establish TCM preparation technology. For example, a T cell preparation technology for improving the proportion of central memory T cells discloses that after sorting and enriching T cells, adding IL-2, IL-7, IL-15 and other cytokines in serum-free medium containing human platelet lysate can induce the up-regulation of TCM proportion. Among them, CD4 + T cell and CD8 + The proportion of TCM is significantly up-regulated by 20% to 30%.

[0007] The in vitro expansion cycle of the traditional TIL culture method is relatively long (4-5 weeks), and high concentrations of IL-2 and T cell stemness-related cytokines (such as IL-7 and IL-15) are added to the system to maintain the expansion rate and stemness of T cells. However, the proportion of memory phenotype T cells in the TIL end product cells obtained from the above culture system is still low, resulting in weak tumor cell killing function of TIL. On the other hand, the traditional TIL end product cells have low memory phenotype T cell content, resulting in short in vivo survival time, weak expansion ability, and dependence on high-dose IL-2 injection for tumor cell killing, and high-dose TIL reinfusion is required to observe the anti-tumor effect.

[0008] SUMMARY

[0009] Therefore, according to various embodiments of the present application, a culture medium for tumor infiltrating lymphocytes and a method for in vitro expansion culture are provided.

[0010] The technical solutions include the following:

[0011] A culture medium for tumor infiltrating lymphocytes, the culture medium comprising a basic culture medium and cell growth factors, the cell growth factors comprising a combination of at least two of IL-2, IL-7, IL-15, and TGF-β.

[0012] In one embodiment, the TGF-β comprises one or more of TGF-β1, TGF-β2, and TGF-β3.

[0013] In one embodiment, in the culture medium, the concentration of TGF-β is 0.5 ng / mL to 10 ng / mL, the concentration of IL-2 is 100 IU / mL to 6000 IU / mL, the concentration of IL-7 is 5 ng / mL to 20 ng / mL, and the concentration of IL-15 is 1 ng / mL to 10 ng / mL.

[0014] In one embodiment, in the culture medium, the concentration of TGF-β is 0.5 ng / mL to 3 ng / mL, the concentration of IL-2 is 600 IU / mL to 1000 IU / mL, the concentration of IL-7 is 5 ng / mL to 10 ng / mL, and the concentration of IL-15 is 5 ng / mL to 10 ng / mL.

[0015] In one embodiment, the culture medium further comprises a serum substitute.

[0016] In one embodiment, in the culture medium, the volume fraction of the serum substitute is 4% to 8%.

[0017] In one embodiment, the basal medium comprises one or more of X-VIVO, AIM-V, OptiVitro, DMEM, and RPMI 1640.

[0018] A method of expanding tumor infiltrating lymphocytes in vitro, the method comprising the steps of culturing tumor infiltrating lymphocytes under a first culture condition and a second culture condition in sequence:

[0019] The first culture medium used in the first culture condition is the medium described above.

[0020] The second culture medium used in the second culture condition does not contain TGF-β relative to the first culture medium.

[0021] In one embodiment, the culturing time of the first culture condition is 9-15 days.

[0022] In one embodiment, the culturing time of the second culture condition is 7-15 days.

[0023] In one embodiment, the culturing time of the first culture condition is 10-11 days.

[0024] In one embodiment, the culturing time of the second culture condition is 10-11 days.

[0025] In one embodiment, a T cell activator is added to the culture system on day 0 of culturing under the first culture condition.

[0026] In one embodiment, the T cell activator comprises an anti-CD3 antibody.

[0027] In one embodiment, the anti-CD3 antibody comprises an anti-CD3 monoclonal antibody with clone number OKT3.

[0028] In one embodiment, under the first culture condition, the concentration of TGF-β in the culture system is maintained by adding TGF-β during culturing.

[0029] In one embodiment, under the first culture condition, the concentration of TGF-β in the culture system is maintained by adding TGF-β through liquid supplement, liquid exchange, or directly adding TGF-β during culturing.

[0030] In one embodiment, under the second culture condition, the cell density is adjusted to be between 2x10 5 / mL and 2x10 6 / mL by liquid supplement or liquid exchange during culturing.

[0031] In one embodiment, the tumor infiltrating lymphocytes are cultured in the presence of feeder cells.

[0032] In one embodiment, the feeder cells are peripheral mononuclear cells.

[0033] In one embodiment, the feeder cells are irradiated feeder cells.

[0034] In one embodiment, the ratio of the tumor infiltrating lymphocytes to the feeder cells is 1:50-1:200.

[0035] In one embodiment, the tumor infiltrating lymphocytes are derived from tumor tissues.

[0036] The cells obtained by the method are used in the preparation of a medicament for preventing, alleviating and / or treating a disease.

[0037] In one embodiment, the disease includes a tumor or a cancer.

[0038] The details of one or more embodiments of the application are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the application will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application or in the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the disclosed drawings.

[0040] FIG. 1 is an effect of different TGF-β concentrations on the cell proliferation rate and phenotype of TILs in one embodiment of the present application. In which, A is the expansion rate curve of TILs cultured with different TGF-β concentrations; B is the detection of CD4 + T cell and CD8 + T cell ratio, and CCR7 + CD45RA - Central memory T cell ratio; Note: the curves in the A graph represent, from top to bottom, 0 ng / ml TGF-β group, 1 ng / ml TGF-β group, 3 ng / ml TGF-β group, and 10 ng / ml TGF-β group.

[0041] Figure 2 shows the effect of different IL-2 concentrations on the proliferation rate and phenotype of TIL in one embodiment of the present application. A shows the expansion rate curve of TIL cultured with different IL-2 concentrations; B shows the proportion of CD8 T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-2 concentrations; C shows the proportion of CCR7 CD45RA T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-2 concentrations; and D shows the proportion of central memory T cells (TCM) detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-2 concentrations. + T cell proportion;

[0042] Figure 3 shows the effect of different IL-7 concentrations on the proliferation rate and phenotype of TIL in one embodiment of the present application. A shows the expansion rate curve of TIL cultured with different IL-7 concentrations; B shows the proportion of CD8 T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-7 concentrations; C shows the proportion of CCR7 CD45RA T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-7 concentrations; and D shows the proportion of central memory T cells (TCM) detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-7 concentrations. + T cell proportion; C shows the proportion of CCR7 CD45RA T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-7 concentrations. + CD45RA - Central memory T cell (TCM) proportion;

[0043] Figure 4 shows the effect of different IL-15 concentrations on the proliferation rate and phenotype of TIL in one embodiment of the present application. A shows the expansion rate curve of TIL cultured with different IL-15 concentrations; B shows the proportion of CD8 T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-15 concentrations; C shows the proportion of CCR7 CD45RA T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-15 concentrations; and D shows the proportion of central memory T cells (TCM) detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-15 concentrations. + T cell proportion; C shows the proportion of CCR7 CD45RA T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with different IL-15 concentrations. + CD45RA - Central memory T cell (TCM) proportion;

[0044] Figure 5 shows the TGF-β expansion memory T cell culture time kinetics analysis in one embodiment of the present application. A shows the expansion rate curve of TIL cultured with or without TGF-β; B shows the proportion of CD8 T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with or without TGF-β; C shows the proportion of CCR7 CD45RA T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with or without TGF-β; and D shows the proportion of central memory T cells (TCM) detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with or without TGF-β. + T cell proportion; C shows the proportion of CCR7 CD45RA T cells detected by flow cytometry at day 3, day 10, and day 21 of TIL cultured with or without TGF-β. + CD45RA - Central memory T cell (TCM) proportion; Batch 1 and Batch 2 are derived from TIL of different liver cancer patients.

[0045] Figure 6A is a graph showing the expansion rate of TILs cultured with different TGF-β addition methods in one embodiment of the present application; wherein, normal culture: only fast expansion without TGF-β addition; TGF-β continuous addition: fast expansion with TGF-β always present in the culture system; TGF-β early addition: fast expansion with TGF-β only present in the culture system for the first 10 days of culture; Figure 6B is a graph showing the proportion of CD4 + T cells and CD8 + T cell ratio, and CCR7 + CD45RA - central memory T cell ratio; wherein, normal culture group: only fast expansion without TGF-β addition; TGF-β continuous addition: fast expansion with TGF-β always present in the culture system; TGF-β early addition: fast expansion with TGF-β only present in the culture system for the first 10 days of culture; Figure 6C is a graph showing the persistent killing effect of TILs on SK-BN105 target cells after 21 days of culture with different TGF-β addition methods in one embodiment of the present application; note: the curves in the figure represent, from top to bottom, the target cell group only, the normal culture group, the TGF-β continuous addition group, and the TGF-β early addition group;

[0046] Figure 7 is a graph showing the cell expansion rate of multiple batches of TILs cultured in the fast expansion system + TGF-β in one embodiment of the present application; note: the curves in the figure represent, from top to bottom, the fast expansion + TGF-β group and the fast expansion group;

[0047] Figure 8 is a graph showing the cell phenotype and killing function verification of multiple batches of TILs cultured in the fast expansion system + TGF-β in one embodiment of the present application; wherein, A is a statistical result of the proportion of central memory T cells (TCM) and terminally differentiated (TD) T cells of TILs cultured from puncture samples of 7 liver cancer patients at the end of culture, detected by flow cytometry using normal expansion and the fast expansion + TGF-β early addition system of the present application (statistics: two-tailed T test); B is a flowchart of the repeated antigen stimulation experiment; C is the expansion fold of TILs in the resting state after each round of antigen stimulation; D is the persistent killing effect of TILs on SK-BN105 target cells after 4 rounds of antigen stimulation and resting, detected by a real-time cell fluorescence imaging system; note: in Figure C, the left column in each round of stimulation represents the fast expansion group, and the right column represents the fast expansion + TGF-β group;

[0048] Figure 9 is an in vitro and in vivo drug efficacy experiment of the rapid expansion system + TGF-β expanded TIL in one embodiment of the present application; wherein A is the sustained killing function of the expanded TIL in the rapid expansion and the rapid expansion + TGF-β groups detected by the real-time cell fluorescence imaging system; B is the flow chart of the in vivo B16F10 experiment; C is the tumor volume growth curve of the in vivo drug efficacy experiment of different TIL reinfusion dose groups (statistics: two-factor variance analysis, Dunnet algorithm correction; ***P<0.001); the curves in figure C represent the rapid expansion group, the solvent control group, and the rapid expansion + TGF-β group from top to bottom, respectively;

[0049] In figures 7-9, the rapid expansion + TGF-β group represents the rapid expansion + TGF-β pre-addition system established by the present application; the rapid expansion group represents the traditional culture system. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0051] 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 the present application belongs. The terminology used in the description of the present application herein only for the purpose of describing specific embodiments of the present application, and is not intended to limit the present application.

[0052] Terminology

[0053] Unless otherwise stated or contradictory, the terms or phrases used herein have the following meanings:

[0054] In the present application, "optionally", "optional", "option" means optional, that is, selected from "have" or "no" two parallel schemes. If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradictory or mutual restrictive relationship, each "optional" is independent. If there is no other description, the present application is described as "optionally includes", "optionally contains" and the like. For example, "optionally includes" means "may include or not include".

[0055] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the selectable numerical values in the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and every numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, the two endpoint integers and every integer between the two endpoints are considered to be directly enumerated. When multiple numerical ranges are provided to describe a characteristic or a property, the numerical ranges can be combined. In other words, unless otherwise specified, numerical ranges disclosed herein are to be understood to include any and all sub-ranges encompassed therein. A "numerical value" in a numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. A "numerical interval" is intended to broadly encompass numerical interval types such as percentage intervals, ratio intervals, and the like.

[0056] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0057] In this application, "one or more" means any one of the listed items or any combination of the listed items, unless otherwise specified. Similarly, "one or more" and the like, when used in a manner other than "one or more of the other items, are to be interpreted to have the same meaning.

[0058] As used in this application, "combinations thereof," "any combination thereof," "any combination manner thereof," and the like include all suitable combinations of any two or more of the listed items.

[0059] In this application, "optionally," "optional," and "optional" mean that the item is optional, i.e., selected from either of the two parallel options "yes" or "no." If there are multiple "optional" in a technical solution, and there is no contradiction or mutual restriction, each "optional" is independent of the other. Unless otherwise specified, "optionally includes," "optionally contains," and the like are described in this application, for example, "optionally includes" means "may include or not include."

[0060] As used in this application, the terms "containing," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members or characteristics. Members or characteristics, such as materials or components, structures, elements, instruments, etc.; non-limiting examples of members or characteristics also include actions, conditions, timing, states, etc.

[0061] In this application, the exemplary descriptions involving "in one embodiment," "in one specific example," and the like can cover, but are not limited to, the following meanings: these options can be combined with other options in a suitable manner to form new technical solutions.

[0062] Transforming growth factor-β (TGF-β) is a superfamily of structure-related multifunctional cytokines, including TGF-β1 / 2 / 3 isoforms, activins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs). It is generally considered a tumor-promoting factor in the tumor microenvironment, inducing epithelial-mesenchymal transition (EMT) and promoting tumor cell invasion and metastasis. Furthermore, TGF-β is a key regulator of T cell responses, playing a crucial role in regulating responses mediated by almost every innate and adaptive immune cell, including dendritic cells, B cells, NK cells, innate lymphocytes, and granulocytes. In advanced tumor stages, most tumor cells secrete TGF-β. Elevated TGF-β levels can block the differentiation of immature T cells into Th1 cells, promote their transformation into Treg subsets, and inhibit the antigen-presenting function of dendritic cells, thereby leading to immune escape by tumor cells. However, TGF-β plays a regulatory role in T cell differentiation and development. Previous studies have indicated that the TGF-β signaling pathway can regulate the expression of IL-7Ra (CD127). Knockout of TGF-βII on T cells significantly downregulates IL-7Ra expression, and the IL-7 signaling axis is crucial for TCM generation. This application found that T cell proliferation is inhibited when TCM is induced by TGF-β.

[0063] Therefore, this application optimizes and establishes a method for in vitro expansion of CD8 based on tumor-infiltrating lymphocytes. + The TCM culture system was developed, and the working concentration and action time of TGF-β in TIL culture were optimized to improve the CD8 content in the final product. + While maintaining the proportion of TCM, the proliferation capacity of T cells is maximized, thus establishing a process culture system for the subsequent clinical translation and treatment of this cell product.

[0064] One embodiment of this application provides a culture medium for culturing tumor-infiltrating lymphocytes, comprising a basal culture medium and cell growth factors, wherein the cell growth factors include at least two combinations of IL-2, IL-7, IL-15 and TGF-β.

[0065] In one specific example, the cell growth factor includes TGF-β, and also includes one or more of IL-2, IL-7 and IL-15.

[0066] In one specific example, the cell growth factors include IL-2, IL-7, IL-15, and TGF-β.

[0067] In one specific example, the concentration of TGF-β in the culture medium is 0.5 ng / mL to 10 ng / mL. In one specific example, the concentration of TGF-β in the culture medium can be, but is not limited to, 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, 7 ng / mL, 7.5 ng / mL, 8 ng / mL, 9.5 ng / mL, 10 ng / mL, or a range between any two of the aforementioned values. Without wishing to be bound by any theory, it is believed that as the working concentration of TGF-β increases, the CD8 + T cells and CD8 + TCM ratio gradually increases, and the expansion rate of TILs significantly decreases. In one specific example, the concentration of TGF-β in the culture medium is 0.5 ng / mL to 3 ng / mL.

[0068] In the conventional in vitro expansion of TILs, a high concentration of IL-2, typically 6000 IU / mL, is used. The addition of IL-2 enhances the proliferative capacity of T cells by activating the JAK-STAT / mTOR / MAPK signaling pathway of T cells. However, the use of a high concentration of IL-2 in vitro to expand TILs can result in the need for high-dose IL-2 concomitant medication after the infusion of the TIL end product into the body, thereby leading to IL-2-related adverse reactions observed in the clinic. In one specific example, the concentration of IL-2 in the culture medium is 100 IU / mL to 6000 IU / mL.

[0069] In one specific example, the concentration of IL-2 in the culture medium can be, but is not limited to, 100 IU / mL, 200 IU / mL, 300 IU / mL, 400 IU / mL, 500 IU / mL, 600 IU / mL, 700 IU / mL, 800 IU / mL, 900 IU / mL, 1000 IU / mL, 1500 IU / mL, 2000 IU / mL, 3000 IU / mL, 4000 IU / mL, 5000 IU / mL, 6000 IU / mL, or a range between any two of the aforementioned values. Without wishing to be bound by any theory, it is believed that in the culture medium of the present application, a lower concentration of IL-2 results in a higher expansion rate of TILs in the system as compared to culturing TILs using a high concentration of IL-2. In one specific example, the concentration of IL-2 in the culture medium is 600 IU / mL to 6000 IU / mL. In one specific example, the concentration of IL-2 in the culture medium is 600 IU / mL to 1000 IU / mL.

[0070] IL-7 is a key cytokine for inducing central memory T cells. Activation of TGF-β signaling pathway can upregulate the expression of CD127 (IL-7Ra) on T cells, enhance the degree of activation of IL-7-IL-7Ra signaling pathway, and thus enhance the ability of IL-7 to induce central memory T cells. However, too high a working concentration of IL-7 can also cause T cells to proliferate excessively, leading to cell exhaustion. In one specific example, the concentration of IL-7 in the culture medium is 5 ng / mL to 20 ng / mL.

[0071] In one specific example, the concentration of IL-7 in the culture medium can be, but is not limited to, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, or a range between any two of the aforementioned values. In one specific example, the concentration of IL-7 in the culture medium is 5 ng / mL to 10 ng / mL.

[0072] The production of central memory T cells is regulated not only by IL-7 through activation, but also by IL-15-IL15Ra signaling pathway. In order to maximize the proliferation rate of T cells and the generation of TCM in the culture system of the present application, the concentration of IL-15 in the culture medium is 1 ng / mL to 10 ng / mL in one specific example.

[0073] In one specific example, the concentration of IL-15 in the culture medium can be, but is not limited to, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, or a range between any two of the aforementioned values. In one specific example, the concentration of IL-15 in the culture system is 5 ng / mL to 10 ng / mL.

[0074] In one specific example, the culture medium further comprises a serum substitute. In one specific example, the volume fraction of the serum substitute in the culture medium is 4% to 8%, which can be, but is not limited to, 4%, 5%, 6%, 7%, 8%, or a range between any two of the aforementioned values.

[0075] In one specific example, the basal medium comprises one or more of X-VIVO, AIM-V, OptiVitro, DMEM, and RPMI 1640.

[0076] One embodiment of the present application provides a method for in vitro expansion of tumor infiltrating lymphocytes, which comprises the steps of culturing the tumor infiltrating lymphocytes under a first culture condition and a second culture condition in sequence; the first culture medium used in the first culture condition comprises the culture medium for culturing tumor infiltrating lymphocytes described above; and the second culture medium used in the second culture condition does not contain TGF-β relative to the first culture medium. The method can improve the CD8 + The proportion of TCM is maximized while the proliferation ability of T cells is maximized, thereby establishing a process culture system for subsequent clinical conversion treatment of the cell product.

[0077] It is found in the present application that the inhibitory effect on the proliferation of TIL is significant after 14 days of adding TGF-β, and the upregulation of TCM is significant after 7 days of adding TGF-β.

[0078] In one specific example, the culture time of the first culture condition is 7 days to 15 days. When the culture time is in the above range, the proliferation effect of TIL is good, and the upregulation of TCM is significant. In one specific example, the culture time of the first culture condition can be selected to be 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or a range between any two of the aforementioned values. In one specific example, the culture time of the first culture condition is 10 days to 11 days.

[0079] In one specific example, the culturing time of the second culturing condition is 7 days to 15 days. In one specific example, the culturing time of the second culturing condition can be selected from 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or a range between any two of the above values. In one specific example, the culturing time of the second culturing condition is 10 days to 11 days.

[0080] In one specific example, the culturing time of the first culturing condition is 10 days, and the culturing time of the second culturing condition is 11 days. Through the first 10 days of culturing, TGF-β is added to the culturing system to induce CD8 + TCM to rise; in the later culturing stage, TGF-β is removed from the culturing system to achieve rapid expansion of TIL, and the maximum proliferative activity of TIL is maintained when the final product cells are harvested.

[0081] In one specific example, the culturing period of the in vitro expansion culture is 16 days to 30 days. In one specific example, the culturing period of the in vitro expansion culture can be 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, or 30 days. It can be understood that in other specific examples, the entire cell culturing period can be extended or shortened by those skilled in the art according to the required number of cells.

[0082] In one specific example, under the first culturing condition, on day 0 of culturing, a T cell activator is added to the culturing system, and the T cell activator includes an anti-CD3 antibody, for example, OKT3. In one specific example, the concentration of the T cell activator in the culturing system is 20 ng / mL to 40 ng / mL, which can be selected from 20 ng / mL, 30 ng / mL, 40 ng / mL, or a range between any two of the above values.

[0083] In one specific example, under the first culturing condition, the concentration of TGF-β in the culturing system is maintained by means of liquid supplementing, liquid changing, or direct addition of TGF-β during the culturing period. Alternatively, the concentration of TGF-β in the culturing system is maintained at, but not limited to, 0.5 ng / mL to 10 ng / mL or 0.5 ng / mL to 3 ng / mL. In one specific example, under the first culturing condition, cell liquid supplementing is performed on day 2 to day 9 of culturing. Alternatively, the number of liquid supplementing is 1 to 3 times. In one specific example, cell liquid supplementing can be performed on day 7 and day 9 of culturing.

[0084] In one specific example, under the second culturing condition, the cell density is adjusted to be between 2 x 10 5 / mL to 2 x 10 6 / mL by means of liquid supplementing or centrifugal liquid changing during the culturing period.

[0085] In one specific example, the method comprises culturing the tumor infiltrating lymphocytes in the presence of feeder cells.

[0086] In one specific example, the feeder cells are peripheral mononuclear cells. In one specific example, the feeder cells are irradiated feeder cells.

[0087] For example, when culturing TILs, the rapid expansion of TILs in vitro is achieved by adding gamma irradiated allogeneic human PBMCs as feeder cells.

[0088] In one specific example, the ratio of tumor infiltrating lymphocytes to feeder cells is 1:50 to 1:200, for example, optionally 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, or a range between any two of the aforementioned values. In one specific example, the ratio of tumor infiltrating lymphocytes to feeder cells is 1:50 to 1:100; in one specific example, the ratio of tumor infiltrating lymphocytes to feeder cells is 1:100 to 1:200.

[0089] In one specific example, the initial seeding density of tumor infiltrating lymphocytes is 5 x 10 3 to 5 x 10 4 per mL; optionally, the initial seeding density of tumor infiltrating lymphocytes is optionally, but not limited to, 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 9 x 10 3 , 1 x 10 4 , 2 x 10 4 , 3 x 10 4 , 4 x 10 4 , 5 x 10 4 , or a range between any two of the aforementioned values.

[0090] In one specific example, the tumor infiltrating lymphocytes are derived from tumor tissue.

[0091] In one specific example, the tumor tissue comprises, but is not limited to, liver cancer tissue. It is understood that in other specific examples, the tumor tissue can also be other tumor tissue, such as stomach cancer tissue, bladder cancer tissue, or breast cancer tissue, etc., and the rapid expansion can also be achieved, which can maximize the proliferation of T cells in the final product cells, while increasing the proportion of CD8 + TCM, for treating tumors.

[0092] The present application optimizes the culture system of traditional TILs, adds TGF-β in the early stage of culture, and removes TGF-β in the later stage of culture, so as to increase the proportion of central memory cells in the final product cells of TILs, thereby significantly enhancing the persistent killing effect of TILs on tumors; the present application can harvest up to 10 11 The above TILs, and at the end of culture, the TILs still maintain an exponential expansion state, to ensure the high-dose reinfusion requirement of subsequent TIL clinical research; the present application realizes that the TILs obtained by culture still have a significant antitumor effect in vivo without IL-2 concomitant medication, thereby solving the toxicity problem of high-dose IL-2 concomitant medication in TIL clinical treatment.

[0093] An embodiment of the present application provides a cell obtained by expanding and culturing by the method.

[0094] An embodiment of the present application provides a pharmaceutical composition, which comprises the cell and a pharmaceutically acceptable excipient.

[0095] An embodiment of the present application provides use of the cell or the pharmaceutical composition in preparation of a drug for preventing, alleviating and / or treating a disease.

[0096] In a specific example, the disease includes a tumor or a cancer. For example, the tumor or the cancer includes, but is not limited to, one or more of liver cancer, gastric cancer, breast cancer and bladder cancer.

[0097] An embodiment of the present application provides a method for preventing and / or treating a tumor, which comprises administering the cell and / or the pharmaceutical composition to a subject in need.

[0098] Embodiments of the present application will be described in detail below with reference to examples. It should be understood that the examples are only used to illustrate the present application and not to limit the scope of the present application. If the specific conditions are not indicated in the following examples, the experimental methods are preferably referred to the indications given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0099] In the following specific examples, the measurement parameters of raw material components may, without specific indication, have slight deviations within the weighing accuracy range. The temperature and time parameters allow acceptable deviations caused by instrument testing accuracy or operation accuracy.

[0100] The main materials involved in the following examples are shown in Table 1:

[0101] Table 1

[0102] The TIL expansion medium and related reagent preparation involved in the following examples include:

[0103] 1) X-VIVO 15 complete medium preparation: X-VIVO 15 medium + 5% SR serum replacement.

[0104] 2) TGF-β working solution preparation: resuspend TGF-β dry powder (25 μg) in 500 μl sterile water for injection, 50 μl / tube, to obtain 50 ng / μl (tube) TGF-β stock solution (stored at -80°C); dilute the TGF-β stock solution with X-VIVO 15 medium to obtain a TGF-β working solution with a concentration of 1 ng / μl (stored below -20°C).

[0105] The main methods involved in the following examples include:

[0106] 1. In vitro culture of TIL

[0107] 1) In vitro cell culture of TIL: take 1 x 10 6 number of TIL (100 mL, with a culture density of 1 x 10 4 TIL / mL) (the T cells are TIL isolated from a puncture sample or a tumor tissue resection sample and expanded in vitro for 7-14 days), and divide them into two groups, one group without the addition of TGF-β and the other group with the addition of TGF-β; the working concentration of TGF-β added is 1-10 ng / mL; at the same time, add 1 x 10 8 number of human PBMCs irradiated by a gamma ray instrument at a ratio of TIL: feeder cells (Feeder cell) of 1:100; and add 30 ng / mL OKT3 to the culture system; in addition, according to different experimental purposes, add different concentrations of IL-2 (600 IU / mL, 6000 IU / mL), different concentrations of IL-7 (5 ng / mL, 10 ng / mL, 20 ng / mL), and different concentrations of IL-15 (1 ng / mL, 5 ng / mL, 10 ng / mL) to the system.

[0108] 2) At the 7th day and the 9th day of culture, supplement the cells with medium, and add the corresponding concentration of TGF-β; in some experiments, completely replace the medium at the 10th day of culture to remove the TGF-β; during the subsequent culture process, maintain the cell density between 2 x 10 5 / mL and 2 x 10 6 / mL by supplementing a certain volume of culture medium. In addition, sample the cells at the 7th day, the 14th day, and the 21st day of culture, respectively, for cell counting and flow cytometric phenotype detection.

[0109] 3) When the cells reach the experimental endpoint, collect the endpoint cells for TIL phenotype detection, in vitro killing experiment, in vitro repeated antigen stimulation experiment, or mouse experiment.

[0110] 2. Flow cytometry detection of TIL phenotype after culture

[0111] Flow cytometry: a) Centrifuge the collected cells at 600g for 5 min and discard the supernatant; b) Resuspend the cells in 1 ml MACS buffer, centrifuge at 600g for 5 min and discard the supernatant; c) Repeat step b); d) Resuspend the cells in 100 μl MACS buffer, add 1 μl each of the flow cytometry antibodies anti-CD3-APC-CY7, anti-CD4-BV650, anti-CD8-PE, anti-CCR7-APC, and anti-CD45RA-BV786, mix well, and incubate at 2–8°C in the dark for 30 min; e) After incubation, resuspend the cells in 1 ml MACS buffer, centrifuge at 600g for 5 min and discard the supernatant; f) Repeat step e); g) Resuspend the cells in 200 μl MACS buffer and perform flow cytometry analysis.

[0112] 3. In vitro sustained killing test

[0113] The SK-Hep-1 hepatocellular carcinoma cell line overexpressing the red fluorescent reporter protein mCherry was used to test the killing effects of rapidly expanded TILs, TILs obtained through rapid expansion + TGF-β, and TILs obtained through continuous TGF-β addition during rapid expansion. 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 density of 1×10⁶ cells / well, and 1×10⁶ cells / well were added according to experimental requirements. 4 / hole or 2×10 4 TILs were co-cultured in / wells. The killing effect was ultimately assessed over a long period (3-7 days) using the IncuCyte live-cell dynamic imaging analysis system, with target cells added every 2-3 days to detect the sustained killing function of TILs. The IncuCyte system analyzes the growth of target cells by detecting their fluorescence signals. Higher fluorescence intensity or area signal values ​​indicate better target cell growth; conversely, lower fluorescence intensity or area signal values ​​indicate decreased target cell viability and a better killing effect of TILs.

[0114] 4. Repeated antigen stimulation test

[0115] The TILs obtained with or without TGF-β culture were co-cultured with the liver cancer cell line SK-Hep-1 expressing mCherry at a ratio of 1:1 overnight. The next day, the upper suspension cells were carefully aspirated, centrifuged, resuspended, counted, and the number of starting cells after antigen stimulation was obtained. The above cells were grouped into TIL expansion medium (X-VIVO 15 medium + 5% SR serum replacement + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15) and rested for 48 hours. After the culture was completed, cell counting was performed to calculate the expansion fold of the resting culture cells after each round of antigen stimulation. The cells of each experimental group were collected, centrifuged, and the TIL expansion medium was removed, resuspended with EMEM + 10% bovine serum complete medium, and co-cultured with SK-Hep-1 at a ratio of 1:1 overnight for the second round of stimulation. The third / fourth round of stimulation was performed in turn, and after four rounds of stimulation and resting, the TILs of each group were subjected to continuous killing experiments.

[0116] 5. In vivo tumor inhibition experiment

[0117] The target cells (SK-Hep-1) in the logarithmic growth phase and in good growth state were collected by trypsin digestion, washed once with normal saline, and the cell density was adjusted to 2x10 7 / mL. 100 μl of cell suspension was subcutaneously injected into the right side near the axillary part of the NOG mouse, i.e. 2x10 6 target cells were inoculated per mouse, and the inoculation day was recorded as day 0.

[0118] On day 7 after inoculation of the target cells (or when the average tumor volume was about 50 mm 3 ), the rapidly expanded TILs (1x10 7 / mouse), the rapidly expanded + TGF-β cultured TILs (1x10 7 / mouse) and the vehicle (400 μl / mouse) were injected through the tail vein, and the injection day was recorded as day 0 of treatment. The tumor size and mouse weight were measured 2-3 times per week.

[0119] Example 1 Effect of different TGF-β concentrations on the cell proliferation rate and phenotype of TILs

[0120] On the basis of the ordinary expansion culture system of TILs (medium system: X-VIVO 15 medium + 5% SR serum replacement + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15), TGF-β was added to induce a higher proportion of CD8 +Central memory T cells (TCM). By adding different TGF-β working concentrations in the culture system, respectively: 0 ng / mL, 1 ng / mL, 3 ng / mL, 10 ng / mL. Detect the cell proliferation and phenotype of TIL to get the best working concentration of TGF-β in the system.

[0121] As can be seen from the results of A in Figure 1 and Table 2, as the working concentration of TGF-β increases, the expansion rate of TILs decreases significantly. By the 21st day of culture, the cell number of the 10 ng / mL TGF-β group was 2.78E+09, which was 4.42 times lower than that of the 0 ng / mL TGF-β group (1.23E+10 cells); the cell number of the 3 ng / mL TGF-β group was 8.92E+09; the cell number of the 1 ng / mL TGF-β group was 9.98E+09, which was slightly decreased by less than 0.5 times. At the same time, according to the results of B in Figure 1, the phenotype of TILs by the 10th day of culture, as the working concentration of TGF-β increases, the proportion of CD8 + T cells gradually increased, respectively: 20.6% (0 ng / mL TGF-β group), 20.1% (1 ng / mL TGF-β group), 24.1% (3 ng / mL TGF-β group), 27.5% (10 ng / mL TGF-β group); correspondingly; the proportion of CD8 + TCM was: 5.10% (0 ng / mL TGF-β group), 4.85% (1 ng / mL TGF-β group), 6.17% (3 ng / mL TGF-β group), 15.4% (10 ng / mL TGF-β group); the proportion of CD4 + TCM was: 4.14% (0 ng / mL TGF-β group), 6.56% (1 ng / mL TGF-β group), 10.0% (3 ng / mL TGF-β group), 7.29% (10 ng / mL TGF-β group).

[0122] Table 2

[0123] According to the above TIL proliferation and phenotype data, when the working concentration of TGF-β is 3 ng / mL and 10 ng / mL, the proliferation of TILs is inhibited, but the proportion of CD8 + T cells and CCR7 + CD45RA + TCM is significantly up-regulated; however, 10 ng / mL TGF-β significantly inhibits TIL proliferation (down-regulation of 4.42 times). Therefore, 3 ng / mL TGF-β working concentration is selected for subsequent culture system optimization experiments.

[0124] Example 2 IL-2 working concentration optimization

[0125] This example optimized the IL-2 working concentration in the TIL expansion culture system (TIL expansion culture system: X-VIVO 15 medium + 5% SR serum replacement + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15 + 3 ng / mL TGF-β) by adjusting the IL-2 concentration in the culture system to 600 IU / mL (low concentration) and 6000 IU / mL (high concentration) respectively for TIL culture. Since IL-2 mainly affects the proliferation of TIL rather than memory cell phenotype. Therefore, this experiment detected the cell expansion rate, as well as the CD8 + T cell ratio.

[0126] As shown in Figure 2A, compared with the high concentration IL-2 culture group, the low concentration IL-2 had a higher expansion rate of TIL in the system, and consistent trends were obtained in two batches of TIL derived from different patients. Further, by analyzing the CD8 + T cell ratio in T cells, it was found that in this culture system, low concentration of IL-2 was more conducive to improving the CD8 + T cell ratio, as shown in Figure 2B.

[0127] In summary of the above results, in the culture system of the present application, low concentration (600 IU / mL) IL-2 is more conducive to the expansion of TIL and the maintenance of CD8 + T cell ratio.

[0128] Example 3 IL-7 working concentration optimization

[0129] This example optimized the IL-7 working concentration (5 ng / mL, 10 ng / mL, 20 ng / mL) in the TIL expansion culture system (TIL expansion culture system: X-VIVO 15 medium + 5% SR serum replacement + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15 + 3 ng / mL TGF-β) to maximize the induction of CD8 + central memory T cells in combination with the use of TGF-β.

[0130] As shown in the results of Figure 3A, different concentrations of IL-7 had no significant effect on the expansion rate of T cells. However, as shown in Figure 3B, compared with the working concentrations of 5 ng / mL IL-7 and 20 ng / mL IL-7, the use of 10 ng / mL IL-7 could significantly improve the ratio of CD8 + T cells; at the same time, as shown in Figure 3C, 10 ng / mL and 20 ng / mL IL-7 had equivalent effects on TCM induction.

[0131] CD8 + T cells and CD8 + TCM ratio data, the optimal working concentration of IL-7 selected by the culture system of the present application is 10 ng / mL.

[0132] Example 4 IL-15 working concentration optimization

[0133] This example optimizes the working concentration (1 ng / mL, 5 ng / mL, 10 ng / mL) of IL-15 in the TIL expansion culture system (TIL expansion culture system: X-VIVO 15 medium + 5% SR serum replacement + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15 + 3 ng / mL TGF-β) on the expansion rate of TIL, the proportion of CD8 + T cells, and the proportion of CD8 + TCM.

[0134] As shown in Figure 4A, using high concentration of IL-15 (10 ng / mL) for TIL expansion showed no significant difference from the low concentration groups (1 ng / mL and 5 ng / mL). As shown in Figure 4B, in terms of maintaining the proportion of CD8 + T cells, compared with the group adding 1 ng / mL IL-15, the groups adding 5 ng / mL and 10 ng / mL IL-15 showed significantly higher proportion of CD8 + T cells, but the proportion of CD8 + T cells between the groups adding 5 ng / mL and 10 ng / mL IL-15 was comparable, suggesting that the working concentration of IL-15 reached 5 ng / mL, i.e. the optimal effect of maintaining the proportion of CD8 + T cells. Further, this experiment detected the effect of different working concentrations of IL-15 on the proportion of CD8 + TCM. As shown in Figure 4C, compared with the group adding 1 ng / mL IL-15, the groups adding 5 ng / mL and 10 ng / mL IL-15 showed significantly higher proportion of CD8 + TCM, but the proportion of CD8 + T cells between the groups adding 5 ng / mL and 10 ng / mL IL-15 was comparable, suggesting that the working concentration of IL-15 reached 5 ng / mL, i.e. the optimal effect of inducing the generation of CD8 + TCM.

[0135] In summary, using 5 ng / mL IL-15 as the working concentration, in the culture system of the present application, the proportion of CD8 + T cells can be maximally maintained and the generation of CD8+ TCM generation.

[0136] Example 5 TGF-β expansion central memory T cell culture time kinetics analysis

[0137] According to the results of Figure 1, the addition of TGF-β induces the production of central memory T cells while inhibiting the proliferation of TILs. The purpose of adding TGF-β is to significantly and stably induce CD8 + TCM. Therefore, this example determines the time point at which the system can stably and significantly induce CD8 + T cells, CD8 + TCM ratio, by detecting the expansion rate of TILs at different time points of TGF-β culture of TILs, and the CD8 + TCM ratio. + TCM.

[0138] This example compares the data with or without the addition of 3 ng / mL TGF-β in the ordinary expansion culture system (X-VIVO 15 medium + 5% SR serum substitute + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15). As shown in Figure 5A, relative to the culture system without TGF-β, the proliferation rate of TILs is significantly inhibited after the addition of TGF-β, and reaches the maximum inhibition degree around 10 days after culture. Further, analyzing the CD8 + T cell ratio at different time points, as shown in Figure 5B, there is no significant effect of TGF-β on the CD8 + T cell ratio at 3 days of culture. However, at 10 days of culture, the addition of TGF-β significantly up-regulates the CD8 + T cell ratio, and at 21 days of culture, the CD8 + T cell ratio is further up-regulated, but the up-regulation amplitude is smaller than that at 10 days of culture. The above results suggest that the addition of TGF-β within 10 days of culture has been able to significantly up-regulate the CD8 + T cell ratio. In the next experiment, the TGF-β removal time point in the culture system can be set to Day 10.

[0139] At the same time, this example analyzes the TGF-β induced CD8 + TCM generation kinetics. Similarly, as shown in Figure 5C, at 3 days of culture, there is no significant effect of TGF-β on the CD8 + TCM ratio; however, at 10 days of culture, the addition of TGF-β significantly up-regulates the CD8+ TCM ratio, and the CD8 + TCM ratio was not further significantly upregulated. The above results suggest that the first 10 days of culture is the point at which the addition of TGF-β has been able to significantly upregulate the CD8 + TCM ratio. This result further demonstrates that the point at which TGF-β can be removed from the culture system can be set as Day 10.

[0140] Example 6 Comparison of data for continuous / early addition of TGF-β expansion of TIL

[0141] According to the results of Example 5, the addition of TGF-β throughout the culture process inhibits the proliferation of TIL, and the addition of TGF-β for 10 days significantly upregulates the TCM ratio in TIL. Therefore, the present application selects the removal of TGF-β from the system at Day 10 of culturing TIL, and verifies through experiments whether the TCM induction can be ensured while the proliferation rate of TIL is maximized to harvest the maximum number of end product cells at the end of the culture. In this example, two batches of TIL from different liver cancer patients were cultured and tested, and the ordinary expansion system (culture medium system: X-VIVO 15 medium + 5% SR serum substitute + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15) and the rapid expansion system (TIL were co-cultured with allogeneic irradiated PBMC at a cell number ratio of 1:100, and the culture medium system was: X-VIVO 15 medium + 5% SR serum substitute + 600 IU / ml IL-2 + 5 ng / ml IL-7 + 5 ng / ml IL-15. 30 ng / mL OKT3 was added on Day 0, and this component was not added again during the subsequent medium exchange / replenishment process) were used as the basis, and TGF-β was added to induce a higher proportion of CD8 + central memory T cells (TCM). During the 21-day culture period, a TGF-β continuous culture group (i.e., TGF-β is added to the culture system throughout the entire culture period) and a TGF-β early addition group (i.e., TGF-β is added for the first 10 days of culture, and there is no TGF-β in the culture system for the last 11 days of culture) were set up to optimize the way in which TGF-β is added.

[0142] As can be seen from the TIL expansion rate results shown in Figure 6A, after the removal of TGF-β on Day 10 of culture, the expansion rate of TIL significantly increased compared to the TGF-β continuous addition group, indicating that the removal of TGF-β can restore the expansion rate of TIL. Further, the detection of the phenotype of TIL cultured for 21 days showed that, as shown in Figure 6B, the CD8 +The ratio can be further up-regulated, which is unexpected. Compared with the ordinary culture group, the TGF-β pre-addition group can significantly induce CD4 + TCM or CD8 + TCM generation, and close to the TGF-β continuous addition group. As shown in the continuous killing results shown in FIG. 6C, compared with the ordinary culture group, the killing effect of the TGF-β addition group is significantly enhanced, and the TGF-β pre-addition group has stronger continuous killing effect than the TGF-β continuous addition group.

[0143] From the above data, it can be seen that in the first 10 days of the culture period, TGF-β is added for TCM induction, and in the later 11 days of the culture period, TGF-β is removed from the culture medium for continuous culture, which can ensure the maximum proliferation rate of TIL while maximizing the generation of TCM.

[0144] Example 7 Verification of TCM generation of multiple batches of TIL by rapid expansion system + TGF-β pre-induction

[0145] In this example, TILs from 7 liver cancer patient puncture samples were used to verify the rapid expansion system + TGF-β pre-addition culture system established in the above examples, and a corresponding TIL in vitro expansion process was developed. As shown in Table 3, after a 21-day culture period, the rapid expansion + TGF-β pre-addition culture process can harvest TIL end product cell number up to 10 11 The above cells are significantly higher than the end product cell number obtained by using the traditional culture (traditional culture medium system: X-VIVO 15 culture medium + 5% SR serum substitute + 600 IU / ml IL-2 + 10 ng / ml IL-7 + 5 ng / ml IL-15). As shown in FIG. 7, the kinetic curve of the rapid expansion + TGF-β pre-addition culture of the present application is closer to the exponential expansion form.

[0146] Table 3 is the end point harvest cell number statistical result of TILs from 7 liver cancer patient puncture samples cultured by using the traditional culture system (rapid expansion group) and the rapid expansion system + TGF-β pre-addition culture system (rapid expansion + TGF-β group)

[0147] The above results show that the rapid expansion system + TGF-β pre-addition culture of the present application can meet the demand of harvesting more than 10 11 The end product cell number, and the TILs still maintain the state of exponential expansion rate at the harvest time point, so as to maximize the cell activity after being reinfused into the patient's body.

[0148] Furthermore, the proportions of memory cells and terminally differentiated T cells in TILs were examined after 21 days of culture. As shown in Figure 8A, compared to the traditional culture group (rapid expansion, no TGF-β addition), the rapid expansion system established in this application combined with the early TGF-β addition process can induce CD8+. + The proportion of TCM in T cells was significantly upregulated, with an average upregulation of 15.34-fold; meanwhile, CD8... + The proportion of TDs in T cells was significantly downregulated, with an average downregulation of 2.72-fold. Since TGF-β can induce the generation of memory T cells, the presence of memory T cells can be manifested in their ability to maintain a killing effect on tumor cells after prolonged antigen stimulation. Therefore, this application used repeated antigen stimulation experiments to detect whether the induced TCMs possess a sustained killing effect (Figure 8B). As shown in Figure 8C, during the first two rounds of antigen stimulation and resting culture, the amplification capacity of TILs cultured with TGF-β was significantly upregulated; and after four rounds of antigen stimulation, as shown in Figure 8D, the sustained killing effect of TGF-β-cultured TILs on target cells SK-BN105 was significantly enhanced.

[0149] The above data demonstrate that the rapid amplification system established in this application, combined with the TGF-β pre-addition system to induce memory cell generation, can significantly increase the TCM ratio in the final TIL culture product, thereby maintaining its sustained anti-tumor function.

[0150] Example 8: In vitro and in vivo efficacy experiment of rapid amplification system + TGF-β pre-addition to amplify TIL.

[0151] This embodiment analyzes the in vitro and in vivo efficacy of TILs obtained by inducing culture using the rapid amplification system established above in this application and TGF-β pre-addition. First, in vitro, the effect of TILs on killing tumor cells was detected by real-time fluorescence imaging. As shown in Figure 9A, TILs obtained by the traditional culture method (rapid amplification group) have a certain anti-tumor killing effect; correspondingly, TILs cultured using the rapid amplification system of this application and TGF-β pre-addition significantly enhanced the sustained killing function of target cells. Meanwhile, in vivo efficacy experiments showed, as shown in Figure 9B, that without IL-2 concomitant administration, the infusion of 5×10⁶ TILs significantly reduced the tumor cell killing effect. 6 and 1×10 7 The TILs obtained from the dosage group and the rapid amplification group did not have significant anti-tumor effects; however, the TILs obtained by culturing using the rapid amplification system of this application with TGF-β pre-addition showed significant inhibitory effects on tumor cell growth, as shown in Figure 9C.

[0152] The above data show that the fast amplification system + TGF-β pre-addition induced memory cell generation system established by the present application can significantly increase the proportion of TCM in the final product of TIL culture, thereby still having a significant anti-tumor effect in in vivo and in vitro test models without IL-2 concomitant medication.

[0153] Each of the technical features of the above-described embodiments can be combined arbitrarily. In order to make the description simple, each of the technical features in the above-described embodiments is not described in all possible combinations, however, as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0154] The above-described embodiments only express several implementation manners of the present application, the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the application. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the patent protection scope of the present application should be subject to the appended claims.

Claims

1. A culture medium for tumor infiltrating lymphocytes, characterized in that, The medium comprises a basal medium and cell growth factors, the cell growth factors comprising a combination of at least two of IL-2, IL-7, IL-15 and TGF-β.

2. The medium of claim 1, wherein, The TGF-β comprises one or more of TGF-β1, TGF-β2 and TGF-β3.

3. The medium according to claim 1 or 2, characterized in that, In the medium, the concentration of the TGF-β is about 0.5 ng / mL to about 10 ng / mL, the concentration of the IL-2 is about 100 IU / mL to about 6000 IU / mL, the concentration of the IL-7 is about 5 ng / mL to about 20 ng / mL, and the concentration of the IL-15 is about 1 ng / mL to about 10 ng / mL.

4. The medium according to claim 3, characterized in that, In the medium, the concentration of the TGF-β is about 0.5 ng / mL to about 3 ng / mL, the concentration of the IL-2 is about 600 IU / mL to about 1000 IU / mL, the concentration of the IL-7 is about 5 ng / mL to about 10 ng / mL, and the concentration of the IL-15 is about 5 ng / mL to about 10 ng / mL.

5. The medium according to any one of claims 1 to 4, characterized in that, The medium further comprises a serum substitute.

6. The medium of claim 5, wherein, In the medium, the volume fraction of the serum substitute is about 4% to about 8%.

7. The medium according to any one of claims 1 to 6, characterized in that, The basal medium comprises one or more of X-VIVO, AIM-V, OptiVitro, DMEM and RPMI 1640.

8. A method of expanding tumor infiltrating lymphocytes in culture in vitro, characterized in that, The method comprises the steps of culturing the tumor infiltrating lymphocytes under a first culture condition and a second culture condition in sequence: The first culture medium used in the first culture condition is the medium according to any one of claims 1 to 7; The second culture medium used in the second culture condition does not contain TGF-β relative to the first culture medium.

9. The method of claim 8, wherein, The culturing time of the first culture condition is 9 days to 15 days.

10. The method of claim 9, wherein, The culturing time of the first culture condition is about 10 days to about 11 days.

11. The method according to any one of claims 8-10, characterized in that, The culturing time of the second culture condition is about 7 days to about 15 days.

12. The method of claim 11, wherein, The culturing time of the second culture condition is about 10 days to about 11 days.

13. The method according to any one of claims 8-10, characterized in that, On day 0 of culturing under the first culture condition, a T cell activator is added to the culture system; Optionally, the T cell activator comprises an anti-CD3 antibody; Optionally, the anti-CD3 antibody comprises an anti-CD3 monoclonal antibody with clone number OKT3.

14. The method of any one of claims 8-10, wherein, Under the first culture condition, TGF-β is added during the culturing to maintain the concentration of TGF-β in the culture system; Optionally, the concentration of TGF-β in the culture system is maintained by adding TGF-β directly, by supplementing or by replacing the medium during the culturing.

15. The method of any one of claims 8-10, wherein, under the second culture condition, the cell density is adjusted to be between about 2 x 10 5 / mL and about 2 x 10 6 / mL during the culture period by feeding or medium exchange.

16. The method of any one of claims 8-10, wherein, The tumor infiltrating lymphocytes are cultured in the presence of feeder cells.

17. The method of claim 16, wherein, The feeder cells are peripheral mononuclear cells; Optionally, the feeder cells are irradiated feeder cells; Optionally, the ratio of the tumor infiltrating lymphocytes to the feeder cells is about 1:50 to about 1:

200.

18. The method of any one of claims 8-10, wherein, The tumor infiltrating lymphocytes are derived from tumor tissue.

19. Use of the cells obtained by the method according to any one of claims 8 to 18 for the preparation of a medicament for preventing, alleviating or treating a disease.

20. The use according to claim 19, characterized in that, The disease comprises a tumor or a cancer.

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