Nouritrophoblast-free one-step culture method of tumor infiltrating lymphocytes and application

Abstract

The invention discloses a trophoblast-free one-step culture method and application of tumor infiltrating lymphocytes, and particularly discloses a one-step amplification method of tumor infiltrating lymphocytes, which comprises the following steps: treating a tumor sample obtained from a subject into tumor segments; the tumor segments are cultured in a culture medium containing a CD3 agonist, a 4-1BB agonist and IL-2, so that an amplified tumor infiltration lymphocyte population is obtained, and the culture medium does not contain trophoblastic cells. Compared with the prior art, the method has the advantages that the required period for amplifying the tumor infiltrating lymphocytes is shorter, the cost is lower, the cell effect function is stronger, and the clinical application range of the tumor infiltrating lymphocyte therapy is widened.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and particularly to a feeder-free one-step culture method and application of tumor-infiltrating lymphocytes. Background Art

[0002] The adoptive transfer therapy of tumor-infiltrating lymphocytes (TILs) has been proven to be an effective means for treating patients with solid tumors with poor prognosis and difficult to cure. However, one of the keys to realizing the adoptive transfer TILs therapy lies in obtaining a sufficient number of functional TILs, which poses high requirements for the in vitro amplification of TILs in terms of technology.

[0003] Currently, the commonly used TILs amplification methods include a preliminary amplification process (pre-REP) based on interleukin-2 (IL-2) and a subsequent "rapid expansion protocol" (REP). This two-step amplification scheme can generate a therapeutically effective number of TILs within a certain period of time and has become the preferred method for TILs amplification. However, this process still has some defects. For example, the REP process requires the use of a large number of irradiated peripheral blood mononuclear cells (PBMCs) from multiple donors as feeder cells. This not only results in a long preparation cycle and high cost of TILs, but also poses high requirements for the tissue volume, T lymphocyte ratio and quantity of the patient's tumor sample, limiting the wide clinical application of this therapy.

[0004] In addition, the existing TILs amplification methods also have other challenges, such as weak amplification ability of TILs from the patient's tumor, difficult control of the proportion of CD8+ cytotoxic T cells, insufficient killing ability against tumor cells, and possible T cell exhaustion caused by culturing with high-concentration IL-2. These factors together affect the effect of the TILs therapy.

[0005] Therefore, there is an urgent need to develop a new TILs amplification process that can shorten the culture cycle, reduce costs and enhance the cell effector function to improve the clinical applicability and efficacy of the TILs therapy. Summary of the Invention

[0006] The present invention provides a method for amplifying tumor-infiltrating lymphocytes. The method provided by the present application shortens the time for amplifying the TIL population by performing a one-step amplification process and does not require feeder cells, thereby providing the clinical application advantages of adoptive TIL transfer therapy. In addition, the method provided by the present application can also be used to enrich the stem cell T cell population and the cytotoxic T cell population.

[0007] As disclosed herein, it has unexpectedly been found that tumor-infiltrating lymphocytes (TILs) can be expanded by providing a combination of a CD3 agonist, a 4-1BB agonist, and IL-2 in the absence of feeder cells. Surprisingly, the feeder-cell-free TIL expansion method described herein allows for the harvesting of a therapeutically effective number of TILs in a shorter period of time and also results in the enrichment of stem-like T cell populations and cytotoxic T cell populations, as well as a reduced proportion of exhausted T cells.

[0008] As disclosed herein, it has also unexpectedly been found that TIL populations can be expanded by a one-step method, thereby eliminating the need for separate pre-REP and REP steps.

[0009] In one aspect, the present invention relates to a method for one-step expansion of tumor-infiltrating lymphocytes, the method comprising culturing a tumor sample obtained from a subject in a culture medium containing a CD3 agonist, a 4-1BB agonist, and IL-2 to obtain an expanded population of tumor-infiltrating lymphocytes, and the culture medium not containing feeder cells.

[0010] In some embodiments, the tumor sample is fragmented prior to culturing.

[0011] In some embodiments, the tumor sample comprises tumor fragments having a size of 0.5 mm 3 to 27 mm 3 In some embodiments, the tumor sample comprises digested tumor fragments.

[0012] In some embodiments, the final concentration of IL-2 in the culture medium is 6000 IU / mL or less, and in some preferred embodiments, the final concentration of IL-2 in the culture medium is 2000 IU / mL or less. In some preferred embodiments, the final concentration of IL-2 in the culture medium is 1000 IU / mL.

[0013] In some embodiments, the CD3 agonist comprises an anti-CD3 antibody and / or an antigen-binding fragment thereof, optionally a humanized anti-CD3 antibody and / or an antigen-binding fragment thereof.

[0014] In some embodiments, the CD3 agonist is OKT3.

[0015] In some embodiments, the final concentration of the CD3 agonist is from 10 ng / mL to 50 ng / mL. In some preferred embodiments, the final concentration of the CD3 agonist is 30 ng / mL.

[0016] In some embodiments, the 4-1BB agonist comprises a ligand-competitive 4-1BB agonistic antibody and / or an antigen-binding fragment thereof, or a ligand-noncompetitive 4-1BB agonistic antibody and / or an antigen-binding fragment thereof. In some preferred embodiments, the 4-1BB agonist is Utomilumab or Urelumab.

[0017] In some embodiments, the final concentration of the 4-1BB agonist is 1-20 μg / mL. In some preferred embodiments, the final concentration of the 4-1BB agonist is 10 μg / mL.

[0018] In some embodiments, the components of the culture medium remain unchanged. In some embodiments, about 30% to about 99% of the culture medium is replaced every 2 to 5 days.

[0019] In some embodiments, wherein replacing the culture medium comprises adding fresh culture to the culture, wherein the fresh culture medium contains IL-2 and does not contain a CD3 agonist and a 4-1BB agonist.

[0020] In some embodiments, wherein the fresh culture medium further comprises a cell basal medium, a serum substitute, L-glutamine or a substitute thereof, and an antibiotic. In some embodiments, the culture medium does not contain serum.

[0021] In some embodiments, the cell basal medium includes but is not limited to X-vivo15 medium, AIM-V medium, RPMI-1640 medium, OpTmizer TM medium or OpTmizer TM Pro medium.

[0022] In some embodiments, wherein the TIL culture lasts for 14-28 days.

[0023] In some embodiments, wherein the TIL culture lasts for at least 14 days.

[0024] In some embodiments, the number of TIL cells harvested at the end of the culture is 1×10 7 cells / fragment or more, preferably 1×10 8 cells / fragment or more, more preferably 1×10 9 cells / fragment or more.

[0025] In some embodiments, the methods herein can salvage TIL samples from previously failed pre-REP amplifications. In some embodiments, the tumor sample is from a subject who has previously submitted a tumor sample for amplification, wherein the previous amplification includes a pre-REP step and wherein the pre-REP step is cultured for 14 days without TIL amplification. In some embodiments, the tumor sample is from a subject who has previously submitted a tumor sample for amplification, wherein the previous amplification includes a pre-REP step and the number of TILs isolated from the pre-REP step at the end point of culturing the pre-REP step is below 1×10 7 cells per fragment.

[0026] In some embodiments, compared to a population of tumor-infiltrating lymphocytes obtained by conventional multi-step amplification, a population of tumor-infiltrating lymphocytes obtained by one-step amplification exhibits improved TIL characteristics.

[0027] In some embodiments, the improved TIL characteristics include one or more selected from the group consisting of: increased TIL cell number and amplification ability, increased proportion of live cells, increased viability, improved proportion of T cell subsets, increased cytokine secretion ability, increased tumor cell killing ability, increased anti-exhaustion ability.

[0028] In some embodiments, the improved proportion of T cell subsets includes one or more selected from the group consisting of: decreased proportion of regulatory T cells, increased proportion of stem-like T cells, increased proportion of cytotoxic T cells, and decreased proportion of exhausted T cells.

[0029] In some embodiments, after culturing the tumor sample obtained from the subject, CD3+ TILs in the amplified population of tumor-infiltrating lymphocytes account for more than about 80% of the total number of cells, preferably more than about 85%, more preferably more than about 90%.

[0030] In some embodiments, after culturing the tumor sample obtained from the subject, CD8+ TILs in the amplified population of tumor-infiltrating lymphocytes account for more than about 50% of CD3+ TILs.

[0031] In some embodiments, after culturing the tumor sample obtained from the subject, CD69- / CD39- TILs in the amplified population of tumor-infiltrating lymphocytes account for more than about 10% of CD8+ TILs.

[0032] In some embodiments, after culturing the tumor sample obtained from the subject, exhausted T cells in the amplified population of tumor-infiltrating lymphocytes account for less than about 50% of CD8+ TILs, preferably less than about 20%.

[0033] In some embodiments, after culturing the tumor sample obtained from a subject, the Foxp3+ TILs in the expanded tumor-infiltrating lymphocyte population account for about 20% or less of the CD4+ TILs.

[0034] In some embodiments, the method further comprises genetically modifying the cells of the expanded tumor-infiltrating lymphocyte population.

[0035] In some embodiments, the cells of the expanded tumor-infiltrating lymphocyte population are modified by a gene editing system. In some embodiments, the cells of the expanded tumor-infiltrating lymphocyte population are modified using RNA interference. In some embodiments, the cells of the expanded tumor-infiltrating lymphocyte population are modified using transcription activator-like effector nucleases (TALENs). In some embodiments, the cells of the expanded tumor-infiltrating lymphocyte population are modified using zinc finger nucleases. In one embodiment, the cells of the expanded tumor-infiltrating lymphocyte population are modified using an RNA-guided nuclease. In some embodiments, the cells of the expanded tumor-infiltrating lymphocyte population are modified using a Cas enzyme and at least one guide RNA. In some embodiments, the Cas enzyme is Cas9.

[0036] In some cases, the cells of the expanded tumor-infiltrating lymphocyte population are genetically modified to comprise a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that targets a tumor-associated antigen.

[0037] In some cases, the cells of the expanded tumor-infiltrating lymphocyte population are modified to comprise a decrease or inhibition of the expression of one or more endogenous genes and / or the function of a protein encoded by a gene. In some embodiments, these endogenous genes include one or more genes selected from the following: NR4A1, NR4A2, NR4A3, RC3H1, and ZC3H12A. In some embodiments, the modification at one or more genes is an insertion, deletion, or mutation of one or more nucleic acids.

[0038] In some embodiments, the endogenous genes of the cells of the expanded tumor-infiltrating lymphocyte population show improved TIL characteristics after being genetically modified compared to TILs with unmodified endogenous genes.

[0039] In some embodiments, the improved TIL characteristics comprise one or more selected from the following group: increased TIL cell number and expansion ability, increased viability, improved T cell subset ratio, increased cytokine secretion ability, increased tumor cell killing ability, increased anti-exhaustion ability.

[0040] In another aspect, the present invention relates to a population of tumor-infiltrating lymphocytes obtained according to the methods disclosed herein.

[0041] In another aspect, the present invention relates to a pharmaceutical composition comprising a population of tumor-infiltrating lymphocytes disclosed herein, and optionally a pharmaceutically acceptable carrier.

[0042] In another aspect, the present invention relates to the use of a population of tumor-infiltrating lymphocytes obtained according to the methods disclosed herein or a population of tumor-infiltrating lymphocytes disclosed herein or a pharmaceutical composition disclosed herein in the preparation of a medicament for preventing and / or treating tumors.

[0043] In some embodiments, the tumor is a solid tumor.

[0044] In some embodiments, the tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0045] In another aspect, the present invention relates to a method for treating a tumor in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a population of tumor-infiltrating lymphocytes obtained according to the methods disclosed herein or a population of tumor-infiltrating lymphocytes disclosed herein or a pharmaceutical composition disclosed herein.

[0046] In some embodiments, the tumor is a solid tumor.

[0047] In some preferred embodiments, the tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0048] Compared with the population of tumor-infiltrating lymphocytes obtained by the trophoblast-free one-step amplification method, the population of tumor-infiltrating lymphocytes obtained by the present invention has the following advantages: 1) the cycle of TILs amplification and culture is shortened; 2) compared with the traditional TIL culture process, the success rate of TILs amplification is increased, and the applicable range of TIL therapy in future clinical practice is improved; 3) the proportion of CD8+ T cells is significantly increased; 4) the killing ability of TIL cells is improved; 5) the risk of cell exhaustion caused by the subsequent reinfusion of a higher concentration of IL-2 is reduced; 6) the proportion of exhausted T cells is reduced; 7) the proportion of stem T cells is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The specific features of the invention involved in this application are shown in the appended claims. The characteristics and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0050] Figure 1 : The number of TILs obtained by amplification using the conventional two-step method (Conventional TILs group) and the one-step method without trophoblast cells (FOST group, with the final concentration of IL-2 indicated in parentheses).

[0051] Figure 2 : The number of TILs obtained by amplification using the conventional two-step method (Conventional group) and the one-step method without trophoblast cells (FOST group, with the final concentration of IL-2 indicated in parentheses), where the FOST 1 group refers to the medium with the 4-1BB agonist Utomilumab, and the FOST 2 group refers to the medium with the 4-1BB agonist Urelumab.

[0052] Figure 3 : The proportion of CD8+ T cells in CD3+ T cells in the TILs population obtained by amplification using the conventional two-step method (Conventional group) and the one-step method without trophoblast cells (F1 group), where the F1-6000 group refers to the medium with the final concentration of IL-2 being 6000 IU / mL, and the F1-1000 group refers to the medium with the final concentration of IL-2 being 1000 IU / mL.

[0053] Figure 4 : The proportion of exhausted T cells in CD8+ T cells in the TILs population obtained by amplification using the conventional two-step method (Conventional group) and the one-step method without trophoblast cells (FOST group). Among them Figure 4 A represents the proportion of TIM-3-positive T cells, which are markers of exhausted T cells, in CD8+ T cells, Figure 4 B represents the proportion of BLIMP-1-positive T cells, which are pro-exhaustion transcription factors, in CD8+ T cells. The FOST-6000 group refers to the medium with the final concentration of IL-2 being 6000 IU / mL, and the FOST-1000 group refers to the medium with the final concentration of IL-2 being 1000 IU / mL.

[0054] Figure 5 : The proportion of CD39- / CD69- stem T cells in CD8+ T cells in the TILs population obtained by amplification using the conventional two-step method (Conventional group) and the one-step method without trophoblast cells (FOST group), where the FOST-6000 group refers to the medium with the final concentration of IL-2 being 6000 IU / mL, and the FOST-1000 group refers to the medium with the final concentration of IL-2 being 1000 IU / mL.

[0055] Figure 6 :Detection results of cytokine ratios of CD8+ T cells in TILs populations amplified using the conventional two-step method (Conven-TIL group) and the feeder-free one-step method (FOST-TIL group). Among them Figure 6 A represents the proportion of interferon γ (IFN-γ), Figure 6 B represents the proportion of granzyme B (GZMB).

[0056] Figure 7 :Cytotoxicity results of TILs populations amplified using the conventional two-step method (Conven group) and the feeder-free one-step method (F1 and F2 groups). Among them Figure 7 A represents a tumor sample derived from lung cancer, Figure 7 B represents a tumor sample derived from ovarian cancer, where the F1-6000 group refers to the medium with 4-1BB agonist Utomilumab and the final concentration of IL-2 being 6000 IU / mL; the F1-1000 group refers to the medium with 4-1BB agonist Utomilumab and the final concentration of IL-2 being 1000 IU / mL; the F2-1000 group refers to the medium with 4-1BB agonist Urelumab and the final concentration of IL-2 being 1000 IU / mL.

[0057] Figure 8 :The number of TILs amplified using the feeder-free one-step method (FOST group) with different final concentrations of IL-2 in the medium, where the FOST-6000 group refers to the medium with the final concentration of IL-2 being 6000 IU / mL, and the FOST-1000 group refers to the medium with the final concentration of IL-2 being 1000 IU / mL.

[0058] Figure 9 :The proportion of CD3+ T cells in the total cell amount in TILs populations amplified using the feeder-free one-step method (FOST group) with different final concentrations of IL-2 in the medium, where the FOST-6000 group refers to the medium with the final concentration of IL-2 being 6000 IU / mL, and the FOST-1000 group refers to the medium with the final concentration of IL-2 being 1000 IU / mL.

[0059] Figure 10 :The proportion of Foxp3+ Treg cells in CD4+ T cells in TILs populations amplified using the feeder-free one-step method with different final concentrations of IL-2 in the medium. Among them Figure 10 A represents a tumor sample derived from renal cell carcinoma, Figure 10B represents a tumor sample derived from ovarian cancer, with the final concentration of IL-2 indicated in parentheses. Group F1 refers to the culture medium with the 4-1BB agonist Utomilumab, group F2 refers to the culture medium with the 4-1BB agonist Urelumab, the Conventional TILs group refers to the TILs cell population obtained by conventional two-step amplification, and the positive control represents Treg cells.

[0060] Figure 11 : Results of the cytotoxicity of TILs populations obtained by one-step amplification using culture media containing different final concentrations of IL-2. Group F1-6000 refers to the culture medium with the 4-1BB agonist Utomilumab and a final concentration of IL-2 of 6000 IU / mL; Group F1-1000 refers to the culture medium with the 4-1BB agonist Utomilumab and a final concentration of IL-2 of 1000 IU / mL;

[0061] Figure 12 : Proportion of CD8+ T cells in CD3+ T cells in TILs populations obtained by one-step amplification using culture media containing 4-1BB agonist or CD28 agonist. Group F1-1000 refers to the culture medium with the 4-1BB agonist (Utomilumab) and a final concentration of IL-2 of 1000 IU / mL; Group F28-6000 refers to the culture medium with the CD28 agonist and a final concentration of IL-2 of 6000 IU / mL; Group F28-1000 refers to the culture medium with the CD28 agonist and a final concentration of IL-2 of 1000 IU / mL. Data are presented as mean ± SEM, and paired T-tests were used. **, p < 0.01, ****, p < 0.0001.

[0062] Figure 13 : Cell numbers of TILs populations obtained by one-step amplification using culture media containing Utomilumab or Urelumab. Group FOST 1 refers to the culture medium with the 4-1BB agonist Utomilumab; Group FOST 2 refers to the culture medium with the 4-1BB agonist Urelumab.

[0063] Figure 14 : Proportion of CD3+ T cells in the total cell mass in TILs populations obtained by one-step amplification using culture media containing Utomilumab or Urelumab. Group FOST 1 refers to the culture medium with the 4-1BB agonist Utomilumab; Group FOST2 refers to the culture medium with the 4-1BB agonist Urelumab.

[0064] Figure 15:The proportion of CD8+ T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab, where the FOST 1 group refers to the medium with the 4-1BB agonist Utomilumab; the FOST2 group refers to the medium with the 4-1BB agonist Urelumab.

[0065] Figure 16 :The proportion of exhausted T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab. Among them Figure 16 A represents the proportion of TIM-3 positive T cells, which are exhausted T cell markers, in CD8+ T cells, Figure 16 B represents the proportion of BLIMP-1 positive T cells, which are pro-exhaustion transcription factors, in CD8+ T cells, where the F1 group refers to the medium with the 4-1BB agonist Utomilumab; the F2 group refers to the medium with the 4-1BB agonist Urelumab.

[0066] Figure 17 :The proportion of CD39- / CD69- stem T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab, where the F1 group refers to the medium with the 4-1BB agonist Utomilumab; the F2 group refers to the medium with the 4-1BB agonist Urelumab.

[0067] Figure 18 :The detection result of the granzyme B proportion of CD8+ T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab, where the F1 group refers to the medium with the 4-1BB agonist Utomilumab; the F2 group refers to the medium with the 4-1BB agonist Urelumab.

[0068] Figure 19 :The number of TILs cells obtained by amplifying tumor samples that failed to be amplified by the conventional two-step method (Conventional TILs group) using a one-step method without trophoblast cells (FOST group, with the final concentration of IL-2 indicated in parentheses). Among them Figure 19 A represents the tumor sample of subject LC002, Figure 19 B represents the tumor sample from subject RC002, where the FOST1 group refers to the medium with the 4-1BB agonist Utomilumab; the FOST 2 group refers to the medium with the 4-1BB agonist Urelumab.

[0069] Figure 20: Amplification curve and viability of TILs population obtained by large-scale TIL production.

[0070] Figure 21 : Phenotypic characteristics of TILs population obtained by large-scale TIL production.

[0071] Figure 22 : Cytokine ratio and secretion ability of TILs population obtained by large-scale TIL production.

[0072] Figure 23 : Target cell killing ability of TILs population obtained by large-scale TIL production. Detailed implementation mode

[0073] To achieve the activation and amplification multiples of TILs sufficient for therapeutic use, the conventional amplification method for general TILs includes at least a separate Pre-REP step and a REP step. In some applications, the Pre-REP step of the conventional method can last for 2 to 6 weeks, and an additional 1 to 3 weeks for REP. In addition, the conventional amplification method requires the use of feeder cells in addition to multiple steps. These two requirements make the conventional amplification method both time-consuming and expensive. Patients who need to use adoptive transfer of TILs for immunotherapy usually have a very poor prognosis. Having an amplified and differentiated TILs population available for treatment more quickly may make the difference between survival and death. Therefore, there is a need for a simpler, faster and lower-cost method for manufacturing TILs.

[0074] To provide an improved, faster and simpler method for generating TILs, the present invention provides a one-step amplification method that uses a more simplified method without using feeder cells. In addition to enriching the amplified TILs population with CD8+ cytotoxic T cell and stem cell-like T cell phenotypes, a composition containing the amplified TILs population is also provided.

[0075] In some aspects, the present disclosure relates to a method for amplifying TILs in a one-step method without using feeder cells, the method comprising culturing a tumor sample obtained from a subject in a medium containing a CD3 agonist, a 4-1BB agonist and IL-2, thereby obtaining an amplified population of tumor-infiltrating lymphocytes, eliminating the need for a Pre-REP step. In certain embodiments, the amplified TILs have a higher percentage of cells with CD8+ cytotoxic T cell and stem cell-like T cell phenotypes compared to TILs isolated using a feeder cell-based method.

[0076] Although the present invention may be embodied in many different forms, specific illustrative embodiments thereof that verify the principles of the present invention are disclosed herein. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. In addition, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0077] Generally, the terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used terms in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook J. & Russell D. M. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Abbas et al., Cellular and Molecular Immunology, 6th ed., W.B. Saunders Company (2010); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The terms and laboratory procedures and techniques related to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used terms in the art.

[0078] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include plural forms and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not limiting. The terms "about" and "approximately" generally refer to a statistically significant range of values. Such a range can be within an order of magnitude of a given value or range, can be within 50%, can be within 20%, can be within 10%, can be within 5%. The allowable variation encompassed by the term "about" or "approximately" can depend on the particular system being studied and can be readily understood by one of ordinary skill in the art. The terms "above", "below", "at most", and "at least" can include the number itself. In addition, the ranges provided in the specification and the appended claims include the endpoints and all values between the endpoints.

[0079] Definition

[0080] For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0081] As used herein, the term "cell population" or "TIL population" refers to a number of cells or TILs having a common trait. Generally, the number of cell populations is generally in the range of 1×10 6 to 1×10 10 and different TIL populations contain different numbers. For example, the initial growth of primary TILs in the presence of IL-2 can result in a large TIL population of approximately 1×10 7 cells. Generally, REP amplification is carried out to provide a TIL population of 1.5×10 9 to 1.5×10 10 cells for infusion.

[0082] As used herein, the term "amplification" or "amplification process" refers to the process of stimulating or activating cells and culturing cells. The amplification process can cause an increase in the total number of desired cells in the cultured cell population, such as an increase in the total number of TILs, after stimulating or activating and culturing the cells. Amplification does not require an increase in the number of all cell types in the cultured cell population. Instead, in some aspects, only the number of a cell subset in the cultured cell population increases during amplification, while the number of other cell types may remain unchanged or may decrease. A process of simply isolating or enriching TILs without substantially increasing the number of TILs is not an amplification process.

[0083] As used herein, the term "agonist" refers to a chemical substance, molecule, macromolecule, molecular complex, or macromolecular complex that binds to a target either on the cell surface or in soluble form. In certain embodiments, when the agonist binds to a target on the cell surface, the agonist activates the target to produce a biological response. Agonists include hormones, neurotransmitters, antibodies, and antibody fragments.

[0084] As used herein, the term "Feeder Cell" refers to a cell that provides extracellular secretions that assist the proliferation of another cell type. In certain embodiments, the Feeder Cell can be a peripheral blood mononuclear cell (PBMC) or an antigen-presenting cell (APC). The Feeder Cell can also be a cell that is not normally used as a Feeder Cell but has been engineered to secrete or express extracellular secretions that assist the proliferation of another cell type.

[0085] As used herein, the term "IL-2" is also referred to as "TCGF", "interleukin 2", "IL-2", and generally refers to a secreted cytokine produced by activated CD4+ and CD8+ T lymphocytes. IL-2 in the present invention includes all forms of IL-2, such as IL-2 derived from humans or other mammals, and includes IL-2 wild-type and mutants, provided that the mutants have activities similar to the wild-type. In the present invention, IL-2 includes recombinant human IL-2.

[0086] As used herein, the term "CD3" refers to a co-stimulatory molecule expressed on the surface of T lymphocytes and is a protein complex. CD3 has five peptide chains, namely the γ chain, δ chain, ε chain, ζ chain, and η chain, and all five chains are transmembrane proteins. The transmembrane region of the CD3 molecule is connected to the transmembrane regions of two peptide chains of the TCR through salt bridges to form a TCR-CD3 complex, which jointly participates in the recognition of antigens by T cells. The activation signal generated by the TCR recognition of antigens is transduced into the T cell by CD3.

[0087] As used herein, the term "anti-CD3 antibody" refers to an antibody or its variant, such as a monoclonal antibody, and includes human antibodies, humanized antibodies, chimeric antibodies, or murine antibodies against the CD3 receptor in the T cell antigen receptor of mature T cells, and its source is not limited. For example, it can be self-developed or commercially available. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UCHT1 clone, also known as T3 and CD3c. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0088] As used herein, the term "CD28" refers to a co-stimulatory molecule expressed on the surface of T lymphocytes and plays an important role in the activation of T cells. For example, CD28 can bind to the B7 molecule on APCs (antigen-presenting cells), mediate co-stimulation of T cells, and promote their survival, proliferation, and cytokine production.

[0089] As used herein, the term "anti-CD28 antibody" refers to an antibody or its variant, such as a monoclonal antibody, and includes human antibodies, humanized antibodies, chimeric antibodies, or murine antibodies against the CD28 receptor in the T cell antigen receptor of mature T cells, and its source is not limited, for example, it can be self-developed or commercially available.

[0090] As used herein, the term "4-1BB", also known as "CD137", generally refers to an inducible co-stimulatory receptor expressed on activated CD4+ and CD8+ T cells, NKT cells, NK cells, DC cells, macrophages, eosinophils, neutrophils, mast cells, and Tregs.

[0091] As used herein, the term "anti-4-1BB antibody" refers to an antibody or its variant, such as a monoclonal antibody, and includes human antibodies, humanized antibodies, chimeric antibodies, or murine antibodies against 4-1BB, and its source is not limited, for example, it can be self-developed or commercially available. In some embodiments, the anti-4-1BB antibody can be used as a 4-1BB ligand. Anti-4-1BB antibodies include Utomilumab and Urelumab.

[0092] As used herein, the term "TIL property" refers to the improved properties of TIL cells after being modified by the preparation method of the present invention. Changes in TIL properties can include: increased TIL proliferation ability, increased TIL cell number, increased survival ability, improved T cell subset ratio, increased cytokine secretion ability, increased granzyme secretion ability, increased tumor cell killing ability, decreased cell exhaustion level, or any combination thereof. The changes of the present invention can be an increase or a decrease.

[0093] As used herein, the term "exhausted cell" generally refers to an immune cell that has gradually lost its effector function due to continuous stimulation by an antigen. For example, the function of an exhausted cell can be reversible or partially reversible. Exhausted cells can have a PD1+, LAG3+, TIM-3+, or BLIMP-1+ phenotype, and for example, exhausted cells can be identified by PD1+, LAG3+, TIM-3+, or BLIMP-1+. Exhausted cells can be characterized by a reduced immune function.

[0094] As used herein, the term "stem cell" generally refers to a class of cells that can have the potential for self-proliferation and / or differentiation (stemness). Stem cells can have a CD69- / CD39- phenotype, for example, stem cells can be identified by CD69- / CD39-. Tumor-specific cells can have a stronger and / or more long-term anti-tumor growth ability compared to normal cells.

[0095] As used herein, the term "regulatory T cell" or "Treg" generally refers to a subset of T cells that control the body's autoimmune reactivity. Regulatory T cells can have a CD4+Foxp3+ phenotype, for example, regulatory T cells can be identified by CD4+ and Foxp3+. Regulatory T cells can have the ability to inhibit the anti-tumor growth of T cells.

[0096] In this application, the term "killing ability" is generally achieved by contacting the cells of this application with an effective amount of a substance to kill target cells. In one embodiment, the substance of this application can be TIL cells. The killing in this application can include killing cells by itself or promoting the CDC, apoptosis, ADCC, and / or phagocytosis of other cells or substances, or by a combination of two or more of these mechanisms.

[0097] In this application, the term "T cell subset ratio" generally refers to the ratio of different T cell subsets in TIL cells or TIL populations. For example, different T cell subsets in this application have different immune activities and / or differentiation abilities. For example, the T cell subsets in this application can be distinguished according to T cell surface markers. For example, cytotoxic T cells can have a CD8+ phenotype. For example, exhausted T cells can have a TIM-3+ phenotype. For example, stem T cells can have a CD69- / CD39- phenotype. For example, regulatory T cells can have a CD4+ / Foxp3+ phenotype.

[0098] As used herein, the term "subject" refers to a person with a tumor, a lymphocyte population that has left the human bloodstream has migrated into the tumor and has been transformed into TIL in the tumor. In some embodiments, this person can be a patient in need of immunotherapy, which involves the patient's own expanded TIL population. In other embodiments, this person can be a patient in need of immunotherapy, which involves the expanded TIL population of another patient.

[0099] As used herein, "administering" refers to physically introducing a therapeutic agent or a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems. Different routes of administration of the therapeutic agents described herein (e.g., TILs cultured as described herein) include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0100] The term "therapeutically effective amount" refers to the amount of an agent (e.g., TILs cultured as described herein) that provides a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, improvement, alleviation, attenuation, delay, and / or mitigation of one or more signs, symptoms, or causes of a disease or any other desired change in a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to cause tumor shrinkage and / or sufficient to reduce the tumor growth rate (e.g., inhibit tumor growth) or sufficient to prevent or delay other undesired cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations.

[0101] As used herein, the terms "tumor cell" or "cancer cell" refer to cells that divide in an uncontrolled manner, form solid tumors, or fill the blood with abnormal cells. Healthy cells stop dividing when no more daughter cells are needed, but tumor cells or cancer cells continue to produce copies. They are also capable of spreading from one part of the body to another in a process called metastasis. Tumor cells can be isolated from a variety of cancer types, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, kidney cancer, lip and oral cavity cancer, liver cancer, melanoma, mesothelioma, lung cancer, non-small cell lung cancer, head and neck cancer, neuroblastoma, glioblastoma multiforme, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, and thyroid cancer. Tumor cells can be isolated from primary tumors and metastases.

[0102] As used herein, the term "tumor sample" refers to tumor cells isolated from a subject. In certain embodiments, the tumor sample is wholly or partially isolated from at least a portion of a solid tumor of a subject suffering from a tumor. Subject tumor samples can be obtained using methods known in the art, generally by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Tumor samples can be isolated from a variety of cancer types, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, kidney cancer, lip and oral cavity cancer, liver cancer, melanoma, mesothelioma, lung cancer, non-small cell lung cancer, head and neck cancer, neuroblastoma, glioblastoma multiforme, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, and thyroid cancer. Tumor samples can be isolated from primary tumors and metastases. "Tumor sample" also includes a tumor sample that has been fragmented into "tumor fragments". Fragmentation can be physical fragmentation, mechanical fragmentation, sonication fragmentation, enzymatic fragmentation, or any combination thereof. Fragmentation can be carried out mechanically and optionally followed by enzymatic digestion of the tumor fragments into a single cell suspension. Mechanical dissociation methods can include chopping or slicing the tumor into smaller tumor fragments, while enzymatic dissociation methods can include treating the tumor fragments with a specific enzyme such as a protease.

[0103] As used herein, the term "culture medium" refers to a liquid or gel designed to support the survival, growth, and / or proliferation of cells in an artificial environment. The formulation of cell culture media is well known in the art. Typically, a culture medium generally contains a defined set of components, such components can include an energy source, growth factors, hormones, stimulants, activators, sugars, salts, vitamins, and / or amino acids, and / or combinations thereof, with the specific components or their concentrations depending on the requirements of the cells to be cultured and / or the desired cell culture parameters.

[0104] As used herein, the term "the components of the culture medium remain unchanged" refers to a culture medium containing a defined set of components (such as specific stimulants and activators), where the identity of the components remains constant, but the concentration of one or more components may vary. In certain embodiments, when cells are cultured in a culture medium, the concentration of one or more components in the culture medium changes over time. However, when the culture medium is replaced, each newly replaced culture medium has the same components.

[0105] As used herein, the term "perfusion" refers to a method of culturing cells such as TIL, in which a portion of the culture medium is continuously replaced with fresh culture medium without removing the cultured cells such as TIL.

[0106] The various aspects described herein are further elaborated in the following subsections.

[0107] Tumor-infiltrating lymphocytes (TIL)

[0108] "Tumor infiltrating lymphocytes" or "TIL" refers to a population of cells initially obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TIL includes, but is not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer (NK) cells, dendritic cells, and M1 macrophages. TIL includes primary TIL and secondary TIL. "Primary TIL" refers to TIL obtained from a subject tissue sample (sometimes referred to as "fresh harvested"), and "secondary TIL" refers to any population of TIL cells that have been expanded or proliferated as discussed herein, including but not limited to bulk TIL and expanded TIL ("pre-REP TIL" or "REP TIL" or "post-REP TIL"). In some embodiments, primary TIL also includes tumor-reactive T cells obtained from a subject's peripheral blood. A population of TIL cells can include genetically modified TIL. "TIL" also refers to a population of lymphocytes that have left the bloodstream of a subject, migrated into a tumor, and then left to re-enter the bloodstream.

[0109] TIL can generally be biochemically defined using cell surface markers or functionally defined by its ability to infiltrate tumors and affect treatment. TIL can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, TCRγδ, CD27, CD28, CD56, CCR7, CD45RA, CD45RO, CD95, PD-1, and CD25. Additionally, or alternatively, TIL can be functionally defined by its ability to infiltrate solid tumors after reintroduction into a patient. TIL can also be characterized by potency, e.g., TIL can be considered potent if interferon gamma (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL upon TCR stimulation.

[0110] Adoptive cell therapy using TIL cultured ex vivo by conventional TIL manufacturing methods involves at least two steps, i.e., there is at least one rapid expansion protocol (REP) step after the Pre-REP step.

[0111] Amplification of TIL

[0112] As generally outlined herein, TILs are typically derived from a sample taken from a subject and manipulated prior to transplantation into the subject to expand their numbers. In some embodiments, the TILs can be genetically manipulated as discussed below. Generally, TILs are initially obtained from a subject's tumor sample and then expanded into a larger population of TILs for further manipulation as described herein, optionally cryopreserved and restimulated, and optionally evaluated for phenotypic and metabolic parameters as an indication of TIL health.

[0113] Subject-derived tumor samples can be obtained using methods known in the art, such as via surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Once the tumor sample is obtained, the sample is typically fragmented by dissection into tumor fragments of about 0.5 mm 3 to 27 mm 3 in size, preferably fragmented into tumor fragments of 0.5 mm 3 to 4 mm 3 in size. Alternatively, a cell suspension can be obtained by combining enzymatic digestion with mechanical dissociation. Generally speaking, the harvested cell suspension is referred to as a "primary cell population" or "freshly harvested" cell population.

[0114] In some embodiments, after fragmenting the tumor sample by dissection, 5, 10, 20, 30, 40, 50, or more tumor fragments are placed in a culture vessel for expansion to obtain an expanded population of TIL cells.

[0115] In some embodiments, the TIL cell population can optionally be cryopreserved after sample harvest and prior to entering the expansion phase.

[0116] Conventional methods for TIL amplification

[0117] In conventional methods of expanding TILs, a multi-step approach is employed in addition to the need to use feeder cells. The multi-step approach includes at least one rapid expansion protocol (REP) step after a separate Pre-REP step.

[0118] The first amplification step in conventional multi-step TIL amplification: Pre-REP

[0119] Conventional multi-step TIL expansion methods begin with Pre-REP. Generally, Pre-REP starts with a tumor sample that has been fragmented and / or enzymatically digested and IL-2 is added thereto to slow down the growth of TILs in the cytokine-driven tumor sample. Generally, IL-2 is the only cytokine or immunomodulator added to Pre-REP. The Pre-REP or the first expansion step may take a time period between 2 weeks and several months.

[0120] In some embodiments, during Pre-REP, tumor tissue or cells from tumor tissue are grown in a standard medium (including but not limited to RPMI 1640 medium) and treated with reagents such as irradiated feeder cells and anti-CD3 antibody to achieve desired effects such as increasing the number of TILs and / or enriching a cell population containing desired cell surface markers or other structural, biochemical, or functional characteristics.

[0121] In some cases, after dissecting or digesting tumor fragments, the resulting cells are cultured in a medium containing IL-2 under conditions favorable for TIL growth over tumor and other cell growth. The tumor digest is incubated in 2 mL wells in a medium containing heat-inactivated human AB serum and 6000 IU / mL of IL-2. In some instances, 300 - 6000 IU / mL of IL-2 is added. During Pre-REP, this primary cell population is cultured for a period of days to months, thereby generating a large population of TILs, generally on the order of about 1×10 8 of a large number of TIL cells.

[0122] In a conventional method that includes a Pre-REP step, once the TILs have undergone expansion in the presence of IL-2 and reached an appropriate cell number required to initiate REP, or have undergone Pre-REP for a predetermined period of time, a demarcation between Pre-REP and REP is made. In various embodiments, depending on the circumstances, when the number of TILs obtained is 1x10 5 、1x10 6 、1x10 7 cells / fragment, Pre-REP amplification can be completed. In another embodiment, when the culture duration reached is 3 to 14 days or 9 to 14 days since fragmentation, Pre-REP amplification can be completed.

[0123] In some cases, the TILs obtained from the Pre-REP step are stored until phenotypic selection is performed. In some cases, the TILs obtained from the Pre-REP step are not stored but are directly subjected to the REP step. In some cases, the TILs obtained from the Pre-REP step are not cryopreserved between Pre-REP amplification and REP amplification. In the case of using genetically modified TILs for treatment, the population of Pre-REP TIL cells obtained after Pre-REP amplification can be genetically modified before performing the REP step.

[0124] The second and subsequent amplification steps in conventional multi-step TIL amplification: REP

[0125] In conventional multi-step TIL expansion, in some cases, after completion of pre-REP expansion, the subsequent large-scale expansion is called the rapid expansion process (REP). REP is generally completed using a culture medium containing multiple components, such as trophoblast cells, cytokines, and anti-CD3 antibodies. In some cases, REP can be carried out for 7 - 14 days or longer.

[0126] In some cases, the second expansion or REP can be carried out using methods known in the art. For example, it can be carried out in a supplemented cell culture medium containing IL-2, OKT-3, and antigen-presenting trophoblast cells. In some cases, the trophoblast cells are PBMCs (peripheral blood mononuclear cells). In some cases, the ratio of TIL to PBMC in the rapid expansion and / or second expansion is 1:25 to 1:500.

[0127] In some cases, REP also includes the step of selecting TILs to obtain excellent tumor reactivity. Any selection method known in the art can be used. Optionally, a cell viability assay can be carried out after REP expansion using standard assays known in the art. In some cases, a cell counter can be used to count and determine the viability of the TIL sample.

[0128] In some cases, multiple REP expansions can be carried out.

[0129] Trophoblast cells

[0130] In many cases, the trophoblast cells used in conventional multi-step trophoblast cell-dependent TIL expansion methods are cells capable of supporting the expansion of lymphocytes or their progeny. Trophoblast cells can secrete or express factors on the cell surface that support the expansion of progenitor cells. An example of trophoblast cells is peripheral blood mononuclear cells (PBMCs). Other non-limiting examples include splenocytes, lymph node cells, and dendritic cells. Trophoblast cells can also be cells that are not normally used as trophoblast cells, such as fibroblasts, which have been engineered to secrete or express factors necessary for supporting the expansion of T cell progenitors on their cell surface. Trophoblast cells can be autologous, allogeneic, syngeneic, artificial, or xenogeneic with respect to lymphocytes and / or the subject.

[0131] Feeder-free one-step technology (FOST)

[0132] The above-mentioned conventional trophoblast cell-dependent multi-step expansion method of TIL requires multiple steps and trophoblast cells, which makes the conventional expansion method time-consuming and expensive. Therefore, there is a need for a simpler, faster, and lower-cost method for manufacturing TIL.

[0133] To provide improved, faster, and simpler methods for generating TILs, the present invention provides a more simplified one-step amplification method that does not use trophoblast cells. In addition to enriching the amplified TIL population with CD8+ cytotoxic T cells and a stem cell-like T cell phenotype, a composition of the amplified TIL population is also provided.

[0134] In one aspect of the methods disclosed herein, the Pre-REP step of a conventional TIL amplification protocol is completely skipped. Surprisingly, in a trophoblast-free one-step amplification method without Pre-REP, a large number of TILs can be obtained within 24 days or less. In some embodiments, the TILs are engineered or genetically modified during the one-step TIL amplification.

[0135] In some embodiments, the methods of the present invention can rescue TIL samples that have failed conventional amplification. In some embodiments, the amplification failure refers to the failure to amplify a sufficient number of TILs after a sufficient culture duration through pre-REP. In some embodiments, pre-REP failure refers to the absence of TIL amplification at day 14 of the pre-REP step. In some embodiments, pre-REP failure refers to the failure to amplify the number of TIL cells isolated from a subject to 1×10 7 cells / fragment at the end point using the pre-REP protocol. In certain embodiments, the methods provided herein are capable of amplifying TIL cells from samples that have experienced pre-REP failure. In some embodiments, the methods described herein are capable of providing a greater number of TILs than conventional amplification techniques. In some embodiments, the methods described herein are capable of providing a clinically useful number of TILs.

[0136] In one aspect of the methods disclosed by the present invention, a method for one-step amplification of tumor-infiltrating lymphocytes includes culturing a tumor sample obtained from a subject in a medium containing a CD3 agonist, a 4-1BB agonist, and IL-2 to obtain an amplified population of tumor-infiltrating lymphocytes, and the medium does not contain trophoblast cells.

[0137] In some embodiments, the tumor sample is fragmented before culturing. In some embodiments, the tumor sample comprises tumor fragments sized from 0.5 mm 3 to 27 mm 3 , preferably fragmented into tumor fragments sized from 0.5 mm 3 to 4 mm 3 , more preferably fragmented into tumor fragments sized from 1 mm 3 to 3 mm 3 . In some embodiments, the tumor sample comprises digested tumor fragments.

[0138] In some embodiments, the final concentration of IL-2 in the medium is 6000 IU / mL or less, and in some preferred embodiments, the final concentration of IL-2 in the medium is 2000 IU / mL or less. In some preferred embodiments, the final concentration of IL-2 in the medium is 1000 IU / mL.

[0139] In some embodiments, the CD3 agonist comprises an anti-CD3 antibody and / or an antigen-binding fragment thereof, optionally a humanized anti-CD3 antibody and / or an antigen-binding fragment thereof.

[0140] In some embodiments, the CD3 agonist is OKT3.

[0141] In some embodiments, the final concentration of the CD3 agonist is from 10 ng / mL to 50 ng / mL. In some preferred embodiments, the final concentration of the CD3 agonist is 30 ng / mL.

[0142] In some embodiments, the 4-1BB agonist comprises a ligand-competitive 4-1BB agonistic antibody and / or an antigen-binding fragment thereof, or a ligand-noncompetitive 4-1BB agonistic antibody and / or an antigen-binding fragment thereof. In some preferred embodiments, the 4-1BB agonist is Utomilumab or Urelumab.

[0143] In some embodiments, the final concentration of the 4-1BB agonist is from 1 to 20 μg / mL. In some preferred embodiments, the final concentration of the 4-1BB agonist is 10 μg / mL.

[0144] In some embodiments, the components of the medium remain unchanged. In some embodiments, about 30% to about 99% of the medium is replaced every 2 to 5 days. In some embodiments, the medium is replaced by perfusion. In some embodiments, perfusion comprises continuous medium replacement at a rate of about 30% to about 99% of the working volume of the culture vessel per 24 hours.

[0145] In some embodiments, wherein replacing the medium comprises adding fresh medium to the culture, wherein the fresh medium contains IL-2 and does not contain a CD3 agonist and a 4-1BB agonist.

[0146] In some embodiments, wherein the fresh medium further comprises a cell basal medium, a serum replacement, L-glutamine or a substitute thereof, and an antibiotic. In some embodiments, the medium does not contain serum.

[0147] In some embodiments, the cell basal medium includes, but is not limited to, X-vivo15 medium, AIM-V medium, RPMI-1640 medium, OpTmizer TM medium or OpTmizer TM Pro medium.

[0148] In some embodiments, TIL expansion lasts for 14 - 28 days in total starting from initial tumor fragmentation. In some embodiments, TIL expansion lasts for at least 14 days starting from initial tumor fragmentation.

[0149] In some embodiments, the number of TIL cells harvested at the end of culture is above 1×10 7 cells per fragment, preferably above 1×10 8 cells per fragment, more preferably above 1×10 9 cells per fragment.

[0150] In some embodiments, the methods herein can salvage TIL samples from a previously failed pre-REP expansion. In some embodiments, the tumor sample is from a subject who has previously submitted a tumor sample for expansion, wherein the previous expansion includes a pre-REP step and wherein the pre-REP step shows no TIL expansion when cultured for 14 days. In some embodiments, the tumor sample is from a subject who has previously submitted a tumor sample for expansion, wherein the previous expansion includes a pre-REP step and the number of TILs isolated from the pre-REP step is below 1×10 7 cells per fragment when the pre-REP step is cultured to the end point.

[0151] In some embodiments, compared with the tumor-infiltrating lymphocyte population obtained by conventional multi-step amplification, the tumor-infiltrating lymphocyte population obtained by one-step amplification shows improved TIL characteristics.

[0152] In some embodiments, the improved TIL characteristics include one or more selected from the following group: increased TIL cell number and expansion ability, increased proportion of live cells, increased viability, improved proportion of T cell subsets, increased cytokine secretion ability, increased tumor cell killing ability, increased anti-exhaustion ability.

[0153] In some embodiments, the improved proportion of T cell subsets includes one or more selected from the following group: decreased proportion of regulatory T cells, increased proportion of stem-like T cells, increased proportion of cytotoxic T cells, and decreased proportion of exhausted T cells.

[0154] In some embodiments, after culturing the tumor sample obtained from a subject, CD3+ TILs in the expanded tumor-infiltrating lymphocyte population account for more than about 80%, preferably more than about 85%, more preferably more than about 90% of the total number of cells.

[0155] In some embodiments, after culturing the tumor sample obtained from a subject, CD8+ TILs in the expanded tumor-infiltrating lymphocyte population account for more than about 50% of CD3+ TILs.

[0156] In some embodiments, after culturing the tumor sample obtained from a subject, CD69- / CD39- TILs in the expanded tumor-infiltrating lymphocyte population account for more than about 10% of CD8+ TILs.

[0157] In some embodiments, after culturing the tumor sample obtained from a subject, exhausted T cells in the expanded tumor-infiltrating lymphocyte population account for less than about 50%, preferably less than about 20% of CD8+ TILs.

[0158] In some embodiments, after culturing the tumor sample obtained from a subject, Foxp3+ TILs in the expanded tumor-infiltrating lymphocyte population account for less than about 20% of CD4+ TILs.

[0159] In some embodiments, the cells of the expanded TIL population are genetically modified.

[0160] In some embodiments, the cell number of the TIL population is amplified to 1×10 6 ~1×10 7 per cell, and the expanded TIL population is genetically modified.

[0161] In some cases, TILs are genetically modified to include additional functions, including but not limited to high-affinity T cell receptors (TCRs), or chimeric antigen receptors (CARs) that bind to tumor-associated antigens.

[0162] In some embodiments, TILs are genetically modified to include TILs with one or more genomic modifications that result in reduced expression and / or function of one or more endogenous target genes, and immune effector cells comprising a gene editing system capable of reducing the expression and / or function of one or more endogenous target genes. In some embodiments, these endogenous genes include NR4A1, NR4A2, NR4A3, RC3H1, and ZC3H12A. In some embodiments, the modified TILs comprise one or more modifications in the genomic DNA sequence of the endogenous target gene, e.g., insertion, deletion, or mutation of one or more nucleic acids, resulting in reduced expression and / or function of the endogenous gene.

[0163] In some embodiments, compared to tumor-infiltrating lymphocytes (TILs) in which the endogenous genes are not genetically modified, the cells of the amplified tumor-infiltrating lymphocyte population show improved TIL characteristics after genetic modification of the endogenous genes.

[0164] In some embodiments, the improved TIL characteristics include one or more selected from the group consisting of: increased TIL cell number and expansion ability, increased viability, improved T cell subset ratio, enhanced cytokine secretion ability, enhanced tumor cell killing ability, and enhanced anti-exhaustion ability.

[0165] In some embodiments, the modified TILs described herein comprise a gene editing system (e.g., a nucleic acid-based gene editing system, a protein-based gene editing system, or a protein / nucleic acid combination-based gene editing system). In such embodiments, the gene editing system comprised in the modified TILs is capable of modifying one or more endogenous target genes.

[0166] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of one or more endogenous target genes and further comprise one or more exogenous transgenes inserted at one or more genomic loci (e.g., genetic “knock-in”). In some embodiments, one or more exogenous transgenes encode a T cell receptor and / or a chimeric antigen receptor.

[0167] In some embodiments, the present invention provides modified TILs that comprise reduced expression and / or function of one, two, or more endogenous target genes. In some embodiments, these endogenous genes include NR4A1, NR4A2, NR4A3, RC3H1, and ZC3H12A, and further comprise a CAR or a recombinant TCR expressed on the cell surface.

[0168] In some embodiments, the present invention provides modified TILs that comprise a gene editing system capable of reducing the expression and / or function of one or more endogenous target genes. In some embodiments, these endogenous genes include NR4A1, NR4A2, NR4A3, RC3H1, and ZC3H12A, and further comprise a CAR or a recombinant TCR expressed on the cell surface.

[0169] Tumor-infiltrating lymphocyte population

[0170] In another aspect, the present invention relates to a population of tumor-infiltrating lymphocytes obtained according to the methods disclosed herein.

[0171] In some embodiments, the CD3+ TILs in the tumor-infiltrating lymphocyte population account for more than about 80% of the total number of cells, preferably more than about 85%, more preferably more than about 90%.

[0172] In some embodiments, the CD8+ TILs in the tumor-infiltrating lymphocyte population account for more than about 50% of the CD3+ TILs.

[0173] In some embodiments, the CD69- / CD39- TILs in the tumor-infiltrating lymphocyte population account for more than about 10% of the CD8+ TILs.

[0174] In some embodiments, the exhausted T cells in the tumor-infiltrating lymphocyte population account for less than about 50% of the CD8+ TILs, preferably less than about 20%.

[0175] In some embodiments, the Foxp3+ TILs in the tumor-infiltrating lymphocyte population account for less than about 20% of the CD4+ TILs.

[0176] Drug compositions, dosages, and administration regimens

[0177] In one embodiment, the tumor-infiltrating lymphocyte population amplified by the method provided by the present invention, and optionally a pharmaceutically acceptable carrier, can be administered to a subject as a pharmaceutical composition. In one embodiment, the tumor-infiltrating lymphocyte population obtained according to the method disclosed herein or the tumor-infiltrating lymphocyte population disclosed herein or the pharmaceutical composition disclosed herein can be used to prepare a drug for preventing and / or treating tumors.

[0178] In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. In some embodiments, the TILs are administered as a single intra-arterial or intravenous infusion, preferably over about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0179] The TILs provided in the pharmaceutical composition of the present invention are effective within a wide dosage range. The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject to be treated, the gender and age of the subject to be treated, the weight of the subject to be treated, and the preference and experience of the attending physician. Clinically determined dosages of TILs can also be used where appropriate. The amount of the pharmaceutical composition administered using the methods herein, such as the dosage of TILs, will depend on the human or mammalian being treated, the severity of the condition or disorder, the rate of administration, the disposition of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

[0180] In some embodiments, the therapeutically effective dose is about 4×10 10 to about 1×10 11 TILs.

[0181] In some embodiments, TIL can be administered in a single dose. Such administration can be by injection, such as intravenous injection. In some embodiments, TIL can be administered in multiple doses. The dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. The dosing can be once per month, once every two weeks, once per week, or once every other day. TIL can be administered continuously as long as necessary.

[0182] An effective amount of TIL can be administered in a single or multiple doses by any of the accepted modes of administration of agents having similar utility, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, topical, by implantation, or by inhalation. In certain embodiments, TIL is administered intravenously.

[0183] In some embodiments, the medicament of the present invention is used for preventing and / or treating solid tumors. Exemplary solid tumors can include, but are not limited to, those selected from melanoma, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0184] Adoptive cell transfer

[0185] Adoptive cell transfer (ACT) is a very effective form of immunotherapy and involves transferring immune cells with anti-tumor activity into a cancer patient. ACT includes identifying lymphocytes with anti-tumor activity in vitro, expanding these cells in large numbers in vitro, and infusing them into a host with cancer. The lymphocytes for adoptive transfer can be derived from the stroma of a resected tumor (tumor-infiltrating lymphocytes or TIL). The population of TIL cells for ACT can be obtained according to the methods disclosed herein.

[0186] In some embodiments, the present invention provides a method for treating a tumor in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a population of tumor-infiltrating lymphocytes obtained according to the methods disclosed herein or a population of tumor-infiltrating lymphocytes disclosed herein or a pharmaceutical composition disclosed herein, and treating cancer by transferring a population of TIL cells that elicits an immune response.

[0187] In some embodiments, the present treatment method is applicable to solid tumors. Exemplary solid tumors can include, but are not limited to, those selected from melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

[0188] Examples

[0189] The present invention generally described herein will be more easily understood by reference to the following examples, from which those skilled in the art can draw on the content herein and appropriately improve process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be within the scope of protection of the present invention. The following examples are provided by way of illustration and are not intended to limit the present invention. These examples are not intended to represent that the following experiments are all or only the experiments conducted.

[0190] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0191] Main materials and reagents

[0192]

[0193]

[0194]

[0195] Culture medium formulation

[0196] (1) Complete medium CM1: 1L Optmizer + 26mL supplement

[0197] (2) Complete medium CM2: CM-1 + 5% mL SR + 10% Glutamax + 1% PS

[0198] (3) Complete medium CM3000: CM-2 + 3000IU / mL IL-2

[0199] Tumor tissue processing

[0200] Receive tumor tissues from subjects, soak the tumor tissue blocks in 10% gentamicin for 5 min, and then soak them in 10% clindamycin for 5 min. Then rinse them twice with CM1 medium. Cut the tissue blocks into small pieces of 1 - 3 mm 3 in size.

[0201] Conventional two-step method for amplifying tumor-infiltrating lymphocytes

[0202] Amplification in the Pre-REP stage

[0203] The obtained tissue pieces were seeded into Grex6 according to the experimental groups for culture. On day 0, 20 mL of CM2 medium was added, and the cells were cultured in a 37 °C, 5% CO2 incubator. On day 5, the medium was supplemented to 40 mL with CM2 medium. Half-medium changes were performed on days 7, 9, and 11. From days 11 to 14, when the cell number reached 5×10 5 or more, pre-REP was completed, and pre-REP TIL cells were harvested.

[0204] Amplification in the REP stage

[0205] The pre-REP TIL cells and trophoblast cells were resuspended with CM3000 medium. 1×10 5 pre-REP TIL cells and 2×10 7 trophoblast cells were inoculated into Grex24, 2 mL of CM3000 medium was added, and then OKT3 was added to a final concentration of 30 ng / mL. The mixture was blown evenly and cultured in a 37 °C, 5% CO2 incubator. On day 2, the medium was supplemented to 8 mL. Half-medium changes were performed on days 5, 7, 9, and 11. When the cells grew to confluence, the cells were transferred to Grex6 for culture. From days 11 to 14, when the cell number reached 5×10 9 or more, REP was completed, and TIL cells were harvested. When the cells were passaged or harvested, samples were taken for subsequent detection.

[0206] Feeder-free one-step method for amplifying tumor-infiltrating lymphocytes

[0207] On day 0, the obtained tissue pieces were seeded into Grex6 according to the experimental groups. 20 mL of CM2 medium containing 10 μg / mL 4-1BB agonist antibody (Utomilumab or Urelumab), 30 ng / mL CD3 agonist antibody (OKT3), IL-2 (500 IU / mL, 1000 IU / mL, 2000 IU / mL, 6000 IU / mL), 1% PS, and 10 μg / mL primocin was added to each well, and 5 tissue pieces were seeded per well. On day 5, the medium was supplemented to 40 mL with CM2 medium. Every 2 - 5 days, when the medium color turned yellow and the cells grew to confluence, half-medium changes and cell counting were performed. When the cell number per well reached 2×10 8 , 1×10 7 -2×10 7 cells were taken for passage. From days 17 to 28, when the cell number reached 5×10 9 or more, amplification was completed, and TIL cells were harvested. When the cells were passaged or harvested, samples were taken for subsequent detection.

[0208] Flow cytometry detection

[0209] After receiving the sample, check the sample information (including: name, quantity, number of cells). After balancing the sample, centrifuge it at 400×g for 5 min in a centrifuge and discard the supernatant. Adjust the cell density to 1×10 7 cells / mL using PBS. Then, take 100 μL of the cell suspension and place it in an EP tube. Centrifuge the sample at 400×g for 5 min in a low-temperature centrifuge and discard the supernatant.

[0210] Cell surface markers

[0211] Add 100 μL of the prepared mixed antibody to the sample tube, incubate it in the dark at room temperature for 20 min, and record the start and end times. After completion, add 200 μL of PBS to the sample tube to terminate the reaction. Centrifuge the sample at 400×g for 5 min in a low-temperature centrifuge, and then discard the supernatant and subject it to detection on the machine.

[0212] Nuclear markers

[0213] Prepare the fixation and permeabilization solution according to the following table:

[0214]

[0215] Prepare 1× washing buffer according to the following table:

[0216]

[0217] Add 200 μL of the prepared fixation and permeabilization solution to the sample tube, incubate it at 4°C for 0.5 h, and record the start and end times.

[0218] Add 100 μL of 1× washing buffer to the sample tube to terminate the reaction. Centrifuge the sample at 1500 g for 15 s in a refrigerated centrifuge and discard the supernatant.

[0219] Add 100 μL of the prepared nuclear antibody to the sample tube and incubate it in the dark at 4°C overnight.

[0220] Add 100 μL of 1× washing buffer to the sample tube to terminate the reaction. Centrifuge the sample at 1500 g for 15 s in a refrigerated centrifuge and discard the supernatant.

[0221] Resuspend the sample in 200 μL of 1× washing buffer and wait for detection on the machine.

[0222] Cytotoxicity detection

[0223] Target cell preparation

[0224] P815 cells were cultured in P815 complete medium (DMEM + 10% FBS) in an incubator at 37°C with 5% CO2. These cells grow in a semi-adherent and semi-suspended manner and are passaged once every 2 - 3 days. They are used within 20 passages after resuscitation.

[0225] P815 cells in the logarithmic growth phase were taken, and the cell suspension medium was collected into a 50 mL centrifuge tube. 3 - 5 mL of trypsin was added to the culture flask and incubated in an incubator at 37°C for about 2 minutes. When the culture flask was shaken and it was observed that the adherent cells were slightly detached, P815 complete medium was immediately added to terminate digestion, and the cells were pipetted and mixed evenly. The digested cells were transferred to the centrifuge tube. Centrifugation was carried out at 400×g for 5 minutes to discard the supernatant, and the cells were resuspended with 4 - 6 mL of P815 complete medium. The cells were counted and the required cell density was adjusted according to the seeding volume to 1×10 5 cells / mL.

[0226] 100 μl of P815 cells were seeded in the cell plate. 100 μl of DPBS or other sterile test solutions were added around to avoid edge effects. It was placed in the incubator and incubated for 3 - 6 hours to allow it to adhere to the wall.

[0227] Effector cell preparation

[0228] The information on the inspection requisition was checked. 10 mL / tube of CM1 medium was taken according to the detection amount and preheated in a water bath at 37°C. The cryopreserved effector cells were resuscitated and transferred to the preheated CM1 medium. Centrifugation was carried out at 400×g for 5 minutes to discard the supernatant. According to the labeled cell number, the cells were resuspended in CM1 medium containing 0.5 μg / mL OKT3 to about 3×10 6 cells / mL and counted. The density was adjusted to 1×10 6 cells / mL respectively, and then serially diluted to 5×10 5 、2.5×10 5 、1.25×10 5 、0.625×10 5 cells / mL.

[0229] Cytotoxicity assay

[0230] The 96 - well plate without added effector cells was put into the instrument for photographing as the 0 h time point. After the photographing was completed, the 96 - well plate was taken out and effector cells were added. 100 μl of effector cells were seeded into the corresponding wells, and each sample had duplicate wells for each effector - target ratio. In the control group without added effector cells, 100 μl of CM1 medium containing 0.5 μg / mL OKT3 was supplemented. After the cell seeding was completed, it was confirmed that there were no bubbles, and it was immediately put into the instrument, and signals were collected at intervals of 3 hours each time.

[0231] Data analysis

[0232] Calculate the killing rate at each time point and effector-to-target ratio according to the following formula.

[0233]

[0234] Using time (h) as the abscissa and the killing rate as the ordinate, plot the killing ability curves under various effector-to-target ratio conditions.

[0235] Detection of interferon-γ secretion level

[0236] Take the TILs cells to be tested, centrifuge at 300×g for 5 min, collect the cell supernatant, dilute it by an appropriate multiple, and then detect it according to the operation instructions of the human interferon-γ ELISA kit (ACROBiosystems, CRS-A017).

[0237] Example 1: One-step amplification without trophoblast cells can improve the proliferation ability of TILs

[0238] Tumor samples from 4 subjects (RC0021, LC002, EC001, LC002) were amplified using the conventional two-step method and the one-step method without trophoblast cells respectively, and cell counts were taken on the 18th day, 14th day, 13th day and 17th day respectively.

[0239] Analysis of the proliferation ability of TILs obtained by one-step amplification without trophoblast cells is as Figure 1 , Figure 2 shown. The TIL culture results from 4 subjects showed that after the same culture time, more TILs cells were obtained by one-step amplification without trophoblast cells, indicating that one-step amplification without trophoblast cells can improve the proliferation ability of TILs and shorten the culture cycle of TILs amplification.

[0240] Example 2: Phenotype of tumor-infiltrating lymphocytes obtained by one-step amplification without trophoblast cells

[0241] Tumor samples from different subjects were amplified using the conventional two-step method and the one-step method without trophoblast cells respectively, and samples were taken at the end of culture to detect the phenotypic characteristics of the TILs population by flow cytometry.

[0242] The results of the phenotypic characteristics of the TILs population obtained by one-step amplification without trophoblast cells are as Figures 3 to 5 shown.

[0243] Figure 3It represents the proportion of CD8+ T cells in the CD3+ T cell population of TILs groups obtained by amplification using the conventional two-step method and the one-step method without trophoblast cells. The results show that, compared with the conventional two-step method, the one-step method without trophoblast cells can significantly increase the proportion of CD8+ T cells in the amplified TILs group.

[0244] Figure 4 It represents the proportion of exhausted T cells in the CD8+ T cell population of TILs groups obtained by amplification using the conventional two-step method and the one-step method without trophoblast cells. Among them Figure 4 A represents the proportion of TIM-3-positive T cells, which are markers of exhausted T cells, in the CD8+ T cell population, Figure 4 B represents the proportion of BLIMP-1-positive T cells, which are pro-exhaustion transcription factors, in the CD8+ T cell population. The results show that, compared with the conventional two-step method, the one-step method without trophoblast cells can significantly reduce the proportion of exhausted T cells in the TILs group, such as a lower proportion of TIM-3-positive T cells and / or BLIMP-1-positive T cells.

[0245] Figure 5 It represents the proportion of CD39- / CD69- stem T cells in the CD8+ T cell population of TILs groups obtained by amplification using the conventional two-step method and the one-step method without trophoblast cells. The results show that, compared with the conventional two-step method, the one-step method without trophoblast cells can significantly increase the proportion of stem T cells in the TILs group.

[0246] Example 3: One-step amplification without trophoblast cells improves the killing ability of tumor-infiltrating lymphocytes

[0247] Tumor samples from different subjects were amplified using the conventional two-step method and the one-step method without trophoblast cells respectively, and samples were taken at the end of culture to detect the cytokine proportion and killing ability of the TILs group.

[0248] Figure 6 It represents the detection results of the cytokine proportion of CD8+ T cells in the TILs group obtained by amplification using the conventional two-step method and the one-step method without trophoblast cells. Among them Figure 6 A represents the proportion of interferon γ, Figure 6 B represents the proportion of granzyme B. The results show that, compared with the conventional two-step method, the one-step method without trophoblast cells can significantly increase the cytokine proportion of CD8+ T cells in the TILs group. For example, a higher proportion of interferon γ or a higher proportion of granzyme B.

[0249] Figure 7 It represents the results of the cell killing ability of the TILs group obtained by amplification using the conventional two-step method and the one-step method without trophoblast cells. Among them Figure 7 A represents a tumor sample from lung cancer, Figure 7B represents a tumor sample derived from ovarian cancer. The results show that compared with the conventional two-step method, the TILs population obtained by one-step amplification without trophoblast cells has stronger target cell killing ability.

[0250] Example 4: Effect of low IL-2 concentration on the phenotype of tumor-infiltrating lymphocytes obtained by one-step amplification

[0251] To further investigate the effect of IL-2 concentration on amplification using the one-step method without trophoblast cells, TILs were amplified by one-step method using media containing different final concentrations of IL-2, and samples were taken at the end of culture to detect the proliferation ability and phenotypic characteristics of the TILs population.

[0252] The results of the proliferation ability and phenotypic characteristics of the TILs population obtained by one-step amplification without trophoblast cells using media containing different final concentrations of IL-2 are as Figures 2 to 5 and Figures 8 to 10 shown.

[0253] Figure 2 and Figure 8 represent the amplification numbers of the TILs population obtained by one-step amplification using media containing different final concentrations of IL-2. The results show that using media with a lower final concentration of IL-2 has no significant effect on the cell number of the TILs population obtained by one-step amplification.

[0254] Figure 9 represents the proportion of CD3+ T cells in the total cell amount of the TILs population obtained by one-step amplification using media containing different final concentrations of IL-2. The results show that using media with a lower final concentration of IL-2 has no significant effect on the proportion of CD3+ cells in the TILs population obtained by one-step amplification.

[0255] Figure 3 represents the proportion of CD8+ T cells in CD3+ T cells of the TILs population obtained by one-step amplification using media containing different final concentrations of IL-2. The results show that using media with a lower final concentration of IL-2 has no significant effect on the proportion of CD8+ T cells in the TILs population obtained by one-step amplification.

[0256] Figure 4 represents the proportion of exhausted T cells in CD8+ T cells of the TILs population obtained by one-step amplification using media containing different final concentrations of IL-2. Among them Figure 4 A represents the proportion of TIM-3-positive T cells, which are markers of exhausted T cells, in CD8+ T cells, Figure 4B represents the proportion of exhausted transcription factor BLIMP-1 positive T cells among CD8+ T cells. The results showed that in the TILs population amplified by the one-step method using a medium with a lower final concentration of IL-2, the proportion of exhausted T cells was lower, such as the proportion of TIM-3 positive T cells and / or BLIMP-1 positive T cells was lower.

[0257] Figure 5 represents the proportion of CD39- / CD69- stem T cells among CD8+ T cells in the TILs population amplified by the one-step method using a medium with different final concentrations of IL-2. The results showed that in the TILs population amplified by the one-step method using a medium with a lower final concentration of IL-2, the proportion of stem T cells was higher.

[0258] Figure 10 represents the proportion of Foxp3+ Treg cells among CD4+ T cells in the TILs population amplified by the one-step method using a medium with different final concentrations of IL-2. Among them Figure 10 A represents a tumor sample derived from renal cell carcinoma, Figure 10 B represents a tumor sample derived from ovarian cancer. The results showed that using a medium with a lower final concentration of IL-2 did not increase the proportion of Treg cells in the TILs population amplified by the one-step method.

[0259] Example 5: Effect of low IL-2 concentration on the killing ability of tumor-infiltrating lymphocytes amplified by the one-step method

[0260] To further study the effect of IL-2 concentration on TIL amplification by the one-step method without feeder cells, TILs were amplified by the one-step method using a medium with different final concentrations of IL-2, and samples were taken at the end of culture to detect the killing ability of the TILs population.

[0261] Figure 11 represents the results of the cell killing ability of the TILs population amplified by the one-step method using a medium with different final concentrations of IL-2. The results showed that the target cell killing ability of the TILs population amplified by the one-step method using a medium with a lower final concentration of IL-2 was similar to that using a medium with a high final concentration of IL-2.

[0262] Example 6: Effect of different T cell co-stimulatory molecule agonists on the proportion of CD8+ cells in tumor-infiltrating lymphocytes amplified by the one-step method

[0263] To further investigate the effects of different T cell costimulatory molecule agonists on the expansion of tumor-infiltrating lymphocytes (TILs) using the feeder cell-free one-step method, TILs were expanded by the one-step method in media containing the 4-1BB agonist or the CD28 agonist (at approximately 50 ng / mL), and samples were taken at the end of the culture to detect the phenotypic characteristics of the TIL population respectively.

[0264] Figure 12 It represents the proportion of CD8+ T cells in CD3+ T cells in the TIL population obtained by one-step expansion in media containing the 4-1BB agonist or the CD28 agonist. The results showed that when the T cell costimulatory molecule agonist was the 4-1BB agonist, the median proportion of CD8+ T cells in CD3+ T cells in the TIL population obtained by one-step expansion was 72.9%; when the T cell costimulatory molecule agonist was the CD28 agonist, the median proportions of CD8+ T cells in CD3+ T cells in the TIL population obtained by one-step expansion were 33.8% (IL-2 concentration was 6000 IU / mL) and 17.4% (IL-2 concentration was 1000 IU / mL), respectively. The above results indicate that when the T cell costimulatory molecule agonist is the 4-1BB agonist, it can significantly increase the proportion of CD8+ T cells in the TIL population obtained by one-step expansion.

[0265] Example 7: Effects of different 4-1BB agonistic antibodies on the phenotype of tumor-infiltrating lymphocytes obtained by one-step expansion

[0266] To further investigate the effects of different 4-1BB agonistic antibodies on the expansion using the feeder cell-free one-step method, TILs were expanded by the one-step method in media containing the 4-1BB agonistic antibodies Utomilumab or Urelumab (IL-2 concentration was 1000 IU / mL), and samples were taken at the end of the culture to detect the proliferation ability, phenotypic characteristics and cytokine ratio of the TIL population.

[0267] The results of the proliferation ability and phenotypic characteristics of the TIL population obtained by one-step expansion using media containing Utomilumab or Urelumab are as Figures 13 to 18 shown.

[0268] Figure 13 It represents the cell number of the TIL population obtained by one-step expansion using media containing Utomilumab or Urelumab. The results showed that using media containing different 4-1BB agonistic antibodies had no significant effect on the cell number of the TIL population obtained by one-step expansion.

[0269] Figure 14It represents the proportion of CD3+ T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab. The results showed that using media containing different 4-1BB agonistic antibodies had no significant effect on the proportion of CD3+ T cells in the TILs population obtained by one-step amplification. In samples from some subjects, using a medium containing Utomilumab could obtain a higher proportion of CD3+ T cells.

[0270] Figure 15 It represents the proportion of CD8+ T cells in the CD3+ T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab. The results showed that using media containing different 4-1BB agonistic antibodies had no significant effect on the proportion of CD8+ T cells in the TILs population obtained by one-step amplification.

[0271] Figure 16 It represents the proportion of exhausted T cells in the CD8+ T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab. Among them Figure 16 A represents the proportion of TIM-3 positive T cells, which are markers of exhausted T cells, in the CD8+ T cells, Figure 16 B represents the proportion of BLIMP-1 positive T cells, which are pro-exhaustion transcription factors, in the CD8+ T cells. The results showed that using media containing different 4-1BB agonistic antibodies had no significant effect on the proportion of exhausted T cells in the TILs population obtained by one-step amplification.

[0272] Figure 17 It represents the proportion of CD39- / CD69- stem T cells in the CD8+ T cells in the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab. The results showed that using media containing different 4-1BB agonistic antibodies had no significant effect on the proportion of stem T cells in the TILs population obtained by one-step amplification.

[0273] Figure 18 It represents the detection result of the proportion of granzyme B in the CD8+ T cells of the TILs population obtained by one-step amplification using a medium containing Utomilumab or Urelumab. The results showed that using media containing different 4-1BB agonistic antibodies had no significant effect on the proportion of granzyme B in the CD8+ T cells of the TILs population obtained by one-step amplification.

[0274] Example 8: One-step amplification of tumor-infiltrating lymphocytes that failed in two-step amplification without trophoblast cells

[0275] Tumor samples from subjects LC002 and RC002 failed during previous amplification using the conventional two-step method. Among them, the tumor sample from subject LC002 had no TIL amplification after 14 days of culture, and the number of cell amplifications of the tumor sample from subject RC002 was less than 5×10 7 cells.

[0276] Tumor samples from this subject were further amplified using a one-step method without trophoblast cells, and samples were taken at the end of culture to detect the number of TILs cells.

[0277] Figure 19 It was shown that the amplification of tumor samples from subjects LC002 and RC002 using the one-step method without trophoblast cells was successful.

[0278] Example 9: Large-scale culture of the one-step method without trophoblast cells

[0279] This example provides an exemplary protocol (batch R1FY16O1) for large-scale production of TIL.

[0280] On day 0, tumor samples obtained from the subject were cut into small pieces about 1-3 mm 3 in size; 100 mL of FOST activation medium and 50 cut tissue pieces were placed into a G-Rex 100M and transferred to a carbon dioxide incubator for culture (temperature: 37±1°C, CO2 concentration: 5±0.5%); counting was performed every three days and FOST amplification medium (100 mL each time) was supplemented. When the total number of live cells was greater than 5×10 8 cells, all the cells were transferred to a cell expansion system (XURI), and the cell density was adjusted to 1×10 6 cells / mL; when the total number of live cells was greater than 5×10 9 cells, the perfusion program of the cell expansion system (XURI) was selected, and the perfusion rate was 1.5 L / day on the first day of perfusion; the perfusion rate was 3.5 L / day on the second day of perfusion; after the third day of perfusion, the perfusion rate was 5.0 L / day for perfusion. When the total number of live cells was greater than 5×10 10 cells, the TILs cell population was harvested.

[0281] Figure 20 The amplification curve and viability of the TILs population obtained through large-scale TIL production were shown. This batch was a 5L system, the amplification took 17 days, and the final harvest of TILs cells reached 4.11×10 10 , and the viability reached 97.3%.

[0282] Figure 21Shows the phenotypic characteristics of TILs populations obtained by large-scale TIL production sampled at three time points: day 10, day 12 of culture, and final harvest. The results show that the proportion of CD3+ T cells remains basically consistent at the three different time points; the proportion of CD8+ T cells decreases slightly with the increase of culture time; the proportion of CD39- / CD69- T cells in both CD4+ and CD8+ T cells increases with the increase of culture time; the proportion of TIM-3+ T cells in CD4+ T cells decreases with the increase of culture time, while the proportion of TIM-3+ T cells in CD8+ T cells increases slightly with the increase of culture time.

[0283] Figure 22 Shows the cytokine proportions and secretion capabilities of TILs populations obtained by large-scale TIL production sampled at three time points: day 10, day 12 of culture, and final harvest. The results show that intracellularly, in CD8+ T cells, the proportion of granzyme B reaches the highest at day 12; in CD8+ T cells, the proportion of perforin decreases slightly with the increase of culture time; the proportion of interferon-γ reaches the highest at harvest; extracellularly, the secretion level of interferon-γ increases with the increase of cell culture time.

[0284] Figure 23 Shows the target cell killing capabilities of TILs populations obtained by large-scale TIL production sampled at three time points: day 10, day 12 of culture, and final harvest. The results show that at an effector-to-target ratio of 0.625:1, the sample sampled on day 12 has the strongest 24-hour killing ability; at an effector-to-target ratio of 1.25:1, the sample sampled on day 12 has the strongest 24-hour killing ability; at an effector-to-target ratio of 2.5:1, the samples at the three time points can all reach a killing rate of over 95% within 24 hours.

[0285] It should be understood that although the present invention has been described by way of example according to its preferred embodiments, it should not be limited to the above embodiments. For those skilled in the art, the present invention can have various modifications and changes. The selection and application of specific technical solutions can be adjusted and changed according to specific needs. Therefore, for those skilled in the art, within the scope of the concept and principle of the present invention, several simple substitutions can still be made, and these should all be included in the protection scope of the present invention.

Claims

1. A method for one-step expansion of tumor infiltrating lymphocytes, the method comprising culturing a tumor sample obtained from a subject in a culture medium containing a CD3 agonist, a 4-1BB agonist and IL-2, thereby obtaining an expanded tumor infiltrating lymphocyte population, and the culture medium does not contain trophoblast cells.

2. The method of claim 1, wherein the tumor sample is fragmented before culturing.

3. The method according to claim 1, wherein the tumor sample comprises a size of 0.5 mm 3 Up to 27mm 3 of tumor fragments.

4. The method of claim 1, wherein the tumor sample comprises digested tumor fragments.

5. The method according to claim 1, wherein the final concentration of IL-2 in the culture medium is 6000 IU / mL or less, preferably 2000 IU / mL or less. The method according to claim 2 , wherein the final concentration of IL-2 in the culture medium is 1000 IU / mL.

7. The method according to claim 1, wherein the CD3 agonist comprises an anti-CD3 antibody and / or an antigen-binding fragment thereof, optionally a humanized anti-CD3 antibody and / or an antigen-binding fragment thereof.

8. The method of claim 7, wherein the CD3 agonist is OKT3.

9. The method according to claim 1, wherein the final concentration of the CD3 agonist is 10 ng / mL to 50 ng / mL.

10. The method according to claim 9, wherein the final concentration of the CD3 agonist is 30 ng / mL.

11. The method according to claim 1, wherein the 4-1BB agonist comprises a ligand-competitive 4-1BB agonist antibody and / or an antigen-binding fragment thereof, or a ligand-noncompetitive 4-1BB agonist antibody and / or an antigen-binding fragment thereof.

12. The method according to claim 11, wherein the 4-1BB agonist is Utomilumab or Urelumab.

13. The method according to claim 1, wherein the final concentration of the 4-1BB agonist is 1-20 μg / mL. The method according to claim 13 , wherein the final concentration of the 4-1BB agonist is 10 μg / mL.

15. The method of claim 1, wherein the culture medium is changed every 2 to 5 days.

16. The method of claim 15, wherein the medium replacement comprises adding fresh medium to the culture, wherein the fresh medium comprises IL-2 and does not comprise a CD3 agonist and a 4-1BB agonist.

17. The method of claim 1, wherein the TIL culture continues for 14-28 days.

18. The method of claim 17, wherein the TIL culture is continued for at least 14 days.

19. The method of claim 1, wherein the tumor sample is from a subject who has previously submitted a tumor sample for expansion, wherein the previous expansion includes a pre-REP step and wherein the pre-REP step cultured to 14 days without TIL expansion or the number of TILs isolated from the pre-REP step is less than 1×10 7 Less than one piece per fragment.

20. The method of claim 1, wherein the tumor infiltrating lymphocyte population obtained by one-step expansion exhibits improved TIL characteristics compared to the tumor infiltrating lymphocyte population obtained by conventional multi-step expansion.

21. The method according to claim 20, wherein the improved TIL characteristics comprise one or more selected from the following groups: increased TIL cell number and expansion capacity, increased proportion of live cells, increased survival capacity, improved T cell subset ratio, increased cytokine secretion capacity, increased tumor cell killing ability, and improved anti-exhaustion ability.

22. The method according to claim 21, wherein the improved T cell subpopulation ratio comprises one or more selected from the following group: a decreased ratio of regulatory T cells, an increased ratio of stem T cells, an increased ratio of cytotoxic T cells, and a decreased ratio of exhausted T cells.

23. The method of claim 22, wherein after culturing the tumor sample obtained from the subject, at least about 50% of the CD3+TILs in the expanded tumor-infiltrating lymphocyte population are CD8+TILs.

24. The method of claim 22, wherein after culturing the tumor sample obtained from the subject, at least about 10% of the CD8+ TILs in the expanded tumor infiltrating lymphocyte population are CD69- / CD39- TILs.

25. The method of claim 1, further comprising genetically modifying cells of the expanded tumor-infiltrating lymphocyte population.

26. The method of claim 25, further comprising genetically modifying cells of the expanded tumor-infiltrating lymphocyte population using a gene editing system, wherein the gene editing system is optionally selected from a gene editing system comprising RNA interference molecules, transcription activator-like effector nucleases, zinc finger nucleases, and RNA-guided nucleases.

27. The method of claim 26, wherein the gene editing system comprises a Cas enzyme, optionally a Cas9 enzyme, and a gRNA.

28. The method of any one of claims 25 to 27, wherein the cells of the expanded tumor infiltrating lymphocyte population comprise a modification, optionally an insertion, deletion, indel or substitution, at one or more endogenous genes selected from the group consisting of NR4A1, NR4A2, NR4A3, RC3H1 and ZC3H12A, optionally wherein the modification results in a reduction or inhibition of expression of the one or more endogenous genes and / or a reduction or inhibition of function of one or more proteins encoded by the one or more endogenous genes.

29. The method of any one of claims 25 to 27, wherein the cells of the expanded tumor infiltrating lymphocyte population are genetically modified to exhibit improved TIL properties compared to TILs that have not been genetically modified.

30. The method according to claim 29, wherein the improved TIL characteristics comprise one or more selected from the following groups: increased TIL cell number and expansion capacity, increased survival capacity, improved T cell subset ratio, increased cytokine secretion capacity, increased tumor cell killing ability, and increased anti-exhaustion ability.

31. A tumor infiltrating lymphocyte population obtained by the method according to any one of claims 1-30.

32. A pharmaceutical composition comprising the tumor infiltrating lymphocyte population according to claim 31, and optionally a pharmaceutically acceptable carrier.

33. Use of the tumor infiltrating lymphocyte population obtained according to any one of claims 1 to 30, the tumor infiltrating lymphocyte population according to claim 31, or the pharmaceutical composition according to claim 32 in the preparation of a drug for preventing and / or treating tumors.

34. The use according to claim 33, wherein the tumor is a solid tumor.

35. The use according to claim 35, wherein the tumor is selected from one or more of the following groups: melanoma, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, and kidney cancer.

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