Trophoblast-free one-step method for culturing tumor infiltrating lymphocytes and use thereof

Abstract

A trophoblast-free one-step method for culturing tumor-infiltrating lymphocytes and the use thereof. Specifically disclosed is a one-step method for expanding tumor-infiltrating lymphocytes. The method comprises processing a tumor sample obtained from a subject into tumor fragments, and culturing the tumor fragments in a culture medium containing a CD3 agonist, a 4-1BB agonist and IL-2, so as to obtain an expanded population of tumor-infiltrating lymphocytes, wherein the culture medium does not contain trophoblast cells. The method requires a shorter period and a lower cost for expanding tumor-infiltrating lymphocytes, and results in a stronger cellular effector function, which helps to broaden the clinical application range of tumor-infiltrating lymphocyte therapy.

Description

A one-step culture method and application of tumor-infiltrating lymphocytes without trophoblast layer Technical Field

[0001] This invention relates to the field of cell therapy, and in particular to a one-step method and application for the culture of tumor-infiltrating lymphocytes without a trophoblast layer. Background Technology

[0002] Adoptive metastasis of tumor-infiltrating lymphocytes (TILs) has proven to be an effective treatment for patients with poor prognosis and refractory solid tumors. However, one of the keys to achieving adoptive metastasis of TILs is obtaining a sufficient number of functional TILs, which places high demands on the in vitro expansion process of TILs.

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

[0004] In addition, existing TILs amplification methods face other challenges, such as the weak amplification capacity of TILs derived from patient tumors, difficulty in controlling the proportion of CD8+ cytotoxic T cells, insufficient killing ability against tumor cells, and the potential for T cell depletion due to high-concentration IL-2 culture. These factors collectively affect the efficacy of TILs therapy.

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

[0006] This invention provides a method for expanding tumor-infiltrating lymphocytes. The method provided in this application shortens the time required to expand TIL populations through a one-step expansion process and eliminates the need for feeder cells, thus offering clinical advantages for adoptive TIL transfer therapy. Furthermore, the method provided in this application can also be used to enrich stem T cell populations and cytotoxic T cell populations.

[0007] As disclosed herein, it was unexpectedly discovered that TILs can be expanded in the absence of trophoblast cells by providing a combination of CD3 agonists, 4-1BB agonists, and IL-2. Surprisingly, the trophoblast-free TIL expansion method described herein yields a therapeutically effective number of TILs in a shorter time and also results in enrichment of stem and cytotoxic T cell populations, as well as a reduced proportion of exhausted T cells.

[0008] As disclosed in this paper, it was also unexpectedly discovered that the TIL group can be amplified in one step, 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, wherein the culture medium does not contain trophoblast cells.

[0010] In some implementations, the tumor sample is fragmented before culture.

[0011] In some embodiments, the tumor sample comprises a sample with a size of 0.5 mm. 3 Up to 27mm 3 The tumor sample contains tumor fragments. 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 below 6000 IU / mL; in some preferred embodiments, the final concentration of IL-2 in the culture medium is below 2000 IU / mL; and 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 its antigen-binding fragment, optionally humanized with the anti-CD3 antibody and / or its antigen-binding fragment.

[0014] In some implementations, 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 agonist antibody and / or its antigen-binding fragment, or a ligand-non-competitive 4-1BB agonist antibody and / or its antigen-binding fragment. 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 composition of the culture medium remains constant. In some embodiments, approximately 30% to approximately 99% of the culture medium is replaced every 2 to 5 days.

[0019] In some embodiments, the replacement of the culture medium includes adding fresh culture to the culture, wherein the fresh culture medium contains IL-2 and does not contain CD3 agonists or 4-1BB agonists.

[0020] In some embodiments, the fresh culture medium further includes 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 basal cell culture medium includes, but is not limited to, X-vivo15 medium, AIM-V medium, RPMI-1640 medium, and OpTmizer. TM Culture medium or OpTmizer TM Pro medium.

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

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

[0024] In some implementations, the number of TIL cells harvested at the end of the culture is 1×10⁻⁶. 7 More than 1 per fragment, preferably 1×10 8 More than 1 per fragment, preferably 1×10 9 More than one per segment.

[0025] In some embodiments, the method described herein can rescue TIL samples from previously failed pre-REP amplification. In some embodiments, the tumor sample is from a subject who previously submitted a tumor sample for amplification, wherein the previous amplification included a pre-REP step and wherein no TIL amplification occurred when the pre-REP step was cultured for 14 days. In some embodiments, the tumor sample is from a subject who previously submitted a tumor sample for amplification, wherein the previous amplification included a pre-REP step and the number of TILs isolated from the pre-REP step was less than 1 × 10⁻⁶ when the pre-REP step was cultured to the endpoint. 7 Below one per segment.

[0026] In some implementations, tumor-infiltrating lymphocyte populations obtained by one-step amplification exhibit improved TIL characteristics compared to those obtained by conventional multi-step amplification.

[0027] In some implementations, the improved TIL properties include one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased proportion of viable cells, increased survival capacity, improved proportion of T cell subsets, enhanced cytokine secretion capacity, enhanced tumor cell killing capacity, and enhanced resistance to exhaustion.

[0028] In some embodiments, the improved T cell subset ratio includes one or more selected from the group consisting of: a reduced ratio of regulatory T cells, an increased ratio of stem T cells, an increased ratio of cytotoxic T cells, and a reduced ratio of exhausted T cells.

[0029] In some embodiments, after culturing the tumor sample obtained from the subject, the expanded tumor-infiltrating lymphocyte population contains CD3+TILs comprising more than 80% of the total number of cells, preferably more than 85%, and more preferably more than 90%.

[0030] In some implementations, after culturing the tumor sample obtained from the subject, the expanded tumor-infiltrating lymphocyte population contains more than 50% CD8+TILs and more than 50% CD3+TILs.

[0031] In some implementations, after culturing the tumor sample obtained from the subject, the expanded tumor-infiltrating lymphocyte population contains CD69- / CD39-TILs comprising more than 10% of CD8+TILs.

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

[0033] In some implementations, after culturing the tumor sample obtained from the subject, the expanded tumor-infiltrating lymphocyte population contains less than 20% Foxp3+TILs and less than 20% CD4+TILs.

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

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

[0036] In some cases, the expanded tumor-infiltrating lymphocyte population is genetically modified to contain T-cell receptors (TCRs) or chimeric antigen receptors (CARs) that target tumor-associated antigens.

[0037] In some embodiments, the expanded tumor-infiltrating lymphocyte population is modified to include reduced or suppressed expression of one or more endogenous genes and / or function of proteins encoded by those genes. In some embodiments, these endogenous genes include one or more genes selected from the group consisting of 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 implementations, the amplified tumor-infiltrating lymphocyte population exhibits improved TIL characteristics after genetic modification of the endogenous gene, compared to TILs with unmodified endogenous genes.

[0039] In some implementations, the improved TIL properties include one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased survival capacity, improved T cell subset ratio, increased cytokine secretion capacity, increased tumor cell killing capacity, and improved resistance to exhaustion.

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

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

[0042] In another aspect, the present invention relates to the use of tumor-infiltrating lymphocyte populations obtained according to the methods disclosed herein, or tumor-infiltrating lymphocyte populations disclosed herein, or pharmaceutical compositions disclosed herein, in the preparation of medicaments for the prevention and / or treatment of tumors.

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

[0044] In some embodiments, the tumor is selected from one or more of the group consisting of: melanoma, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, stomach 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, the method comprising administering to the subject a therapeutically effective amount of a 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.

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

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

[0048] Compared with tumor-infiltrating lymphocytes obtained by conventional multi-step expansion, the tumor-infiltrating lymphocyte population obtained by the one-step expansion method of the present invention has the following advantages: 1) The culture cycle of TILs expansion is shortened; 2) Compared with the traditional TIL culture process, the success rate of TIL expansion is improved, and the applicability of TIL therapy in future clinical practice is expanded; 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 high concentrations of IL-2 in subsequent reinfusion is reduced; 6) The proportion of exhausted T cells is reduced; 7) The proportion of stem T cells is increased. Attached Figure Description

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

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

[0051] Figure 2: Number of TILs obtained by amplification using the conventional two-step method (Conventional group) and the one-step method without feeder cells (FOST group, with the final IL-2 concentration indicated in parentheses). In FOST group 1, the 4-1BB agonist in the culture medium is Utomilumab, and in FOST group 2, the 4-1BB agonist in the culture medium is Urelumab.

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

[0053] Figure 4: The proportion of exhausted T cells to CD8+ T cells in the TILs population obtained by the conventional two-step method (Conventional group) and the one-step method without feeder cells (FOST group). Figure 4A shows the proportion of TIM-3 positive T cells to CD8+ T cells, and Figure 4B shows the proportion of BLIMP-1 positive T cells to CD8+ T cells. The FOST-6000 group refers to a final IL-2 concentration of 6000 IU / mL in the culture medium, and the FOST-1000 group refers to a final IL-2 concentration of 1000 IU / mL in the culture medium.

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

[0055] Figure 6: Cytokine ratio detection results of CD8+ T cells obtained by TILs population amplification using the conventional two-step method (Conven-TIL group) and the one-step method without feeder cells (FOST-TIL group). Figure 6A shows the interferon-γ (IFN-γ) ratio, and Figure 6B shows the granzyme B (GZMB) ratio.

[0056] Figure 7: Cell-killing ability of TIL populations obtained by expansion using the conventional two-step method (Conven group) and the one-step method without feeder cells (F1 and F2 groups). Figure 7A shows a tumor sample from lung cancer, and Figure 7B shows a tumor sample from ovarian cancer. In the F1-6000 group, the 4-1BB agonist in the culture medium is Utomilumab, and the final IL-2 concentration is 6000 IU / mL; in the F1-1000 group, the 4-1BB agonist in the culture medium is Utomilumab, and the final IL-2 concentration is 1000 IU / mL; in the F2-1000 group, the 4-1BB agonist in the culture medium is Urelumab, and the final IL-2 concentration is 1000 IU / mL.

[0057] Figure 8: Number of TILs obtained by one-step amplification of cells without feeder layer (FOST group) using medium containing different final concentrations of IL-2. The FOST-6000 group refers to the final concentration of IL-2 in the medium of 6000 IU / mL, and the FOST-1000 group refers to the final concentration of IL-2 in the medium of 1000 IU / mL.

[0058] Figure 9: The proportion of CD3+ T cells in the total cell mass of TILs population obtained by one-step amplification of TILs without feeder layer cells (FOST group) using culture medium containing different final concentrations of IL-2. Among them, the FOST-6000 group refers to the final concentration of IL-2 in the culture medium of 6000 IU / mL, and the FOST-1000 group refers to the final concentration of IL-2 in the culture medium of 1000 IU / mL.

[0059] Figure 10: The proportion of Foxp3+ Treg cells to CD4+ T cells in TILs populations obtained by one-step expansion without feeder cells using medium containing different final concentrations of IL-2. Figure 10A shows a tumor sample from renal cell carcinoma, and Figure 10B shows a tumor sample from ovarian cancer. The final IL-2 concentration is indicated in parentheses. Group F1 indicates that the 4-1BB agonist in the medium is Utomilumab, and group F2 indicates that the 4-1BB agonist in the medium is Urelumab. The Conventional TILs group refers to the TILs cell population obtained by conventional two-step expansion. The positive control represents Treg cells.

[0060] Figure 11: Cell-killing ability of TIL populations obtained by one-step amplification using medium containing different final concentrations of IL-2. In the F1-6000 group, the 4-1BB agonist in the medium was Utomilumab, and the final concentration of IL-2 was 6000 IU / mL; in the F1-1000 group, the 4-1BB agonist in the medium was Utomilumab, and the final concentration of IL-2 was 1000 IU / mL.

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

[0062] Figure 13: Cell number of TILs populations obtained by one-step amplification using medium containing Utomilumab or Urelumab, where FOST 1 group refers to the medium containing Utomilumab as the 4-1BB agonist; and FOST 2 group refers to the medium containing Urelumab as the 4-1BB agonist.

[0063] Figure 14: The proportion of CD3+ T cells in the total cell mass of TILs population obtained by one-step amplification using medium containing Utomilumab or Urelumab. In FOST 1 group, the 4-1BB agonist in the medium is Utomilumab; in FOST 2 group, the 4-1BB agonist in the medium is Urelumab.

[0064] Figure 15: The proportion of CD8+ T cells to CD3+ T cells in TILs populations obtained by one-step amplification using medium containing Utomilumab or Urelumab. In FOST group 1, the 4-1BB agonist in the medium is Utomilumab; in FOST group 2, the 4-1BB agonist in the medium is Urelumab.

[0065] Figure 16: The proportion of exhausted T cells to CD8+ T cells in TILs populations obtained by one-step amplification using medium containing Utomilumab or Urelumab. Figure 16A shows the proportion of TIM-3 positive T cells (a marker of exhausted T cells) to CD8+ T cells, and Figure 16B shows the proportion of BLIMP-1 positive T cells (a pro-exhaustion transcription factor) to CD8+ T cells. In Group F1, the 4-1BB agonist in the medium is Utomilumab; in Group F2, the 4-1BB agonist in the medium is Urelumab.

[0066] Figure 17: The proportion of CD39- / CD69- stem T cells to CD8+ T cells in TILs populations obtained by one-step amplification using medium containing Utomilumab or Urelumab. Group F1 refers to the culture medium containing Utomilumab as the 4-1BB agonist; Group F2 refers to the culture medium containing Urelumab as the 4-1BB agonist.

[0067] Figure 18: Granulase B ratio detection results of CD8+ T cells with TILs populations obtained by one-step amplification using medium containing Utomilumab or Urelumab. In F1 group, the 4-1BB agonist in the medium is Utomilumab; in F2 group, the 4-1BB agonist in the medium is Urelumab.

[0068] Figure 19: Number of TILs population cells obtained by amplifying tumor samples that failed to be amplified using the one-step method without feeder cells (FOST group, with the final IL-2 concentration indicated in parentheses). Figure 19A represents tumor samples from subject LC002, and Figure 19B represents tumor samples from subject RC002. In FOST group 1, the 4-1BB agonist in the culture medium is Utomilumab; in FOST group 2, the 4-1BB agonist in the culture medium is Urelumab.

[0069] Figure 20: Amplification curves and viability of TIL populations obtained through large-scale TIL production.

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

[0071] Figure 22: Cytokine ratios and secretion capacity of TIL populations obtained through large-scale TIL production.

[0072] Figure 23: Target cell killing ability of TIL populations obtained through large-scale TIL production. Detailed Implementation

[0073] To achieve sufficient activation and expansion folds of TILs for therapeutic use, conventional TIL expansion methods typically include at least separate Pre-REP and REP steps. In some applications, the Pre-REP step in conventional methods can last 2 to 6 weeks, followed by an additional 1 to 3 weeks of REP. Furthermore, in addition to the multi-step process, conventional expansion methods require the use of feeder cells. These two requirements make conventional expansion methods both time-consuming and expensive. Patients requiring immunotherapy with adoptive TIL transfer often have very poor prognoses, and a faster availability of a therapeutically viable population of expanded and differentiated TILs can make or break a patient's life. Therefore, there is a need for simpler, faster, and lower-cost TIL manufacturing methods.

[0074] To provide an improved, faster, and simpler method for generating TILs, this invention provides a simplified one-step expansion method that does not use feeder cells. In addition to expanded TIL populations enriched with CD8+ cytotoxic T cells and stem T cell phenotypes, compositions containing expanded TIL populations are also provided.

[0075] In some aspects, this disclosure relates to a method for expanding tumor-infiltrating lymphocytes (TILs) in a one-step process without using feeder cells. The method 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 expanded population of tumor-infiltrating lymphocytes, eliminating the need for a pre-REP step. In some embodiments, the expanded TILs have a higher percentage of cells exhibiting CD8+ cytotoxic T cell and stem T cell phenotypes compared to TILs isolated using feeder cell-based methods.

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

[0077] Generally, the terms and techniques used in relation 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 in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods 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. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Abbas et al., Cellular and Molecular Immunology, 6th ed., WBSaunders Company (2010); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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 performed according to the manufacturer's instructions, as is commonly practiced in the art or as described herein. The terms, laboratory procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used in the field.

[0078] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, 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 explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, 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 numerical values. Such a range may be within an order of magnitude of a given value or range, and may include within 50%, within 20%, within 10%, or within 5%. Permissible variations included in the terms “about” or “approximately” may depend on the specific system being studied and will be readily understood by one of ordinary skill in the art. The terms “above,” “below,” “at most,” and “at least” may include the stated number. Furthermore, the scope provided in the specification and appended claims includes all values ​​between endpoints.

[0079] definition

[0080] To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0081] As used herein, the term "cell population" or "TIL population" refers to a large number of cells or TILs that share a common trait. Generally, the size of a cell population is typically 1 × 10⁻⁶. 6 Up to 1×10 10 Within this range, different TIL groups contain different amounts. For example, the initial growth of primary TILs in the presence of IL-2 can result in approximately 1 × 10⁻⁶ TILs. 7 A large population of TILs per cell. REP amplification is typically performed to provide 1.5 × 10⁶ cells. 9 Up to 1.5×10 10 The TIL population of 1 cell was used for infusion.

[0082] As used herein, the term "amplification" or "amplification process" refers to the process of stimulating or activating cells and cultured cells. An amplification process can cause an increase in the desired total number of cells in a cultured cell population, such as an increase in the total number of TILs, after stimulation or activation and culture of cells. Amplification does not require an increase in the number of all cell types in the cultured cell population. Rather, in some respects, only the number of cell subgroups in the cultured cell population increases during amplification, while the number of other cell types may remain unchanged or may decrease. A process that merely isolates or enriches 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 on the cell surface or in a soluble form. In some embodiments, when an 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 aid the proliferation of another cell type. In some embodiments, the feeder cell may be a peripheral blood mononuclear cell (PBMC) or an antigen-presenting cell (APC). The feeder cell may also be a cell engineered to secrete or express extracellular secretions that aid the proliferation of another cell type, and is not normally used as a feeder cell.

[0085] As used herein, the term "IL-2," also known as "TCGF," "interleukin 2," or simply "interleukin-2," generally refers to a secreted cytokine produced by activated CD4+ and CD8+ T lymphocytes. The IL-2 used in this invention includes all forms of IL-2, such as IL-2 derived from humans or other mammals, and comprises both wild-type and mutant IL-2, provided that the mutant has similar activity to the wild-type. In this invention, the IL-2 comprises recombinant human IL-2.

[0086] As used herein, the term "CD3" refers to a co-stimulatory molecule expressed on the surface of T lymphocytes, which is a protein complex. CD3 has five peptide chains: γ, δ, ε, ζ, and η, all of which are transmembrane proteins. The transmembrane region of the CD3 molecule connects to the transmembrane regions of the two peptide chains of the TCR via salt bridges, forming the TCR-CD3 complex, which jointly participates in the recognition of antigens by T cells. The activation signal generated by 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 a variant thereof, such as a monoclonal antibody, and includes human antibodies, humanized antibodies, chimeric antibodies, or mouse antibodies targeting the CD3 receptor in the T-cell antigen receptor on mature T cells, regardless of their origin, and may be either proprietary 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 in this article, the term "CD28" refers to a co-stimulatory molecule expressed on the surface of T lymphocytes that plays an important role in T cell activation. For example, CD28 can bind to the B7 molecule on APCs (antigen-presenting cells), mediating T cell co-stimulation and promoting their survival, proliferation, and cytokine production.

[0089] As used herein, the term "anti-CD28 antibody" refers to an antibody or a variant thereof, such as a monoclonal antibody, and includes human antibodies, humanized antibodies, chimeric antibodies, or mouse antibodies targeting the CD28 receptor in the T-cell antigen receptor on mature T cells, which are not limited in origin and may be, for example, self-developed or commercially available.

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

[0091] As used herein, the term "anti-4-1BB antibody" refers to an antibody or a variant thereof, such as a monoclonal antibody, and includes human antibodies, humanized antibodies, chimeric antibodies, or mouse antibodies against 4-1BB, which are not limited in origin and may be, for example, self-developed or commercially available. In some embodiments, anti-4-1BB antibodies may be used as 4-1BB ligands. Anti-4-1BB antibodies include utomilumab and urelumab.

[0092] As used herein, the term "TIL properties" refers to the improved properties of TIL cells after modification by the preparation method of the present invention. Changes in TIL properties may include: increased TIL proliferation capacity, increased TIL cell number, increased survival capacity, improved T cell subset ratio, enhanced cytokine secretion capacity, enhanced granzyme secretion capacity, enhanced tumor cell killing capacity, reduced cell exhaustion level, or any combination thereof. The changes of the present invention can be either improvements or reductions.

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

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

[0095] As used herein, the terms "regulatory T cells" or "Treg" generally refer to a subset of T cells that control autoimmune responses in the body. Regulatory T cells can have a CD4+Foxp3+ phenotype, and can be identified by their CD4+ and Foxp3+ phenotypes. Regulatory T cells can also possess the ability to suppress the anti-tumor growth of other T cells.

[0096] In this application, the term "killing ability" generally refers to the ability to kill target cells by exposing the cells of this application to an effective amount of a substance. In one embodiment, the substance of this application may be TIL cells. The killing ability of this application may include killing cells by itself or by promoting 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 proportion of different T cell subsets within TIL cells or TIL populations. For example, different T cell subsets in this application have different immune activities and / or differentiation capabilities. For example, the T cell subsets in this application can be distinguished based on T cell surface markers. For example, cytotoxic T cells may have a CD8+ phenotype. For example, exhausted T cells may have a TIM-3+ phenotype. For example, stem T cells may have a CD69- / CD39- phenotype. For example, regulatory T cells may have a CD4+ / Foxp3+ phenotype.

[0098] As used herein, the term "subject" refers to a person with a tumor in which a population of lymphocytes that has left the bloodstream has migrated to the tumor and transformed into TILs within it. In some embodiments, this person may be a patient requiring immunotherapy involving the expansion of the patient's own TIL population. In other embodiments, this person may be a patient requiring immunotherapy involving the expansion of another patient's own TIL population.

[0099] As used herein, “administration” means the physical introduction of a therapeutic agent or a composition containing 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 administration routes, such as by injection or infusion.

[0100] The term "therapeutic effective dose" refers to the amount of an agent (e.g., TIL cultured as described herein) that provides a desired biological, therapeutic, and / or preventative outcome. This outcome can be a reduction, improvement, mitigation, attenuation, delay, and / or alleviation of one or more signs, symptoms, or causes of disease or any other desired change in a biological system. In the context of solid tumors, an effective dose 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 undesirable cell proliferation. In some respects, an effective dose is an amount sufficient to delay tumor development. In some respects, an effective dose is an amount sufficient to prevent or delay tumor recurrence. An effective dose can be administered in a single or multiple applications.

[0101] As used in this article, the terms "tumor cell" or "cancer cell" refer to cells that divide uncontrollably, form solid tumors, or fill the bloodstream 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 can also spread from one site of the body to another in a process called metastasis. Tumor cells can be isolated from a wide variety of cancer types, including bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral 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 metastatic tumors.

[0102] As used herein, the term "tumor sample" refers to tumor cells isolated from a subject. In some embodiments, a tumor sample is at least a portion, wholly or partially, isolated from a solid tumor of a subject suffering from a tumor. Tumor samples from subjects 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 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 metastatic tumors. "Tumor sample" also includes tumor samples that have been fragmented into "tumor fragments." Fragmentation can be physical fragmentation, mechanical fragmentation, ultrasound fragmentation, enzymatic fragmentation, or any combination thereof. Fragmentation can be performed mechanically, and optionally the tumor fragments can then be enzymatically digested into a single-cell suspension. Mechanical degradation methods may include chopping or slicing the tumor into smaller fragments, while enzymatic degradation methods may include treating the tumor fragments with specific enzymes such as proteases.

[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, culture media typically contain a defined set of components, which may include energy sources, growth factors, hormones, stimulants, activators, sugars, salts, vitamins, and / or amino acids, and / or combinations thereof, depending on the requirements of the cells to be cultured and / or the desired cell culture parameters.

[0104] As used herein, the term "constant composition of the culture medium" 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 concentrations of one or more components may vary. In some embodiments, the concentrations of one or more components in the culture medium change over time as cells are cultured. However, when the culture medium is changed, the new medium has the same composition each time.

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

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

[0107] Tumor-infiltrating lymphocytes (TILs)

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

[0109] TILs are typically defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. TILs are generally 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. Alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into the patient. TILs can also be characterized by potency; for example, a TIL is considered effective if it releases more than about 50 pg / mL, more than about 100 pg / mL, more than about 150 pg / mL, or more than about 200 pg / mL upon TCR stimulation.

[0110] Adoptive cell therapy using TILs cultured in vitro via conventional TIL manufacturing methods involves at least two steps: a Pre-REP step followed by at least one Rapid Expansion Protocol (REP) step.

[0111] TIL amplification

[0112] As generally outlined herein, tumor-associated lymphoid tissue (TILs) are typically derived from samples taken from subjects and manipulated to expand their numbers prior to transplantation into the subjects. In some embodiments, TILs may be genetically manipulated as discussed below. Generally, TILs are initially obtained from tumor samples taken from subjects and then expanded into larger TIL populations for further manipulation as described herein, optionally cryopreservation and restimulation, and optionally evaluation of phenotypic and metabolic parameters as indicators of TIL health.

[0113] Subject-derived tumor samples can be obtained using methods known in the art, such as surgical resection, biopsy, or other methods for obtaining samples containing a mixture of tumor and TIL cells. Once a tumor sample is obtained, it is typically fragmented to approximately 0.5 mm by dissection. 3 Up to 27mm 3 The tumor fragments are preferably fragmented into 0.5mm segments. 3 Up to 4mm 3 Tumor fragments. Or, a combination of enzymatic digestion and mechanical dissociation can be used to obtain a cell suspension. Generally speaking, the harvested cell suspension is called a "primary cell population" or a "freshly harvested" cell population.

[0114] In some implementations, after dissecting and fragmenting the tumor sample, 5, 10, 20, 30, 40, 50 or more tumor fragments are placed in a culture vessel for amplification to obtain an expanded TIL cell population.

[0115] In some implementations, the TIL cell population may optionally be cryopreserved after sample harvest and before entering the amplification phase.

[0116] Conventional methods for TIL amplification

[0117] Conventional methods for expanding TILs, in addition to requiring feeder cells, employ a multi-step approach. This multi-step approach includes at least one rapid expansion protocol (REP) step following a separate Pre-REP step.

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

[0119] A standard multi-step TIL amplification method begins with Pre-REP. Generally, Pre-REP starts with a tumor sample that has been fragmented and / or enzymatically digested, and IL-2 is added to it to slow TIL growth in the cytokine-driven tumor sample. Typically, IL-2 is the only cytokine or immunomodulator added to Pre-REP. Pre-REP, or the first amplification step, can take anywhere from two weeks to several months.

[0120] In some implementations, during Pre-REP, tumor tissue or cells derived from tumor tissue are grown in standard culture media (including, but not limited to, RPMI 1640 medium) and treated with reagents such as irradiated feeder cells and anti-CD3 antibodies to achieve desired effects, such as increasing the number of TILs and / or enriching cell populations 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 IL-2-containing medium under conditions that favor TIL growth over tumor and other cell growth. Tumor digests are incubated in 2 mL wells of medium containing inactivated human AB serum and 6000 IU / mL IL-2. In some instances, 300–6000 IU / mL IL-2 is added. During the pre-REP period, this primary cell population is cultured for several days to several months, resulting in a large TIL population, typically approximately 1 × 10⁻⁶. 8 A large number of TIL cells.

[0122] In conventional methods that include a Pre-REP step, a boundary is established between Pre-REP and REP once the TILs have expanded in the presence of IL-2 and reached the appropriate cell number required to initiate REP, or once a predetermined period of Pre-REP has been completed. In various implementations, depending on the specific circumstances, when the number of TILs obtained is 1x10⁻⁶… 5 1x10 6 1x10 7 Pre-REP amplification can be completed when the cell count is equal to the fragment count. In another embodiment, Pre-REP amplification can be completed when the culture duration reaches 3 to 14 days or when 9 to 14 days have elapsed since fragmentation.

[0123] In some cases, TILs obtained from the Pre-REP step are stored until phenotypic selection is performed. In other cases, TILs obtained from the Pre-REP step are not stored and are directly subjected to the REP step. In some cases, TILs obtained from the Pre-REP step are not cryopreserved after pre-REP amplification and before REP amplification. In cases where genetically modified TILs are used for treatment, the pre-REP TIL cell population obtained after pre-REP amplification can be genetically modified before the REP step.

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

[0125] In conventional multi-step TIL amplification, in some cases, a further large-scale amplification process that begins after pre-REP amplification is called rapid amplification (REP). REP is generally performed using a culture medium containing multiple components, such as feeder cells, cytokines, and anti-CD3 antibodies. In some cases, REP can last 7–14 days or longer.

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

[0127] In some cases, REP also includes a step of selecting TILs to obtain superior tumor reactivity. Any selection method known in the art can be used. Optionally, cell viability assays can be performed after REP amplification 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 samples.

[0128] In some cases, multiple REP amplifications can be performed.

[0129] trophoblast cells

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

[0131] Feeder-free one-step technology (FOST) for cell expansion.

[0132] The conventional trophoblast-dependent multi-step expansion method for TILs described above requires multiple steps and trophoblast cells, making it both time-consuming and expensive. Therefore, a simpler, faster, and lower-cost TIL manufacturing method is needed.

[0133] To provide an improved, faster, and simpler method for generating TILs, this invention provides a simplified one-step expansion method that does not use feeder cells. In addition to expanding TIL populations enriched with CD8+ cytotoxic T cells and stem T cell phenotypes, compositions of expanded TIL populations are also provided.

[0134] In one aspect of the method disclosed herein, the Pre-REP step of conventional TIL amplification protocols is completely skipped. Surprisingly, large quantities of TILs can be obtained in 24 days or less in this one-step amplification method for feederless cells without Pre-REP. In some embodiments, the TILs are engineered or genetically modified during the one-step TIL amplification process.

[0135] In some embodiments, the method of the present invention can salvage TIL samples that have failed to amplify using conventional methods. In some embodiments, amplification failure refers to the failure to amplify a sufficient number of TILs after a sufficient culture duration via pre-REP. In some embodiments, pre-REP failure refers to the absence of TIL amplification by 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 the subject to 1 × 10⁻⁶ cells at the endpoint using the pre-REP protocol. 7 Cells per fragment. In some embodiments, the methods provided herein are capable of amplifying TILs 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 usable number of TILs.

[0136] In one aspect of the method disclosed in this invention, a method for one-step expansion of tumor-infiltrating lymphocytes includes 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, wherein the culture medium does not contain trophoblast cells.

[0137] In some embodiments, the tumor sample is fragmented prior to culture. In some embodiments, the tumor sample comprises tumors with a size of 0.5 mm. 3 Up to 27mm 3 The tumor fragments are preferably fragmented into 0.5mm segments. 3 Up to 4mm 3 The tumor fragment, more preferably fragmented into 1mm 3 Up to 3mm 3 The tumor sample contains tumor fragments. In some embodiments, the tumor sample comprises digested tumor fragments.

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

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

[0140] In some implementations, 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 agonist antibody and / or its antigen-binding fragment, or a ligand-non-competitive 4-1BB agonist antibody and / or its antigen-binding fragment. 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 1-20 μg / mL. In some preferred embodiments, the final concentration of the 4-1BB agonist is 10 μg / mL.

[0144] In some embodiments, the composition of the culture medium remains constant. In some embodiments, approximately 30% to approximately 99% of the culture medium is replaced every 2 to 5 days. In some embodiments, the culture medium is replaced by perfusion. In some embodiments, perfusion includes continuous culture medium replacement at a rate of approximately 30% to approximately 99% of the working volume of the culture vessel every 24 hours.

[0145] In some embodiments, the replacement of the culture medium includes adding fresh culture to the culture, wherein the fresh culture medium contains IL-2 and does not contain CD3 agonists or 4-1BB agonists.

[0146] In some embodiments, the fresh culture medium further includes 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.

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

[0148] In some implementations, TIL amplification continues for 14-28 days from the initial tumor fragmentation. In some implementations, TIL amplification continues for at least 14 days from the initial tumor fragmentation.

[0149] In some implementations, the number of TIL cells harvested at the end of the culture is 1×10⁻⁶. 7 More than 1 per fragment, preferably 1×10 8 More than 1 per fragment, preferably 1×10 9 More than one per segment.

[0150] In some embodiments, the method described herein can rescue TIL samples from previously failed pre-REP amplification. In some embodiments, the tumor sample is from a subject who previously submitted a tumor sample for amplification, wherein the previous amplification included a pre-REP step and wherein no TIL amplification occurred when the pre-REP step was cultured for 14 days. In some embodiments, the tumor sample is from a subject who previously submitted a tumor sample for amplification, wherein the previous amplification included a pre-REP step and the number of TILs isolated from the pre-REP step was less than 1 × 10⁻⁶ when the pre-REP step was cultured to the endpoint. 7 Below one per segment.

[0151] In some implementations, tumor-infiltrating lymphocyte populations obtained by one-step amplification exhibit improved TIL characteristics compared to those obtained by conventional multi-step amplification.

[0152] In some implementations, the improved TIL properties include one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased proportion of viable cells, increased survival capacity, improved proportion of T cell subsets, enhanced cytokine secretion capacity, enhanced tumor cell killing capacity, and enhanced resistance to exhaustion.

[0153] In some embodiments, the improved T cell subset ratio includes one or more selected from the group consisting of: a reduced ratio of regulatory T cells, an increased ratio of stem T cells, an increased ratio of cytotoxic T cells, and a reduced ratio of exhausted T cells.

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

[0155] In some implementations, after culturing the tumor sample obtained from the subject, the expanded tumor-infiltrating lymphocyte population contains more than 50% CD8+TILs and more than 50% CD3+TILs.

[0156] In some implementations, after culturing the tumor sample obtained from the subject, the expanded tumor-infiltrating lymphocyte population contains CD69- / CD39-TILs comprising more than 10% of CD8+TILs.

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

[0158] In some implementations, after culturing the tumor sample obtained from the subject, the expanded tumor-infiltrating lymphocyte population contains less than 20% Foxp3+TILs and less than 20% CD4+TILs.

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

[0160] In some implementations, the number of cells used to expand the TIL population using a one-step method can reach 1×10⁻⁶. 6 ~1×10 7 When the number of TILs per cell is increased, 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, the TIL is genetically modified to include one or more genomic modifications that result in reduced expression and / or function of one or more endogenous target genes, and to include immune effector cells of 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 TIL includes one or more modifications to the genomic DNA sequence of the endogenous target gene, such as the insertion, deletion, or mutation of one or more nucleic acids, resulting in reduced expression and / or function of the endogenous gene.

[0163] In some implementations, the cells of the expanded tumor-infiltrating lymphocyte population exhibit improved TIL characteristics after the endogenous genes of the cells are genetically modified, compared to TILs with unmodified endogenous genes.

[0164] In some implementations, the improved TIL properties include one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased survival capacity, improved T cell subset ratio, increased cytokine secretion capacity, increased tumor cell killing capacity, and improved resistance to exhaustion.

[0165] In some embodiments, the modified TIL described herein includes 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 gene editing system). In such embodiments, the gene editing system included in the modified TIL is capable of modifying one or more endogenous target genes.

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

[0167] In some embodiments, the present invention provides a modified TIL comprising 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 also comprise CARs or recombinant TCRs expressed on the cell surface.

[0168] In some embodiments, the present invention provides a modified TIL 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, and also contain a CAR or recombinant TCR expressed on the cell surface.

[0169] Tumor-infiltrating lymphocyte population

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

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

[0172] In some implementations, CD8+TILs account for more than 50% of the tumor-infiltrating lymphocyte population, which is composed of CD3+TILs.

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

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

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

[0176] Drug composition, dosage and dosing regimen

[0177] In one embodiment, a tumor-infiltrating lymphocyte population amplified using the methods provided in this invention, along with optionally pharmaceutically acceptable carriers, can be administered to a subject as a pharmaceutical composition. In one embodiment, the tumor-infiltrating lymphocyte population obtained according to the methods disclosed herein, or the tumor-infiltrating lymphocyte population or pharmaceutical composition disclosed herein, can be used to prepare a medicament for the prevention and / or treatment of tumors.

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

[0179] The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage will depend on the route of administration, the form in which the compound is administered, the subject being treated, the sex and age of the subject being treated, the weight of the subject being treated, and the preferences and experience of the attending physician. Clinically determined doses of TIL may also be used where appropriate. The amount of pharmaceutical composition administered using the methods described herein, such as the dose of TIL, will depend on the person or mammal being treated, the severity of the condition or symptom, the rate of administration, the disposal of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

[0180] In some implementation schemes, the effective therapeutic dose is approximately 4 × 10⁻⁶. 10 To approximately 1×10 11 TIL.

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

[0182] An effective amount of TIL can be administered in single or multiple doses via any of the accepted routes of administration of agents with similar efficacy, including intranasal and percutaneous routes, intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, local, implantation, or inhalation. In some embodiments, TIL is administered intravenously.

[0183] In some embodiments, the medicament of the present invention is used for the prevention and / or treatment of solid tumors. Exemplary solid tumors may include, but are not limited to, melanoma, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer, and kidney cancer.

[0184] Adoptive cell transfer

[0185] Adoptive cell transfer (ACT) is a highly effective form of immunotherapy and involves transferring immune cells with anti-tumor activity into a cancer patient. ACT involves identifying lymphocytes with anti-tumor activity in vitro, expanding these cells in large quantities in vitro, and infusing them into a host carrying cancer. Lymphocytes used for adoptive transfer can be derived from the stromal tissue of a resected tumor (tumor-infiltrating lymphocytes or TILs). TIL cell populations used 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, the method comprising administering to the subject a therapeutically effective amount of a tumor-infiltrating lymphocyte population obtained according to the method disclosed herein, or a tumor-infiltrating lymphocyte population disclosed herein, or a pharmaceutical composition disclosed herein, to treat the cancer by transferring a population of TIL cells that elicit an immune response.

[0187] In some implementations, this treatment method is applicable to solid tumors. Exemplary solid tumors may include, but are not limited to, 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] Example

[0189] The invention generally described herein will be more readily understood by referring to the following embodiments. Those skilled in the art can use this content to appropriately modify the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications will be obvious to those skilled in the art and are considered to be within the scope of protection of this invention. The following embodiments are provided by way of illustration and are not intended to limit the invention. These embodiments are not intended to represent all or only the experiments conducted below.

[0190] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0191] Main materials and reagents

[0192] Culture medium formulation

[0193] (1) Complete culture medium CM1: 1L Optmizer + 26mL supplement

[0194] (2) Complete culture medium CM2: CM-1 + 5% mL SR + 10% Glutamax + 1% PS

[0195] (3) Complete culture medium CM3000: CM-2 + 3000 IU / mL IL-2

[0196] Tumor tissue treatment

[0197] Tumor tissue from the subjects was received, and the tissue blocks were soaked in 10% gentamicin for 5 minutes, followed by soaking in 10% clindamycin for 5 minutes. Then, the tissue blocks were rinsed twice with CM1 culture medium. The tissue blocks were cut into 1–3 mm pieces using sterile scissors. 3 Small pieces.

[0198] Conventional two-step method for expanding tumor-infiltrating lymphocytes

[0199] Pre-REP stage amplification

[0200] The obtained tissue fragments were seeded into Grex6 culture media according to the experimental groups. On day 0, 20 mL of CM2 medium was added, and the culture was carried out in a 37°C, 5% CO2 incubator. On day 5, the medium was replenished to 40 mL using CM2 medium. Half-medium medium changes were performed on days 7, 9, and 11. From days 11 to 14, when the cell count reached 5 × 10⁶ cells / mL... 5 At this point, pre-REP is complete, and pre-REP TIL cells are harvested.

[0201] REP stage amplification

[0202] Pre-REP TIL cells and trophoblast cells were resuspended in CM3000 medium, and 1×10⁻⁶ cells were added. 5 10 Pre-REP TIL cells and 2×10 7 One layer of feeder cells was seeded into Grex24 culture media, and CM3000 medium was added to a final concentration of 2 mL. Then, OKT3 was added to a final concentration of 30 ng / mL, and the mixture was thoroughly mixed. The cells were then incubated in a 37°C, 5% CO2 incubator. On day 2, the medium was increased to 8 mL. Half-medium replacements were performed on days 5, 7, 9, and 11. When the cells reached confluence, they were transferred to Grex6 culture media and cultured from day 11 to 14 until the cell count reached 5 × 10⁶ cells / mL. 9 At this point, REP is complete, and TIL cells are harvested. Samples are taken for further analysis during cell passage or harvest.

[0203] One-step expansion of tumor-infiltrating lymphocytes using non-trophoblast cells

[0204] On day 0, the obtained tissue fragments were seeded into Grex6 culture media 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, with 5 tissue fragments per well. On day 5, the medium was replenished to 40 mL using CM2 medium. Every 2–5 days, when the medium turned yellow and the cells reached confluence, a half-medium replacement and cell counting were performed. Cells were counted when the cell count reached 2 × 10⁶ cells per well. 8 At that time, take 1×10 7 ~2×10 7 The cells were passaged. Between days 17 and 28, when the cell number reached 5 × 10⁶ cells / year... 9 At this point, amplification is complete, and TIL cells are harvested. Samples are taken for further testing during cell passage or harvesting.

[0205] Flow cytometry detection

[0206] Upon receiving the samples, verify the sample information (including: name, quantity, and cell count). After balancing the samples, centrifuge at 400×g for 5 minutes and discard the supernatant. Adjust the cell density to 1×10⁻⁶ cells using PBS. 7 After the cells / mL were measured, 100 μL of cell suspension was transferred to an EP tube, and the sample was centrifuged at 400 × g for 5 min in a low-temperature centrifuge before discarding the supernatant.

[0207] Membrane surface markers

[0208] Add 100 μL of the prepared mixed antibody to a sample tube and incubate at room temperature in the dark for 20 min, recording the start and end times. After incubation, add 200 μL of PBS to the sample tube to terminate the incubation. Centrifuge the sample at 400 × g for 5 min in a low-temperature centrifuge, discard the supernatant, and then perform the analysis.

[0209] Intranuclear markers

[0210] Prepare the fixative membrane-breaking solution according to the table below:

[0211] Prepare 1× washing buffer according to the table below:

[0212] Add 200 μL of the prepared fixation and membrane rupture solution to the sample tube, incubate at 4°C for 0.5 h, and record the start and end times.

[0213] Add 100 μL of 1× wash buffer to the sample tube to stop the reaction. Centrifuge the sample in a refrigerated centrifuge at 1500g for 15 seconds and discard the supernatant.

[0214] Add 100 μL of the prepared intranuclear antibody to the sample tube and incubate overnight at 4°C in the dark.

[0215] Add 100 μL of 1× wash buffer to the sample tube to stop the reaction. Centrifuge the sample in a refrigerated centrifuge at 1500g for 15 seconds and discard the supernatant.

[0216] Add 200 μL of 1× wash buffer to the sample tube to resuspend it, ready for instrumental analysis.

[0217] Lethality detection

[0218] Target cell preparation

[0219] P815 cells were cultured in P815 complete medium (DMEM + 10% FBS) at 37°C in a 5% CO2 incubator. The cells were grown in a semi-attached and semi-suspended manner, and were passaged every 2-3 days. After cell resuscitation, the cells were used within 20 passages.

[0220] Collect P815 cells in the logarithmic growth phase and transfer the cell suspension to a 50 mL centrifuge tube. Add 3–5 mL of trypsin to the culture flask and incubate at 37°C for approximately 2 minutes. When slight detachment of adherent cells is observed after shaking the flask, immediately add P815 complete culture medium to stop digestion and mix thoroughly by pipetting. Transfer the digested cells to a centrifuge tube. Centrifuge at 400 × g for 5 minutes, discard the supernatant, and resuspend the cells in 4–6 mL of P815 complete culture medium. Count the cells and adjust the cell density to 1 × 10⁶ cells according to the desired plating volume. 5 cells / mL.

[0221] Seed 100 μl of P815 cells into a cell culture plate. Add 100 μl of DPBS or other sterile reagent around the cells to avoid edge effects. Incubate in an incubator for 3–6 hours to allow them to adhere.

[0222] Effector cell preparation

[0223] Verify the test request information, and take 10 mL / vial of CM1 medium according to the test volume, preheating it in a 37°C water bath. Revive the frozen effector cells and transfer them to the preheated CM1 medium. Centrifuge at 400×g for 5 min and discard the supernatant. Resuspend the cells in CM1 medium containing 0.5 μg / mL OKT3 to approximately 3×10⁻⁶ cells, according to the labeled cell count. 6 Cells / mL, counted. Density adjusted to 1×10⁻⁶. 6 cells / mL, then serially diluted to 5×10 5 2.5×10 5 1.25×10 5 0.625×10 5 cells / mL.

[0224] Lethality Detection

[0225] Place a 96-well plate without effector cells into the instrument for imaging at the 0h point. After imaging, remove the plate and add effector cells. Seed 100 μl of effector cells into each well, with duplicate wells for each effector cell of the same sample. Add 100 μl of CM1 medium containing 0.5 μg / mL OKT3 to the control group without effector cells. After cell seeding, confirm the absence of air bubbles and immediately place the plate into the instrument. Acquire signals at 3-hour intervals.

[0226] Data Analysis

[0227] The kill rate at each time point and under the effective-to-target ratio condition is calculated using the following formula.

[0228] Plot the lethality curves under various effective-to-target ratio conditions with time (h) on the x-axis and lethality rate on the y-axis.

[0229] Interferon-gamma secretion level detection

[0230] Take the TILs cells to be tested, centrifuge at 300×g for 5 min, collect the cell supernatant, dilute it appropriately, and perform the test according to the instructions of the human interferon-γ ELISA kit (ACROBiosystems, CRS-A017).

[0231] Example 1: One-step expansion of trophoblast-free cells can improve the proliferation capacity of TILs.

[0232] Tumor samples from four subjects (RC0021, LC002, EC001, and LC002) were amplified using a conventional two-step method and a one-step method without feeder cells, respectively. Cell counts were performed on days 18, 14, 13, and 17.

[0233] The proliferative capacity analysis of TILs obtained by one-step expansion without a feeder layer is shown in Figures 1 and 2. The culture results of TILs from four subjects showed that, after the same culture time, the number of TILs obtained by one-step expansion without a feeder layer was greater, indicating that one-step expansion without a feeder layer can improve the proliferative capacity of TILs and shorten the culture period for TIL expansion.

[0234] Example 2: Phenotype of tumor-infiltrating lymphocytes obtained by one-step expansion of trophoblast-free cells

[0235] Tumor samples from different subjects were amplified using a conventional two-step method and a one-step method without feeder cells, and samples were taken at the end of the culture to detect the phenotypic characteristics of the TIL population by flow cytometry.

[0236] The phenotypic characteristics of the TILs population obtained by one-step expansion of non-trophoblast cells are shown in Figures 3 to 5.

[0237] Figure 3 shows the proportion of CD8+ T cells to CD3+ T cells in the TIL populations obtained by the conventional two-step method and the one-step method without feeder cells. The results show that, compared with the conventional two-step method, the one-step method without feeder cells can significantly increase the proportion of CD8+ T cells in the expanded TIL populations.

[0238] Figure 4 shows the proportion of exhausted T cells to CD8+ T cells in the TILs population obtained using the conventional two-step method and the one-step method without feeder cells. Figure 4A shows the proportion of TIM-3 positive T cells (a marker of exhausted T cells) to CD8+ T cells, and Figure 4B shows the proportion of BLIMP-1 positive T cells (a pro-exhaustion transcription factor) to CD8+ T cells. The results show that, compared with the conventional two-step method, the one-step method without feeder cells can significantly reduce the proportion of exhausted T cells in the TILs population, for example, the proportion of TIM-3 positive T cells and / or BLIMP-1 positive T cells is lower.

[0239] Figure 5 shows the proportion of CD39- / CD69- stem T cells to CD8+ T cells in the TIL populations obtained using the conventional two-step method and the one-step method without feeder cells. The results show that the one-step method without feeder cells can significantly increase the proportion of stem T cells in the TIL populations compared with the conventional two-step method.

[0240] Example 3: One-step expansion of trophoblast-free cells enhances the killing ability of tumor-infiltrating lymphocytes

[0241] Tumor samples from different subjects were amplified using a conventional two-step method and a one-step method without feeder cells, and samples were taken at the end of the culture to detect the cytokine ratio and killing ability of the TILs population.

[0242] Figure 6 shows the cytokine ratios of CD8+ T cells from TIL populations amplified using the conventional two-step method and the one-step method without feeder cells. Figure 6A shows the interferon-γ ratio, and Figure 6B shows the granzyme B ratio. The results indicate that, compared to the conventional two-step method, the one-step method without feeder cells significantly increases the cytokine ratios of CD8+ T cells from TIL populations. For example, it results in a higher interferon-γ ratio or a higher granzyme B ratio.

[0243] Figure 7 shows the cell-killing ability of TIL populations obtained by expansion using the conventional two-step method and the one-step method without feeder cells. Figure 7A shows a tumor sample from lung cancer, and Figure 7B shows a tumor sample from ovarian cancer. The results show that, compared with the conventional two-step method, the TIL populations obtained by expansion using the one-step method without feeder cells have a stronger target cell-killing ability.

[0244] Example 4: Effect of low IL-2 concentration on the phenotype of tumor-infiltrating lymphocytes obtained by one-step amplification

[0245] To further investigate the effect of IL-2 concentration on one-step amplification using feederless cells, TILs were amplified using a one-step method with culture media containing different final concentrations of IL-2, and samples were taken at the end of the culture to detect the proliferation capacity and phenotypic characteristics of the TIL population.

[0246] The proliferation capacity and phenotypic characteristics of TIL populations obtained by one-step amplification of cells without a feeder layer using culture media containing different final concentrations of IL-2 are shown in Figures 2 to 5 and Figures 8 to 10.

[0247] Figures 2 and 8 show the amplification numbers of TIL populations obtained by one-step amplification using medium containing different final concentrations of IL-2. The results show that using medium containing a lower final concentration of IL-2 has no significant effect on the number of cells in the TIL populations obtained by one-step amplification.

[0248] Figure 9 shows the proportion of CD3+ T cells in the total cell mass of TILs populations obtained by one-step expansion using medium containing different final concentrations of IL-2. The results show that using medium containing a lower final concentration of IL-2 has no significant effect on the proportion of CD3+ cells in the TILs populations obtained by one-step expansion.

[0249] Figure 3 shows the proportion of CD8+ T cells to CD3+ T cells in TIL populations obtained by one-step amplification using medium containing different final concentrations of IL-2. The results show that using medium containing a lower final concentration of IL-2 has no significant effect on the proportion of CD8+ T cells in TIL populations obtained by one-step amplification.

[0250] Figure 4 shows the proportion of exhausted T cells to CD8+ T cells in TIL populations obtained by one-step expansion using media containing different final concentrations of IL-2. Figure 4A shows the proportion of TIM-3 positive T cells (a marker of exhausted T cells) to CD8+ T cells, and Figure 4B shows the proportion of BLIMP-1 positive T cells (a pro-exhaustion transcription factor) to CD8+ T cells. The results indicate that the proportion of exhausted T cells, such as TIM-3 positive T cells and / or BLIMP-1 positive T cells, is lower in TIL populations obtained by one-step expansion using media containing lower final concentrations of IL-2.

[0251] Figure 5 shows the proportion of CD39- / CD69- stem T cells to CD8+ T cells in TIL populations obtained by one-step amplification using medium containing different final concentrations of IL-2. The results show that the proportion of stem T cells is higher in TIL populations obtained by one-step amplification using medium containing lower final concentrations of IL-2.

[0252] Figure 10 shows the proportion of Foxp3+ Treg cells to CD4+ T cells in TILs populations obtained by one-step expansion using medium containing different final concentrations of IL-2. Figure 10A shows a tumor sample from renal cell carcinoma, and Figure 10B shows a tumor sample from ovarian cancer. The results indicate that using medium containing lower final concentrations of IL-2 does not increase the proportion of Treg cells in the TILs populations obtained by one-step expansion.

[0253] Example 5: Effect of low IL-2 concentration on the killing ability of tumor-infiltrating lymphocytes obtained by one-step amplification

[0254] To further investigate the effect of IL-2 concentration on TIL amplification using a one-step method with untrophobic cells, TILs were amplified using a one-step method with culture media containing different final concentrations of IL-2, and samples were taken at the end of the culture to detect the killing ability of the TIL population.

[0255] Figure 11 shows the cell-killing ability of TIL populations obtained by one-step amplification using medium containing different final concentrations of IL-2. The results show that the target cell-killing ability of TIL populations obtained by one-step amplification using medium containing lower final concentrations of IL-2 is similar to that using medium containing higher final concentrations of IL-2.

[0256] Example 6: Effects of different T cell co-stimulatory molecular agonists on the proportion of CD8+ cells in tumor-infiltrating lymphocytes obtained by one-step expansion

[0257] To further investigate the effects of different T cell co-stimulatory molecule agonists on TIL amplification using a one-step method with feederless cells, TILs were amplified using a one-step method with culture medium containing 4-1BB agonist or CD28 agonist (approximately 50 ng / mL). Samples were taken at the end of the culture to detect the phenotypic characteristics of the TIL populations.

[0258] Figure 12 shows the proportion of CD8+ T cells to CD3+ T cells in TIL populations obtained by one-step expansion using culture medium containing 4-1BB or CD28 agonists. The results showed that when the T cell co-stimulatory molecule agonist was 4-1BB, the median proportion of CD8+ T cells to CD3+ T cells in the TIL populations obtained by one-step expansion was 72.9%; when the T cell co-stimulatory molecule agonist was CD28, the median proportions of CD8+ T cells to CD3+ T cells in the TIL populations obtained by one-step expansion were 33.8% (IL-2 concentration of 6000 IU / mL) and 17.4% (IL-2 concentration of 1000 IU / mL), respectively. These results indicate that using 4-1BB agonists as the T cell co-stimulatory molecule agonist can significantly increase the proportion of CD8+ T cells in the TIL populations obtained by one-step expansion.

[0259] Example 7: Effects of different 4-1BB agonist antibodies on the phenotype of tumor-infiltrating lymphocytes obtained by one-step amplification

[0260] To further investigate the effect of different 4-1BB agonist antibodies on one-step amplification using feederless cells, TILs were amplified in a one-step process using culture medium with 4-1BB agonist antibodies Utomilumab or Urelumab (IL-2 concentration of 1000 IU / mL). Samples were taken at the end of the culture to detect the proliferation capacity, phenotypic characteristics, and cytokine ratios of the TIL population.

[0261] The proliferation capacity and phenotypic characteristics of TIL populations obtained by one-step expansion of feeder-free cells using a medium containing Utomilumab or Urelumab are shown in Figures 13 to 18.

[0262] Figure 13 shows the cell numbers of TIL populations obtained by one-step amplification using medium containing Utomilumab or Urelumab. The results show that using medium containing different 4-1BB agonist antibodies has no significant effect on the cell numbers of TIL populations obtained by one-step amplification.

[0263] Figure 14 shows the proportion of CD3+ T cells in the total cell mass of TILs obtained by one-step amplification using media containing Utomilumab or Urelumab. The results showed that using media containing different 4-1BB agonist antibodies had no significant effect on the proportion of CD3+ T cells in the TILs obtained by one-step amplification; however, a higher proportion of CD3+ T cells was obtained using media containing Utomilumab in some samples from subjects.

[0264] Figure 15 shows the proportion of CD8+ T cells to CD3+ T cells in the TIL population obtained by one-step amplification using medium containing Utomilumab or Urelumab. The results show that using medium containing different 4-1BB agonist antibodies has no significant effect on the proportion of CD8+ T cells in the TIL population obtained by one-step amplification.

[0265] Figure 16 shows the proportion of exhausted T cells to CD8+ T cells in the TILs population obtained by one-step amplification using media containing Utomilumab or Urelumab. Figure 16A shows the proportion of TIM-3 positive T cells (a marker of exhausted T cells) to CD8+ T cells, and Figure 16B shows the proportion of BLIMP-1 positive T cells (a pro-exhaustion transcription factor) to CD8+ T cells. The results indicate that using media containing different 4-1BB agonist antibodies had no significant effect on the proportion of exhausted T cells in the TILs population obtained by one-step amplification.

[0266] Figure 17 shows the proportion of CD39- / CD69- stem T cells to CD8+ T cells in the TILs population obtained by one-step amplification using medium containing Utomilumab or Urelumab. The results show that using medium containing different 4-1BB agonist antibodies has no significant effect on the proportion of stem T cells in the TILs population obtained by one-step amplification.

[0267] Figure 18 shows the granzyme B ratio of CD8+ T cells with TILs populations obtained by one-step amplification using medium containing Utomilumab or Urelumab. The results show that using medium containing different 4-1BB agonist antibodies has no significant effect on the granzyme B ratio of CD8+ T cells with TILs populations obtained by one-step amplification.

[0268] Example 8: One-step expansion of tumor-infiltrating lymphocytes that failed to expand using a two-step method without a trophoblast layer

[0269] Tumor samples derived from subjects LC002 and RC002 failed to expand previously using the conventional two-step method. Specifically, tumor samples derived from subject LC002 showed no TIL expansion after 14 days of culture, while tumor samples derived from subject RC002 showed cell expansion of less than 5 × 10⁻⁶ cells after 24 days of culture. 7 indivual.

[0270] Tumor samples from the subject were further amplified using a one-step method with no feeder cells, and samples were taken at the end of the culture to detect the number of TILs population cells.

[0271] Figure 19 shows that the one-step amplification of tumor samples derived from LC002 and RC002 in the subject was successful using a non-trophoblast cell method.

[0272] Example 9: Large-scale culture of feeder-free cells in one step

[0273] This embodiment provides an example scheme (batch R1FY16O1) for large-scale production of TILs.

[0274] On day 0, tumor samples obtained from the subject were cut into pieces approximately 1-3 mm in size. 3 Small pieces of tissue were placed in a G-Rex 100M container with 100 mL of FOST activation medium and transferred to a CO2 incubator for culture (temperature: 37±1℃, CO2 concentration: 5±0.5%). FOST amplification medium was added every three days (100 mL each time). When the total number of viable cells exceeded 5 × 10⁻⁶, the culture was complete. 8 At this time, all cells were transferred to the cell expansion system (XURI) and cultured at a rate of 1 × 10⁻⁶. 6 Cell density was adjusted by cells / mL; when the total number of viable cells was greater than 5 × 10⁻⁶, the cell density was adjusted accordingly. 9 At this time, the perfusion program of the XURI cell expansion system was selected, and the perfusion rate was 1.5 L / day on the first day of perfusion; 3.5 L / day on the second day of perfusion; and 5.0 L / day thereafter. When the total number of viable cells was greater than 5 × 10⁻⁶, the perfusion was continued. 10 At that time, the TILs cell population was harvested.

[0275] Figure 20 shows the expansion curves and viability of the TIL population obtained through large-scale TIL production. This batch was a 5L system, with an expansion time of 17 days, and the final TIL cell yield reached 4.11 × 10⁻⁶. 10 The survival rate reached 97.3%.

[0276] Figure 21 shows the phenotypic characteristics of TIL populations obtained through large-scale TIL production, sampled at three points: day 10, day 12 of culture, and final harvest. The results show that the proportion of CD3+ T cells remained relatively consistent across the three points; the proportion of CD8+ T cells decreased slightly with increasing culture time; the proportions of CD39- / CD69- T cells in both CD4+ T cells and CD8+ T cells increased with increasing culture time; the proportion of TIM-3+ T cells in CD4+ T cells decreased with increasing culture time, while the proportion of TIM-3+ T cells in CD8+ T cells increased slightly with increasing culture time.

[0277] Figure 22 shows the cytokine proportions and secretion capacity of TIL populations obtained through large-scale TIL production, sampled at three points: day 10, day 12 of culture, and final harvest. The results showed that intracellularly, in CD8+ T cells, the proportion of granzyme B reached its highest level on day 12; in CD8+ T cells, the proportion of perforin decreased slightly with increasing culture time; the proportion of interferon-γ reached its highest level at harvest; extracellularly, the level of interferon-γ secretion increased with increasing cell culture time.

[0278] Figure 23 shows the target cell killing ability of TILs obtained through large-scale TIL production, sampled at three points: day 10, day 12 of culture, and final harvest. The results show that the sample taken on day 12 exhibited the strongest killing ability at 24 hours when the effector-to-target ratio was 0.625:1; the sample taken on day 12 also showed the strongest killing ability at 24 hours when the effector-to-target ratio was 1.25:1; and at an effector-to-target ratio of 2.5:1, samples at all three points achieved a killing rate of over 95% at 24 hours.

[0279] 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. Various modifications and variations can be made to the present invention by those skilled in the art. The selection and application of specific technical solutions can be adjusted and changed according to specific needs. Therefore, those skilled in the art can make several simple substitutions without departing from the concept and principles of the present invention, and these should all be included within 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 to obtain an expanded population of tumor-infiltrating lymphocytes, wherein the culture medium does not contain trophoblast cells.

2. The method of claim 1, wherein the tumor sample is fragmented prior to culture.

3. The method according to claim 1, wherein the tumor sample comprises a size of 0.5 mm. 3 Up to 27mm 3 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 less than 6000 IU / mL, preferably less than 2000 IU / mL.

6. The method according to claim 2, wherein the final concentration of IL-2 in the culture medium is 1000 IU / mL.

7. The method of claim 1, wherein the CD3 agonist comprises an anti-CD3 antibody and / or its antigen-binding fragment, optionally humanized with the anti-CD3 antibody and / or its antigen-binding fragment.

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

9. The method according to claim 1, wherein the final concentration of the CD3 agonist is from 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 of claim 1, wherein the 4-1BB agonist comprises a ligand-competitive 4-1BB agonist antibody and / or its antigen-binding fragment, or a ligand-non-competitive 4-1BB agonist antibody and / or its antigen-binding fragment.

12. The method of 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.

14. 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 culture medium replacement comprises adding fresh culture to the culture, wherein the fresh culture medium contains IL-2 and does not contain a CD3 agonist or a 4-1BB agonist.

17. The method according to claim 1, wherein the TIL culture lasts for 14-28 days.

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

19. The method of claim 1, wherein the tumor sample is derived from a subject who has previously submitted a tumor sample for amplification, wherein the previous amplification includes a pre-REP step and wherein no TIL amplification or the number of TILs isolated from the pre-REP step is less than 1 × 10⁻⁶ when the pre-REP step is cultured for 14 days. 7 Below one per segment.

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

21. The method of claim 20, wherein the improved TIL properties comprise one or more selected from the group consisting of: increased TIL cell number and expansion capacity, increased viable cell ratio, increased survival capacity, improved T cell subset ratio, enhanced cytokine secretion capacity, enhanced tumor cell killing capacity, and enhanced resistance to exhaustion.

22. The method of claim 21, wherein the improved T cell subset ratio comprises one or more selected from the group consisting of: a reduced ratio of regulatory T cells, an increased ratio of stem T cells, an increased ratio of cytotoxic T cells, and a reduced 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 according to claim 1, further comprising genetically modifying the cells of the expanded tumor-infiltrating lymphocyte population.

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

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

28. The method according to any one of claims 25 to 27, wherein the cells of the expanded tumor-infiltrating lymphocyte population contain modifications at one or more endogenous genes selected from the group consisting of NR4A1, NR4A2, NR4A3, RC3H1, and ZC3H12A, wherein optionally the modifications result in a reduction or inhibition of the expression of the one or more endogenous genes and / or a reduction or inhibition of the function of one or more proteins encoded by the one or more endogenous genes.

29. The method according to any one of claims 25 to 27, wherein the cells of the expanded tumor-infiltrating lymphocyte population exhibit improved TIL characteristics after genetic modification compared with unmodified TIL.

30. The method of claim 29, wherein the improved TIL properties comprise one or more selected from the group consisting of: increased TIL cell number and proliferative capacity, increased survival capacity, improved T cell subset ratio, increased cytokine secretion capacity, increased tumor cell killing capacity, and increased resistance to exhaustion.

31. A tumor-infiltrating lymphocyte population, said 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 of claim 31, and optionally a pharmaceutically acceptable carrier.

33. The use of the tumor-infiltrating lymphocyte population obtained by the method of any one of claims 1-30, or the tumor-infiltrating lymphocyte population of claim 31, or the pharmaceutical composition of claim 32, in the preparation of a medicament for the prevention and / or treatment of tumors.

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

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

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