Method for improving autologous tumor antigen reactivity of tumor infiltrating lymphocytes and application

Abstract

The invention provides a method for improving autologous tumor antigen reactivity of tumor infiltrating lymphocytes and application, and belongs to the field of cell therapy. The method comprises the following steps: culturing a tumor sample obtained from a subject in a culture medium containing a first factor and a second factor to obtain an amplified tumor infiltrating lymphocyte population, and the culture medium does not contain trophoblast cells; wherein the first factor comprises a CD3 agonist, a 4-1BB agonist and an IL-2 (Interleukin-2); and the second factor comprises at least one of IL-7, IL-15, TGF (Transforming Growth Factor) beta, IL-27 and CD25-bias IL-2. The culture medium containing the first factor and the second factor is used for carrying out one-step amplification on the tumor infiltrating lymphocytes, so that the autologous tumor antigen reactivity of the tumor infiltrating lymphocytes can be effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell therapy, in particular, to a method for improving the autologous tumor antigen reactivity of tumor infiltrating lymphocytes and applications thereof. BACKGROUND

[0002] Adoptive transfer therapy of tumor infiltrating lymphocytes (TILs) has been proven to be an effective means for treating patients with poor prognosis and difficult-to-cure solid tumors.

[0003] Tumor infiltrating lymphocytes (TILs) are required for the treatment of tumors by adoptive autologous transfer, and the TILs need to have sufficient number and high cell function, but the tumor antigen reactivity of TILs obtained by in vitro expansion is insufficient, which affects the therapeutic effect of TILs therapy. SUMMARY

[0004] The present application aims to at least partially solve one of the technical problems in the related art. To this end, the present application provides a method for expanding tumor infiltrating lymphocytes, which can effectively improve the autologous tumor antigen reactivity of tumor infiltrating lymphocytes by using a culture medium containing a first factor and a second factor for one-step expansion of tumor infiltrating lymphocytes.

[0005] Specifically, the technical solution of the present application is as follows:

[0006] In a first aspect, the present application provides a method for expanding tumor infiltrating lymphocytes. According to embodiments of the present application, the method comprises: culturing a tumor sample obtained from a subject in a culture medium containing a first factor and a second factor, thereby obtaining an expanded population of tumor infiltrating lymphocytes, and the culture medium does not contain feeder cells; wherein the first factor comprises: a CD3 agonist, a 4-1BB agonist, and IL-2; and the second factor comprises: at least one of IL-7, IL-15, TGFβ, IL-27, and CD25-bias IL-2 (i.e., IL-2Rα-bias IL-2).

[0007] In some examples of the present application, the tumor sample is fragmented before being cultured.

[0008] In some examples of the present application, the tumor sample comprises tumor fragments having a size of 0.5mm 3 to 27mm 3 In some examples of the present application, the tumor sample comprises digested tumor fragments.

[0009] In some examples of the application, the CD3 agonist comprises an anti-CD3 antibody and / or antigen binding fragment thereof, optionally a humanized anti-CD3 antibody and / or antigen binding fragment thereof. In some preferred examples of the application, the CD3 agonist is OKT3.

[0010] In some examples of the application, the CD3 agonist has a final concentration of 10 ng / mL - 30 ng / mL. It will be appreciated that the CD3 agonist has a final concentration of 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL or 30 ng / mL. In some preferred examples of the application, the CD3 agonist has a final concentration of 30 ng / mL.

[0011] In some examples of the application, the 4-1BB agonist comprises a ligand-competitive 4-1BB agonistic antibody and / or antigen binding fragment thereof, or a ligand- noncompetitive 4-1BB agonistic antibody and / or antigen binding fragment thereof. In some preferred examples of the application, the 4-1BB agonist is selected from Utomilumab or Urelumab.

[0012] In some examples of the application, the 4-1BB agonist has a final concentration of 1 pg / mL - 10 pg / mL. It will be appreciated that the 4-1BB agonist has a final concentration of 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, 6 pg / mL, 7 pg / mL, 8 pg / mL, 9 pg / mL or 10 pg / mL. In some preferred examples of the application, the 4-1BB agonist has a final concentration of 10 pg / mL.

[0013] In some examples of the application, the IL-2 has a final concentration of 1000 IU / mL - 3000 IU / mL in the medium. It will be appreciated that the IL-2 has a final concentration of 1000 IU / mL, 1200 IU / mL, 1400 IU / mL, 1600 IU / mL, 1800 IU / mL, 2000 IU / mL, 2200 IU / mL, 2400 IU / mL, 2600 IU / mL, 2800 IU / mL or 3000 IU / mL in the medium.

[0014] In some examples of the application, the second factor comprises: IL-7 and IL-15.

[0015] In some examples of the application, the second factor comprises: IL-27 and CD25-bias IL-2.

[0016] In some examples of the application, the second factor comprises: CD25-bias IL-2.

[0017] In some examples of the application, the second factor comprises: TGFβ.

[0018] In some preferred examples of the application, the second factor is from human origin.

[0019] In some examples of the application, the final concentration of IL-7 in the culture medium is 5 ng / mL - 15 ng / mL. It will be appreciated that the final concentration of IL-7 in the culture medium is 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL. In some preferred examples of the application, the final concentration of IL-7 in the culture medium is 10 ng / mL.

[0020] In some examples of the application, the final concentration of IL-27 in the culture medium is 20 ng / mL - 30 ng / mL. It will be appreciated that the final concentration of IL-27 in the culture medium is 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL or 30 ng / mL. In some preferred examples of the application, the final concentration of IL-15 in the culture medium is 25 ng / mL.

[0021] In some examples of the application, the final concentration of CD25-bias IL-2 in the culture medium is 500 IU / mL - 1500 IU / mL. It will be appreciated that the final concentration of CD25-bias IL-2 in the culture medium is 500 IU / mL, 600 IU / mL, 700 IU / mL, 800 IU / mL, 900 IU / mL, 1000 IU / mL, 1100 IU / mL, 1200 IU / mL, 1300 IU / mL, 1400 IU / mL or 1500 IU / mL. In some preferred examples of the application, the final concentration of CD25-bias IL-2 in the culture medium is 1000 IU / mL.

[0022] In some examples of the application, the final concentration of TGFp in the culture medium is between 1 ng / mL and 5 ng / mL. It will be appreciated that the final concentration of TGFp in the culture medium is 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL or 5 ng / mL. In some preferred examples of the application, the final concentration of TGFp in the culture medium is 2 ng / mL.

[0023] In some examples of the application, the components of the culture medium remain constant. In some examples of the application, between about 30% and about 99% of the culture medium is replaced every 2 to 5 days.

[0024] In some examples of the application, wherein the replacing the culture medium comprises adding fresh culture medium to the culture, wherein the fresh culture medium comprises IL-2 and does not comprise a CD3 agonist and a 4-1 BB agonist.

[0025] In some examples of the application, wherein the fresh culture medium further comprises a cell basal medium, a serum replacement, L-glutamine or a substitute thereof, an antibiotic. In some examples of the application, the culture medium does not comprise serum.

[0026] In some examples of the application, the cell basal medium comprises, but is not limited to, X-vivo 15 medium, AIM-V medium, RPMI-1640 medium, OpTmizer™ medium or OpTmizer™ Pro medium.

[0027] In some examples of the application, the tumor infiltrating lymphocyte (TIL) culture is for a duration of 14-26 days.

[0028] In some examples of the application, the tumor infiltrating lymphocyte culture is for a duration of at least 14 days.

[0029] In some examples of the application, the number of TIL cells harvested at the end of the culture is above 1 x 10 7 preferably above 1 x 10 8 more preferably above 1 x 10 9 per fragment.

[0030] In some examples of the application, wherein after culturing the tumor sample obtained from the subject, the CD3 + TILs are above about 80%, preferably above about 85%, more preferably above about 90% of the total number of cells in the expanded tumor infiltrating lymphocyte population.

[0031] In some examples of the application, wherein after culturing the tumor sample obtained from the subject, the CD8 +TILs are CD3 + More than about 50% of TILs.

[0032] In some examples of the application, wherein following culturing of the tumor sample obtained from the subject, the CD69- / CD39- TILs in the expanded population of tumor infiltrating lymphocytes are more than about 50% of CD8 + More than about 10% of TILs.

[0033] In some examples of the application, wherein following culturing of the tumor sample obtained from the subject, the exhausted T cells in the expanded population of tumor infiltrating lymphocytes are more than about 20% of CD8 + Less than about 50% of TILs, preferably less than about 20%.

[0034] In some examples of the application, wherein following culturing of the tumor sample obtained from the subject, the Foxp3 + TILs are CD4 + Less than about 20% of TILs.

[0035] In some examples of the application, the method further comprises genetically modifying the cells of the expanded population of tumor infiltrating lymphocytes.

[0036] In some examples of the application, the method further comprises genetically modifying the cells of the expanded population of tumor infiltrating lymphocytes using a gene editing system, optionally selected from the group consisting of RNA interference molecules, transcription activator-like effector nucleases, zinc finger nucleases, and RNA-guided nucleases.

[0037] In one embodiment, the modification at one or more genes is an insertion, deletion, or mutation of one or more nucleic acids.

[0038] In some examples of the application, the endogenous genes of the cells of the expanded population of tumor infiltrating lymphocytes, following genetic modification, exhibit improved TIL properties as compared to TILs that are not genetically modified.

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

[0040] According to embodiments of the present application, after adding the first factor, the tumor infiltrating lymphocyte population obtained by one-step expansion shows improved TIL properties compared to the tumor infiltrating lymphocyte population obtained by conventional multi-step expansion. 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 T cell subset proportion, increased cytokine secretion capacity, increased tumor cell killing capacity, and increased anti-exhaustion capacity. The improved T cell subset proportion includes one or more selected from the group consisting of: decreased proportion of regulatory T cells, increased proportion of stem T cells, increased proportion of killer T cells, and decreased proportion of exhausted T cells.

[0041] According to embodiments of the present application, on the basis of the first factor, a second factor is further added, and the tumor infiltrating lymphocyte population obtained by the method has the following advantages:

[0042] 1) the secretion level of IFN-γ in TILs is improved; 2) the proportion of CD8 + CD137 + TILs is improved; 3) the absolute number of CD8 + CD137 + TILs is improved; 4) the proportion of CD8 + CD25 + TILs is improved; 5) the absolute number of CD8 + CD25 + TILs is improved; 6) the killing capacity of TILs on autologous organoids is improved; 7) the infiltration capacity of TILs in autologous organoids is improved; 8) the proportion of CD8 + CD25 + cells in TILs at the time of harvest is improved; 9) the proportion of CD8 + CD137 + cells in TILs at the time of harvest is improved; 10) the proportion of CD8 + CD103 + cells in TILs at the time of harvest is improved; 11) the expansion speed during the culture of TILs is not significantly affected; 12) the proportion of CD3 + cells in TILs at the time of harvest is not significantly affected; 13) the proportion of CD8 + cells in TILs at the time of harvest is not significantly affected; 14) the proportion of CD4 + cells in TILs at the time of harvest is not significantly affected; 15) the proportion of Treg cells in TILs at the time of harvest is not significantly affected; 16) the stem regulatory transcription factor TCF-1 +the proportion of CD4+ T cells had no significant effect; 17) the transcription factor BCL6 + the proportion of CD4+ T cells had no significant effect; 18) the TILs terminal exhaustion marker TIM-3 + BLIMP1 + the proportion of CD4+ T cells had no significant effect.

[0043] In a second aspect, the present application provides a tumor infiltrating lymphocyte population. According to embodiments of the present application, the tumor infiltrating lymphocyte population is obtained by the methods disclosed herein. The tumor infiltrating lymphocyte population of the present application is capable of effectively boosting the antigenic reactivity against autologous tumors.

[0044] In a third aspect, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the tumor infiltrating lymphocyte population disclosed herein, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition of the present application is capable of achieving effective killing of autologous tumors.

[0045] In some examples of the present application, the pharmaceutical composition is a suspension of TILs in a sterile buffer. In some embodiments, the TILs are administered as a single intra-arterial or intravenous infusion, preferably lasting about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0046] The TILs provided in the pharmaceutical composition of the present application are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is employed, the subject to be treated, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the judgment of the attending physician. Clinically determined dosages of TILs can also be used where appropriate.

[0047] The amount of the pharmaceutical composition, such as the dosage of TILs, administered using the methods herein will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients, and the judgment of the prescribing physician.

[0048] In some examples of the present application, the therapeutically effective dose is about 4 x 10 10 to about 1 x 10 11 TILs.

[0049] In some examples of the present application, the TILs can be administered in a single dose. Such administration can be performed by injection, for example, intravenous injection.

[0050] In some examples of the present application, the TILs can be administered in multiple doses.

[0051] The administration can be once, twice, three times, four times, five times, six times, or more than six times per year. The administration can be once per month, once per two weeks, once per week, or once every other day. The TILs can be administered continuously as necessary.

[0052] An effective amount of TILs can be administered in a single dose or in multiple doses by any of the accepted modes of administration of an agent of like efficacy, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, by implantation, or by inhalation. In certain embodiments, the TILs are administered intravenously.

[0053] In some examples of the present application, the medicament of the present application is used for preventing and / or treating a solid tumor. Exemplary solid tumors can include, but are not limited to, one or more selected from 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, gastric cancer, colorectal cancer, and kidney cancer.

[0054] In a fourth aspect, use of the tumor infiltrating lymphocyte population obtained according to the method disclosed herein or the tumor infiltrating lymphocyte population disclosed herein or the pharmaceutical composition disclosed herein in the manufacture of a medicament for preventing and / or treating a tumor.

[0055] In some examples of the present application, the tumor is a solid tumor.

[0056] In some examples of the present application, 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, gastric cancer, colorectal cancer, and kidney cancer.

[0057] In a fifth aspect, the present application provides a method for treating a tumor in a subject in need thereof. According to embodiments of the present application, the method comprises administering to the subject a therapeutically effective amount of the tumor infiltrating lymphocyte population obtained according to the method disclosed herein or the tumor infiltrating lymphocyte population disclosed herein or the pharmaceutical composition disclosed herein. By administering to the subject a therapeutically effective amount of the tumor infiltrating lymphocyte population or the pharmaceutical composition, effective killing of the tumor in the subject can be achieved.

[0058] In some examples of the present application, the tumor is a solid tumor.

[0059] In some examples of the present application, 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, gastric cancer, colorectal cancer, and kidney cancer.

[0060] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those having ordinary skill in the art upon examination of the following or can be learned from practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0061] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0062] Figure 1 A schematic diagram of the detection result of the secretion level of IFN-γ in TILs provided by an embodiment of the present application is shown in the figure; wherein, A is a tumor sample from subject LC003; B is a tumor sample from subject LC006; C is a tumor sample from subject LC008;

[0063] Figure 2 A schematic diagram of the detection result of the proportion of CD8 + CD137 + TILs provided by an embodiment of the present application is shown in the figure; wherein, A is a tumor sample from subject LC003; B is a tumor sample from subject LC006; C is a tumor sample from subject LC008;

[0064] Figure 3 A schematic diagram of the detection result of the absolute number of CD8 + CD137 + TILs provided by an embodiment of the present application is shown in the figure; wherein, A is a tumor sample from subject LC003; B is a tumor sample from subject LC006;

[0065] Figure 4 A schematic diagram of the detection result of the proportion of CD8 + CD25 + TILs provided by an embodiment of the present application is shown in the figure; wherein, A is a tumor sample from subject LC003; B is a tumor sample from subject LC006; C is a tumor sample from subject LC008;

[0066] Figure 5 A schematic diagram of the detection result of the absolute number of CD8 + CD25 + TILs provided by an embodiment of the present application is shown in the figure; wherein, A is a tumor sample from subject LC003; B is a tumor sample from subject LC006;

[0067] Figure 6A schematic diagram of the killing ability of TILs to organoids provided by an embodiment of the present application; wherein A is the killing ability of TILs to organoids when the effector target ratio is 3:1; B is the killing ability of TILs to organoids under a high-content imaging system, the scale is 500 μm; C is the killing ability of TILs to organoids when co-cultured for 48 hours;

[0068] Figure 7 A schematic diagram of the infiltration ability of TILs in autologous organoids provided by an embodiment of the present application; wherein A is the infiltration ability of TILs in autologous organoids under a high-content imaging system; B is the statistical result of the infiltration ability of TILs in autologous organoids;

[0069] Figure 8 A schematic diagram of the proportion detection result of CD8 + CD25 + cells of TILs when harvested provided by an embodiment of the present application; wherein A is a tumor sample from subject LC003; B is a tumor sample from subject LC006; C is a tumor sample from subject LC008;

[0070] Figure 9 A schematic diagram of the proportion detection result of CD8 + CD137 + cells of TILs when harvested provided by an embodiment of the present application; wherein A is a tumor sample from subject LC003; B is a tumor sample from subject LC004; C is a tumor sample from subject LC008;

[0071] Figure 10 A schematic diagram of the proportion detection result of CD8 + CD103 + cells of TILs when harvested provided by an embodiment of the present application; wherein A is a tumor sample from subject OC002; B is a tumor sample from subject LC004; C is a tumor sample from subject LC008;

[0072] Figure 11 A schematic diagram of the expansion speed detection result of TILs in the culture process provided by an embodiment of the present application; wherein A is a tumor sample from subject LC002; B is a tumor sample from subject LC003; C is a tumor sample from subject LC008; D is a tumor sample from subject LC009; E is a tumor sample from subject OC002;

[0073] Figure 12 A schematic diagram of the proportion detection result of CD3 +Ratio detection results of cells; wherein, A is a tumor sample derived from LC002 of a subject; B is a tumor sample derived from LC003 of a subject; C is a tumor sample derived from LC004 of a subject; D is a tumor sample derived from LC005 of a subject; E is a tumor sample derived from OC002 of a subject; F is a tumor sample derived from LC008 of a subject;

[0074] Figure 13 CD8 TILs at the time of harvest for one embodiment provided by the present application + Ratio detection results of cells; wherein, A is a tumor sample derived from LC002 of a subject; B is a tumor sample derived from LC003 of a subject; C is a tumor sample derived from LC004 of a subject; D is a tumor sample derived from LC005 of a subject;

[0075] Figure 14 CD4 TILs at the time of harvest for one embodiment provided by the present application + Ratio detection results of cells; wherein, A is a tumor sample derived from LC002 of a subject; B is a tumor sample derived from LC003 of a subject; C is a tumor sample derived from LC004 of a subject; D is a tumor sample derived from LC005 of a subject;

[0076] Figure 15 Ratio detection results of Treg cells at the time of harvest for one embodiment provided by the present application; wherein, A is a tumor sample derived from LC001 of a subject; B is a tumor sample derived from LC004 of a subject; C is a tumor sample derived from LC005 of a subject; D is a tumor sample derived from LC008 of a subject;

[0077] Figure 16 Ratio detection results of TILs stemness regulatory transcription factor TCF-1 for one embodiment provided by the present application + ; wherein, A is a tumor sample derived from LC003 of a subject; B is a tumor sample derived from LC006 of a subject; C is a tumor sample derived from LC008 of a subject;

[0078] Figure 17 Ratio detection results of transcription factor BCL6 regulating early exhaustion of TILs for one embodiment provided by the present application + ; wherein, A is a tumor sample derived from LC002 of a subject; B is a tumor sample derived from LC006 of a subject; C is a tumor sample derived from LC009 of a subject;

[0079] Figure 18 TIM-3 BLIMP1 + BLIMP1 +A schematic diagram of the ratio detection results; wherein A is a tumor sample derived from subject LC003; B is a tumor sample derived from subject LC006; C is a tumor sample derived from subject LC009. DETAILED DESCRIPTION

[0080] In order to solve the problem that the TILs obtained by the existing tumor infiltrating lymphocyte expansion method have low autologous tumor antigen reactivity, the present application provides a one-step method for expanding TILs without using feeder cells, which comprises culturing a tumor sample obtained from a subject in a culture medium containing a first factor and a second factor, thereby obtaining an expanded tumor infiltrating lymphocyte population; wherein the first factor comprises: a CD3 agonist, a 4-1BB agonist and IL-2; and the second factor comprises: at least one of IL-7, IL-15, TGFβ, IL-27 and CD25-bias IL-2. The method significantly improves the autologous tumor antigen reactivity of the obtained TILs.

[0081] While the application can be susceptible to various modifications and alternative forms, specific embodiments are shown by way of example in the drawings and are described in detail herein. It should be understood that the application is not intended to be limited to the particular implementations disclosed. Additionally, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0082] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated. See e.g., Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Abbas et al., Cellular and Molecular Immunology, 6th ed., W.B. Saunders Company (2010); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0083] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the term "or" means "and / or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms such as "include," "includes," and "included" is not limiting. The terms "about" and "approximately" generally mean values within a statistically meaningful range. Such ranges can be within one order of magnitude of a given value or range, can be within 50%, can be within 20%, can be within 10%, can be within 5%. The allowable variation encompassed by "about" or "approximately" can depend upon the particular system being studied, and can be readily appreciated by one of ordinary skill in the art. The terms "above," "below," "up to," and "at least" can include the recited number. Furthermore, ranges provided in the specification and the appended claims are inclusive of the recited endpoints and all values between them.

[0084] Definitions

[0085] For better understanding of the present application, definitions and explanations of relevant terms are provided as follows.

[0086] As used herein, the term "cell population" or "TIL population" refers to a number of cells or TILs that share common traits. Generally, the number of cells in a cell population is generally in the range of 1 x 10 6 to 1 x 10 10 Different TIL populations comprise different numbers.

[0087] As used herein, the term "expansion" or "expansion process" refers to a process of stimulating or activating cells and culturing the cells. An expansion process can result in an increase in the total number of desired cells in a cultured cell population, e.g., an increase in the total number of TILs, after stimulating or activating and culturing the cells. Expansion does not require an increase in the number of all cell types in the cultured cell population. Rather, in some aspects, only a subset of the cells in the cultured cell population increases in number during expansion, while the number of other cell types can remain unchanged or can decrease. A process that isolates or enriches TILs without substantially increasing the number of TILs is not an expansion process.

[0088] As used herein, the term "agonist" refers to a chemical, a molecule, a macromolecule, a molecular complex, or a macromolecular complex that binds to a target at the cell surface or in soluble form. In certain 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.

[0089] As used herein, the term“feeder cell” refers to a cell that is used to provide extracellular secretions that aid in the proliferation of another cell type. In certain embodiments, the feeder cell can be a peripheral blood mononuclear cell (PBMC) or an antigen presenting cell (APC). The feeder cell can also be a cell that is not normally used as a feeder cell that is engineered to secrete or express extracellular secretions that aid in the proliferation of another cell type.

[0090] As used herein, the term“IL-2” also known as“TCGF,”“interleukin 2,”“IL-2,” generally refers to a cytokine produced by activated CD4 + and CD8 + T lymphocytes that secretes a cytokine. IL-2 in the present invention includes all forms of IL-2, such as IL-2 derived from humans or other mammals, and includes IL-2 wild type and mutant, as long as the mutant has similar activity as the wild type. In the present invention, IL-2 includes recombinant human IL-2.

[0091] As used herein, the term“CD3” refers to a costimulatory molecule expressed on the surface of T lymphocytes that is a protein complex. CD3 has five peptide chains, a gamma chain, a delta chain, an epsilon chain, a zeta chain, and an eta chain, all of which are transmembrane proteins. The transmembrane region of the CD3 molecule links the transmembrane regions of the two peptide chains of the TCR via salt bridges, forming a TCR-CD3 complex that collectively participates in the recognition of antigen by T cells. The activation signal generated by TCR recognition of antigen is transduced into the T cell by CD3. As used herein, the term“anti-CD3 antibody” refers to an antibody or variant thereof, such as a monoclonal antibody, and includes a human antibody, a humanized antibody, a chimeric antibody, or a murine antibody directed against the CD3 receptor in the T cell antigen receptor of a mature T cell, the source of which is not limited, e.g., can be in-house 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, e.g., otelixizumab, teplizumab, and visilizumab.

[0092] As used herein, the term“4-1BB” also known as“CD137,” generally refers to an inducible costimulatory receptor expressed on activated CD4 + and CD8 + T cells, NKT, NK cells, DC cells, macrophages, eosinophils, neutrophils, and mast cells, as well as Tregs.

[0093] As used herein, the term "anti-4-1BB antibody" refers to an antibody or a variant thereof, e.g., a monoclonal antibody, and includes a human antibody, a humanized antibody, a chimeric antibody, or a murine antibody against 4-1BB, the origin of which is not limited, e.g., can be self-developed or commercially available. In some embodiments, an anti-4-1BB antibody can be used as a 4-1BB ligand. Anti-4-1BB antibodies include Utomilumab and Urelumab.

[0094] As used herein, the term "IL-7", also known as "interleukin-7", refers to a cytokine produced by bone marrow stromal cells, thymic epithelial cells, etc. It mainly plays a role by binding to IL-7 receptor (IL-7Rα / CD127). IL-7 plays an important role in maintaining T cell homeostasis, promoting early T cell development, and enhancing T cell survival, and is widely used in immune cell in vitro expansion systems.

[0095] As used herein, the term "IL-15", also known as "interleukin-15", refers to a cytokine produced by monocytes, dendritic cells, etc. It mediates signal transduction through the IL-15 receptor complex (IL-15Rα / IL-2Rβ / γc), and plays an important role in maintaining natural killer cells (NK cells) and memory CD8 + IL-15 plays a key role in the proliferation and survival of T cells. IL-15 is used to enhance the effector function of T cells or NK cells in various immunotherapy strategies.

[0096] As used herein, the term "TGFβ" refers to a class of cytokines of the Transforming Growth Factor-beta (TGFβ) family, which usually includes TGFβ1, TGFβ2, and TGFβ3 subtypes, and has the function of regulating cell proliferation, differentiation, apoptosis, and immune response. In the tumor immune microenvironment, TGFβ can inhibit T cell activation and infiltration, induce immune tolerance, and is widely considered to be an immunosuppressive factor.

[0097] As used herein, the term "IL-27", also known as "interleukin-27", refers to a heterodimeric cytokine secreted by dendritic cells and macrophages, composed of EBI3 subunit and p28 subunit. IL-27 can activate STAT1 and STAT3 signaling pathways through IL-27 receptor, has a dual immune regulation effect, can induce T cell activation, and can also promote the production of immunosuppressive factors (such as IL-10), and is widely involved in the process of inflammation and tumor immune regulation.

[0098] As used herein, the term "CD25-bias IL-2" refers to a recombinant human IL-2 molecule that is structurally or functionally engineered to preferentially bind to the IL-2 high affinity receptor (CD25, i.e., IL-2Ra). In comparison to native IL-2, CD25-bias IL-2 selectively expands CD25highexpressing cells (e.g., regulatory T cells or activated CD8 + T cells) on the basis of not significantly activating the CD122 (IL-2Rβ) / CD132 (IL-2Rγ) intermediate affinity complex, for immunomodulation or to enhance the specificity and safety of T cell therapy.

[0099] As used herein, the term "TIL property" refers to a property of TIL cells that is improved after the TIL cells are modified by the methods of the application. Changes in TIL properties can include: increased TIL proliferation capacity, increased TIL cell number, increased persistence, improved T cell subpopulation ratio, increased cytokine secretion capacity, increased granzyme secretion capacity, increased tumor cell killing capacity, decreased level of cell exhaustion, or any combination thereof. Changes in the application can be increases or decreases.

[0100] As used herein, the term "T cell subpopulation ratio" generally refers to the proportion of different T cell subpopulations in TIL cells or a TIL population. For example, different T cell subpopulations of the application have different immunological activities and / or differentiation capacities. For example, T cell subpopulations of the application can be distinguished according to T cell surface markers. For example, cytotoxic T cells can have a CD8 + phenotype.

[0101] In the present application, the term "killing capacity" generally refers to the ability to kill target cells by contacting the cells with an effective amount of an agent of the application. In one embodiment, the agent of the application can be a TIL cell. Killing by the agent of the application can include killing cells by CDC, apoptosis, ADCC, and / or phagocytosis by the agent itself or by facilitating other cells or agents, or by a combination of two or more of these mechanisms.

[0102] As used herein, the term "subject" refers to a human who has a tumor into which a population of lymphocytes that have left the human bloodstream has migrated and transformed into TILs. In some embodiments, the human can be a patient in need of an immunotherapy involving an expanded population of the patient's own TILs. In other embodiments, the human can be a patient in need of an immunotherapy involving an expanded population of another patient's own TILs.

[0103] As used herein, the term "administering" refers to the physical introduction of a therapeutic agent or a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems. Different routes of administration of the therapeutic agents described herein (e.g., TILs cultured as described herein) include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion.

[0104] The term "therapeutically effective amount" refers to the amount of an agent (e.g., TILs cultured as described herein) that provides the desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired change in a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to cause tumor shrinkage and / or to reduce the rate of tumor growth (e.g., inhibit tumor growth) or to prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations.

[0105] As used herein, the term "tumor cell" or "cancer cell" refers to a cell that divides in an uncontrolled manner, forms a solid tumor, or fills the blood with abnormal cells. When there is no longer a need for more daughter cells, healthy cells stop dividing, but tumor or cancer cells continue to make copies. They are also able to spread from one part of the body to another part in a process called metastasis. Tumor cells can be isolated from a variety of cancer types, including bladder cancer, breast cancer, cervical cancer, colon and rectum cancer, endometrial cancer, kidney cancer, lip and oral cavity cancer, liver cancer, melanoma, mesothelioma, lung cancer, non-small cell carcinoma lung cancer, head and neck cancer, neuroblastoma, glioblastoma multiforme, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, and thyroid cancer. Tumor cells can be isolated from primary tumors and metastases.

[0106] As used herein, the term "tumor sample" refers to tumor cells isolated from a subject. In certain embodiments, a tumor sample is at least a portion of a solid tumor isolated in whole or in part from a subject having a tumor. A subject tumor sample can be obtained using methods known in the art, generally by surgical resection, punch biopsy, or other means for obtaining a mixture containing tumor and TIL cells. Tumor samples can be isolated from a variety of cancer types, including bladder cancer, breast cancer, cervical cancer, colon and rectum cancer, endometrial cancer, kidney cancer, lip and oral cavity cancer, liver cancer, melanoma, mesothelioma, lung cancer, non-small cell carcinoma lung cancer, head and neck cancer, neuroblastoma, glioblastoma multiforme, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, and thyroid cancer. Tumor samples can be isolated from primary tumors and metastases. "Tumor sample" also includes tumor samples that have been fragmented into "tumor fragments." Fragmentation can be physical fragmentation, mechanical fragmentation, ultrasonic fragmentation, enzymatic fragmentation, or any combination thereof. Fragmentation can be performed mechanically and, optionally, the tumor fragments are subsequently digested into a single cell suspension by enzymes. Mechanical disintegration methods can include mincing or slicing the tumor into smaller tumor fragments, while enzymatic disintegration methods can include treating the tumor fragments with specific enzymes such as proteases.

[0107] As used herein, the term "culture medium" refers to a liquid or gel designed to support the survival, growth, and / or proliferation of cells in an artificial environment. The formulation of cell culture media is well known in the art. Typically, a culture medium generally comprises a defined set of components, such components can include energy sources, growth factors, hormones, stimulants, activators, sugars, salts, vitamins, and / or amino acids, and / or combinations of these, the particular components or concentrations thereof depending on the requirements of the cells to be cultured and / or the desired cell culture parameters.

[0108] As used herein, the term "culture medium with constant components" refers to a culture medium comprising a defined set of components, such as specific stimulants and activators, in which the identity of the components remains constant, but the concentration of one or more components can vary. In certain embodiments, the concentration of one or more components in the culture medium changes over time as the cells are cultured in the medium. However, the new medium that is exchanged each time has the same components.

[0109] The scheme of the present application will be explained below with reference to Examples. Those skilled in the art will appreciate that the following Examples are intended to be illustrative only and should not be viewed as limiting the scope of the present application. Unless otherwise indicated, the techniques and conditions in the Examples are in accordance with those described in the literature or as per the manufacturer's instructions. Unless otherwise indicated, the reagents or instruments used are conventional products that can be obtained commercially.

[0110] The main materials and reagents used in the following examples are shown in Table 1.

[0111] Table 1

[0112]

[0113]

[0114] The general method steps used in the following examples are as follows:

[0115] 1. TILs expansion culture

[0116] 1.1 Culture medium

[0117] Complete medium (CM): 1 L Optmizer (base medium), 26 mL supplement (supplement), 5% mL SR, 10% Glutamax and 100 μg / mL primocine (antibiotic);

[0118] Complete medium (CM1): 1 L Optmizer (base medium) and 26 mL supplement (supplement);

[0119] 1.2 Tumor tissue processing

[0120] The tumor tissue block was soaked with 10% gentamicin for 5 min, followed by 10% clindamycin for 5 min. Then rinse with CM1 for 2 times. The tissue block was cut into 1-3 mm 3 small pieces with sterile scissors.

[0121] 1.3 One-step culture method without feeder cells (FOST) to expand tumor infiltrating lymphocytes, the specific steps are as follows:

[0122] On day 0, the obtained tissue small pieces were plated into Grex6 according to experimental grouping, 20 mL of Optmizer medium containing 10 μg / mL 4-1BB agonist (Utomilumab), 30 ng / mL CD3 agonist (OKT3), 1000 IU / mL IL-2, 1% PS and 10 μg / mL primocin were added to each well, and 15 tissue blocks were plated per well. On day 5, the medium was supplemented to 40 mL. According to the cell growth: every 2-3 days, half of the medium was replaced and cell counting was performed. When the number of cells per well reached 2×10 8 , 1×10 7 -2×10 7 cells were taken for subculture. On day 17-26, when the number of cells reached more than 5×10 9 , the expansion was completed, and the TIL cells were harvested, and samples were taken for subsequent detection.

[0123] 2. Flow detection

[0124] 2.1 After receiving the sample, check the sample information (including: name, quantity, cell number). After the sample is adjusted, centrifuge at 400g for 5 min, and discard the supernatant. After adjusting the cell density to 1x10 7 cells / mL, take 100 μL of the cell suspension into an EP tube, and centrifuge at 400g for 5 min after placing the sample in a low-temperature centrifuge, and discard the supernatant.

[0125] 2.2 Take 100 μL of the prepared mixed antibody and add it to the sample tube, incubate at room temperature for 20 min in the dark, and record the start and end times.

[0126] 2.3 (Membrane surface marker) Add 200 μL of PBS to the sample tube to terminate, centrifuge at 400g for 5 min after placing the sample in a low-temperature centrifuge, and discard the supernatant for detection.

[0127] 2.4 The preparation of the membrane-breaking solution is shown in Table 2.

[0128] Table 2

[0129]

[0130] 1x Washing Buffer (1x Washing Buffer) is prepared as shown in Table 3.

[0131] Table 3

[0132] Permeabilization Buffer 10x Deionized water Total 1.5 mL 13.5 mL 15 mL

[0133] 2.5 (Nuclear marker) Add 100 μL of PBS to the sample well to terminate, centrifuge at 1500g for 15 s after placing the sample in a refrigerated centrifuge, and discard the supernatant.

[0134] 2.6 Add 200 μL of the prepared membrane-breaking solution to the sample well, and incubate at 4°C for 0.5 h, and record the start and end times.

[0135] 2.7 Add 100 μL of 1x Washing Buffer to the sample well to terminate, centrifuge at 1500g for 15 s after placing the sample in a refrigerated centrifuge, and discard the supernatant.

[0136] 2.8 Add 100 μL of the prepared nuclear antibody to the sample well, and incubate at 4°C in the dark overnight.

[0137] 2.9 Add 100 μL of 1x Washing Buffer to the sample well to terminate, centrifuge at 1500g for 15 s after placing the sample in a refrigerated centrifuge, and discard the supernatant.

[0138] 2.10 Add 200 μL 1 x Washing Buffer to resuspend the sample and run on the machine.

[0139] 3. Autologous tumor cell suspension activation

[0140] 3.1 Autologous tumor cell suspension preparation

[0141] Tumor tissue pieces were soaked in 10% gentamicin for 5 min, followed by 10% clindamycin for 5 min. Then the tissue pieces were washed twice with CM. The tissue pieces were cut into small pieces with sterile scissors and placed in a 70 μm cell strainer. The strainer was placed in a petri dish containing 5 mL of CM and the small tumor tissue pieces were pressed through the strainer with a syringe tail for 2-10 min. The homogenate outside the strainer was collected and transferred to a centrifuge tube and stored frozen after centrifugation.

[0142] 3.2 Autologous tumor cell suspension plating

[0143] The frozen autologous tumor cell suspension was thawed and added to 10 ml of pre-warmed 1640 complete medium. The cells were spun down and resuspended in an appropriate volume of 1640 complete medium. The cell density was adjusted to 1 x 10 6 cells / ml based on the cell count. 100 μl of the tumor cell suspension was added to each well of a 96-well cell plate. Anti-human HLA-ABC and anti-human HLA-DP / DQ / DR were added to the negative control wells at a concentration of 5 μg per well, i.e., HLA-I + II blocking group. The blank control wells were added with 100 μl of 1640 complete medium.

[0144] 3.3 TIL cell preparation and addition

[0145] The thawed TIL cells were added to 10 ml of pre-warmed OpTmizer complete medium. The cells were spun down and resuspended in complete medium to a concentration of about 5 x 10 6 cells / ml based on the labeled cell count. The cell density was adjusted to 1 x 10 6 cells / ml. 100 μl of the TIL cells were added to each well of a 96-well cell plate. After plating, the cell plate was checked for air bubbles and incubated in an incubator for about 24 hours. After incubation, the cell plate was removed and spun down at 300 g for 5 minutes. The supernatant was carefully aspirated and tested for IFN-γ content. The cell pellet in the cell plate was tested by flow cytometry.

[0146] 4. Elisa detection of IFN-γ secretion

[0147] Take the microplate from the equilibrated sealed bag at room temperature, add the sample to be tested and serially diluted standards to the wells, 100 μL / well. Seal the plate with the plate sealer, incubate at room temperature for 1 h in the dark. Wash the plate: carefully remove the plate sealer, discard the liquid in the wells, add 300 μL of 1 x Washing Buffer to each well, wash the plate 3 times, and tap dry on absorbent paper. Add 100 μL of Biotin-Anti-IFN-γ Antibody Working Solution to each well. Seal the plate with the plate sealer, incubate at room temperature for 1 h in the dark. Repeat the washing step. Add 100 μL of Streptavidin-HRP Working Solution to each well. Seal the plate with the plate sealer, incubate at room temperature for 30 min in the dark. Repeat the washing step. Add 100 μL of Substrate Solution to each well. Use the plate sealer to seal the plate, incubate at room temperature for about 20 min in the dark. The color development time can be adjusted according to the color development. Add 50 μL of Stop Solution to each well, gently shake the plate to mix well. Measure the absorbance of each well at 450 nm and 630 nm using a microplate reader. Read the plate within 10 min after stopping the reaction.

[0148] 5. TILs killing and infiltration detection of autologous tumor organoids

[0149] 5.1 Organoid construction

[0150] Place the fresh sample in tissue preservation solution, and transport it to the laboratory at low temperature. After washing the tissue with tissue washing solution, use ophthalmic scissors to break the tissue into 1-2 mm3, collect it into a centrifuge tube, add tissue enzyme solution, and incubate it at 37°C on a constant temperature horizontal shaking incubator for 30-60 min. After resuspending the cell suspension with organoid washing solution, pass it through a 100 μm cell mesh, collect the cell suspension after passing, centrifuge to obtain a cell pellet, and count it using a counting instrument. After resuspending with Matrigel, seed it in the form of a gel droplet (2-5 w cells / 50 μL gel droplet) in a 24-well cell culture plate, and after solidification in the incubator, add 500 μL of lung cancer organoid culture medium. The organoid culture medium should be supplemented with anti-apoptotic factors 2 days before culture, and the medium should be completely replaced every 3-4 days, and the organoids should be passaged every 7-14 days according to the culture conditions. During the culture process, record the bright field state photos of the organoids using a microscope.

[0151] 5.2 TILs and lung cancer organoid co-culture killing experiment

[0152] IBAC O2 chip-based tumor organoid-TIL killing model was constructed. Tumor organoid spheres (4200 cells / well) were inoculated on the surface of Matrigel, and TIL cells were added for contact co-culture at an effector-target ratio of 3: 1 (12600 cells / well) after stable culture. Before co-culture experiment, TIL cells were labeled with tracer dye and incubated at 37°C for 40 min. The concentration of tracer dye was 5 μM, and the concentration of apoptosis fluorescent dye added in the culture system was 1 μM. High content imaging was performed, and apoptosis fluorescent signal and TIL cell tracer fluorescent signal in the organoid were photographed at 0 h, 24 h, 48 h, and 72 h, respectively. MetaXPress high content analysis software was used to select the apoptosis fluorescent signal and TIL cell tracer fluorescent signal, respectively. By subtracting the T cell signal from the apoptosis signal image signal, only the signal representing the apoptosis of the organoid was obtained. The apoptosis fluorescent intensity of the corresponding image part was quantified to obtain the total fluorescence intensity of the organoid apoptosis. The efficacy was evaluated by comparing the total fluorescence intensity of the organoid apoptosis under different treatment conditions.

[0153] 5.3 TILs infiltration evaluation

[0154] IBAC O2 chip-based tumor organoid-TIL killing model was constructed. Tumor organoid spheres (4200 cells / well) were inoculated on the surface of Matrigel, and TIL cells were added for contact co-culture at an effector-target ratio of 3: 1 (12600 cells / well) after stable culture. Before co-culture experiment, TIL cells were labeled with tracer dye and incubated at 37°C for 40 min. The concentration of tracer dye was 5 μM, and the concentration of apoptosis fluorescent dye added in the culture system was 1 μM. High content imaging was performed, and apoptosis fluorescent signal and TIL cell tracer fluorescent signal in the organoid were photographed at 0 h, 24 h, 48 h, and 72 h, respectively. MetaXPress high content analysis software was used to select the apoptosis fluorescent signal and TIL cell tracer fluorescent signal, respectively. By subtracting the T cell signal from the apoptosis signal image signal, only the signal representing the apoptosis of the organoid was obtained. The apoptosis fluorescent intensity of the corresponding image part was quantified to obtain the total fluorescence intensity of the organoid apoptosis. The efficacy was evaluated by comparing the total fluorescence intensity of the organoid apoptosis under different treatment conditions.

[0155] It should be noted that the "method of the present application" described in the following examples is based on the addition of a second factor to the one-step culture method without trophoblast cells (FOST). The specific addition scheme is shown in Table 4.

[0156] Table 4

[0157]

[0158] Note: All cytokines are selected from human sources.

[0159] Example 1: The method of this application increases the secretion level of IFN-γ in TILs stimulated by autologous tumor cells.

[0160] Non-small cell lung cancer tumor samples from three subjects (LC003, LC006, and LC008) were amplified using both the one-step method without feeder cells (FOST) and the method described in this application, and co-stimulation was performed using different methods. The secretion level of IFN-γ in TILs stimulated with autologous tumor cells was detected by ELISA. The co-stimulation methods included: TILs... + Tumor group, anti-MHC group, and TIL only group. TIL + The Tumor group was co-stimulated with TIL and autologous tumor cell suspension; the anti-MHC group was co-stimulated with TIL and autologous tumor cell suspension, with the addition of an HLA blocker; the TIL only group contained only TIL and no tumor cells.

[0161] The results of the detection of IFN-γ secretion levels in TILs stimulated by autologous tumor cells using FOST and the method of this application are as follows: Figure 1 As shown. Among them, Figure 1 In this context, A represents the IFN-γ secretion levels of LC003 sample, FOST, and the method of this application (IL-7 / 15) under different co-stimulation modes; Figure 1 In this context, B represents the IFN-γ secretion levels of LC006 sample, FOST, and the methods of this application (Alpha, Beta) under different co-stimulation modes; Figure 1 In the diagram, C represents the IFN-γ secretion levels of the LC008 sample, FOST, and the methods of this application (Beta, A27) under different co-stimulation modalities. TIL levels in different samples... + The comparison of the Tumor group showed that, compared with the FOST method, the method of this application significantly increased the IFN-γ secretion level in TILs stimulated with autologous tumor cells. Results from different co-stimulation treatments within the same group also showed that stimulation with autologous tumor cells significantly increased the IFN-γ secretion level.

[0162] Example 2: The method of this application enhances CD8 after stimulation by autologous tumor cells. + CD137 + The proportion of TILs

[0163] Using the same amplification method and stimulation mode as in Example 1, flow cytometry was employed to detect CD8 in the test samples. + CD137 + The proportion of TILs.

[0164] FOST and the method of the present application after autologous tumor cell stimulation CD8 + CD137 + TILs ratio detection results of TILs are shown in Figure 2 Among them, Figure 2 A in LC003 sample, FOST and the method of the present application (Alpha, IL-7 / 15) CD8 + CD137 + TILs ratio under different co-stimulation modes; Figure 2 B in LC006 sample, FOST and the method of the present application (Alpha, Beta) CD8 + CD137 + TILs ratio under different co-stimulation modes; Figure 2 C in LC008 sample, FOST and the method of the present application (Alpha, Beta, A27) CD8 + CD137 + TILs ratio. By comparing TIL+Tumor groups in different samples, it can be seen that compared with the FOST method, the method of the present application can significantly improve the CD8 + CD137 + TILs ratio after autologous tumor cell stimulation. From the results of different co-stimulation processing modes in the same group, it can be seen that autologous tumor cell stimulation can significantly improve the CD8 + CD137 + TILs ratio.

[0165] Example 3: The method of the present application improves the absolute number of CD8 + CD137 + TILs after autologous tumor cell stimulation. The absolute number of CD8 + CD137 + TILs in the test sample was counted.

[0166] FOST and the method of the present application after autologous tumor cell stimulation CD8 + CD137 + TILs absolute number detection results are shown in Figure 3 Among them, Figure 3 A in LC003 sample, FOST and the method of the present application (Alpha, IL-7 / 15) CD8 + CD137 + TILs absolute number under different co-stimulation modes; Figure 3 B in LC006 sample, FOST and the method of the present application (Alpha, Beta) CD8+ CD137 + The absolute number of TILs was determined. Results show that, compared to the FOST method, the method in this application significantly improves CD8 efficiency. + CD137 + The absolute number of TILs.

[0167] Example 4: The method of this application enhances CD8 after stimulation with autologous tumor cells. + CD25 + The proportion of TILs

[0168] Using the same amplification method and stimulation mode as in Example 1, flow cytometry was employed to detect CD8 in the test samples. + CD25 + The proportion of TILs.

[0169] FOST and CD8 after stimulation with autologous tumor cells according to the method of this application + CD25 + The proportion detection results of TILs are as follows: Figure 4 As shown. Among them, Figure 4 In this context, A represents the OC003 (ovarian cancer) sample. FOST and the methods of this application (Alpha, Beta) under different co-stimulation modalities, CD8... + CD25 + The proportion of TILs; Figure 4 In this context, B represents the LC006 sample, and FOST and the methods of this application (Alpha, Beta) under different co-stimulation modalities, CD8 + CD25 + The proportion of TILs; Figure 4 In this context, C represents the LC008 sample, and FOST and the methods of this application (Alpha, Beta, A27) under different co-stimulatory modalities, CD8 + CD25 + The proportion of TILs was measured. Results showed that, compared to the FOST method, the method described in this application significantly improved CD8 efficiency. + CD25 + The proportion of TILs.

[0170] Example 5: The method of this application enhances CD8 after stimulation by autologous tumor cells. + CD25 + The absolute number of TILs

[0171] Using the same amplification method and stimulation mode as in Example 1, CD8 in the test sample was... + CD25 + TILs are counted in absolute terms.

[0172] FOST and CD8 after stimulation with autologous tumor cells according to the method of this application + CD25 + The absolute number of TILs detection results are as follows: Figure 5 As shown. Among them, Figure 5 In this context, A represents the LC004 sample, and FOST and the methods of this application (Alpha, IL-7 / 15) under different co-stimulatory modalities, CD8 + CD25 + The absolute number of TILs; Figure 5 In this context, B represents the LC006 sample, and FOST and the methods of this application (Alpha, Beta) under different co-stimulation modalities, CD8 + CD25 + The absolute number of TILs was determined. Results show that, compared to the FOST method, the method in this application significantly improves CD8 efficiency. + CD25 + The absolute number of TILs.

[0173] Example 6: The method of this application improves the killing ability of TILs against autologous organoids.

[0174] The autologous organoid killing ability of TILs in the test samples was tested using the same amplification method as in Example 1.

[0175] The results of the FOST and the method of this application on the organoid killing ability test are as follows: Figure 6 As shown. Among them, Figure 6 In the figures, A represents the results of TILs' organ-killing ability detection at an effector-to-target ratio of 3:1; B represents the results of TILs' organ-killing ability detection under a high-content imaging system, with a scale bar of 500 μm; and C represents the results of TILs' organ-killing ability detection after 48 hours of co-culture. The results indicate that, compared to the PDO-only group and the allogeneic FOST group, FOST and the method of this application have stronger organ-killing ability against autologous organoids; specifically for autologous organoids, the method of this application has a stronger organ-killing ability compared to the FOST method.

[0176] Example 7: The method of this application improves the infiltration ability of TILs in autologous organoids.

[0177] The same amplification method as in Example 1 was used to detect the autologous organoid infiltration capacity of TILs in the test samples.

[0178] The results of the infiltration ability test of TILs in autologous organoids are as follows: Figure 7As shown in the figure, A represents the detection results of the infiltration ability of TILs in autologous organoids under a high-content imaging system; B represents the statistical results of the infiltration ability of TILs in autologous organoids. The results show that, compared with the allogeneic FOST group, FOST and the method of this application have stronger infiltration ability for autologous organoids; for autologous organoids, the method of this application has stronger infiltration ability than the FOST method.

[0179] Example 8: The method of this application improves CD8 at TILs harvest time. + CD25 + Cell ratio

[0180] The same amplification method as in Example 1 was used to perform CD8 amplification on the TILs of the test samples. + CD25 + Cell proportion detection.

[0181] TILs harvest CD8 + CD25 + Cell proportion detection results as follows Figure 8 As shown in the figure. A represents a tumor sample from subject LC003; B represents a tumor sample from subject LC006; and C represents a tumor sample from subject LC008. The results indicate that, compared to the FOST method, the TILs obtained by the method described in this application have higher CD8 content at harvest time across different test samples. + CD25 + The proportion of cells is higher.

[0182] Example 9: The method of this application improves CD8 at TILs harvest time. + CD137 + Cell ratio

[0183] The same amplification method as in Example 1 was used to perform CD8 amplification on the TILs of the test samples. + CD137 + Cell proportion detection.

[0184] TILs harvest CD8 + CD137 + Cell proportion detection results as follows Figure 9 As shown in the figure. A represents a tumor sample from subject LC003; B represents a tumor sample from subject LC004; and C represents a tumor sample from subject LC008. The results indicate that, compared to the FOST method, the TILs obtained by the method described in this application have higher CD8 counts at harvest time across different test samples. + CD137 + The proportion of cells is higher.

[0185] Example 10: The method of this application improves CD8 at TILs harvest time. + CD103 + Cell ratio

[0186] The same amplification method as in Example 1 was used to perform CD8 amplification on the TILs of the test samples. + CD103 + Cell proportion detection.

[0187] TILs harvest CD8 + CD103 + Cell proportion detection results as follows Figure 10 As shown in the figure. A represents a tumor sample from subject OC002; B represents a tumor sample from subject LC004; and C represents a tumor sample from subject LC008. The results indicate that, compared to the FOST method, the TILs obtained by the method described in this application have higher CD8 content at harvest time across different test samples. + CD103 + The proportion of cells is higher.

[0188] Example 11: The method of this application has no significant effect on the amplification rate during the culture of TILs.

[0189] The amplification rate of TILs during the culture process was detected using the same amplification method as in Example 1.

[0190] The amplification rate detection results during the TILs culture process are as follows: Figure 11 As shown in the figure, A represents a tumor sample from subject LC002; B represents a tumor sample from subject LC003; C represents a tumor sample from subject LC008; D represents a tumor sample from subject LC009; and E represents a tumor sample from subject OC002. The results indicate that, among different test samples, there was no significant difference in the amplification rate of TILs during the culture process between this application and the FOST method.

[0191] Example 12: The method of this application for CD3 during TILs harvest + The proportion of cells was not significantly affected.

[0192] Using the same amplification method as in Example 1, CD3 at harvest time of the test sample was... + The proportion of cells was measured.

[0193] TILs harvest CD3 + Cell proportion detection results as follows Figure 12As shown in the figure. A represents a tumor sample from subject LC002; B represents a tumor sample from subject LC003; C represents a tumor sample from subject LC004; D represents a tumor sample from subject LC005; E represents a tumor sample from subject OC002; and F represents a tumor sample from subject LC008. The results indicate that in different test samples, this application and the FOST method, at harvest time, [show that] CD3 [is present / are ... + There was no significant difference in the proportion of cells.

[0194] Example 13: The method of this application for CD8 during TILs harvest + The proportion of cells was not significantly affected.

[0195] Using the same amplification method as in Example 1, CD8+ was harvested from the test samples. + The proportion of cells was measured.

[0196] TILs harvest CD8 + Cell proportion detection results as follows Figure 13 As shown in the figure. A represents a tumor sample from subject LC002; B represents a tumor sample from subject LC003; C represents a tumor sample from subject LC004; and D represents a tumor sample from subject LC005. The results indicate that, in different test samples, this application and the FOST method, at harvest time, [show that] CD8 [is present / are ... + There was no significant difference in the proportion of cells.

[0197] Example 14: CD4 during TILs harvesting using the method of this application + The proportion of cells was not significantly affected.

[0198] Using the same amplification method as in Example 1, CD4+ was harvested from the test samples. + The proportion of cells was measured.

[0199] TILs harvest CD4 + Cell proportion detection results as follows Figure 14 As shown in the figure. A represents a tumor sample from subject LC002; B represents a tumor sample from subject LC003; C represents a tumor sample from subject LC004; and D represents a tumor sample from subject LC005. The results indicate that, in different test samples, this application and the FOST method, at harvest time, [show that] CD4 [is present]. + There was no significant difference in the proportion of cells.

[0200] Example 15: The method of this application has no significant effect on the proportion of Treg cells at the time of TIL harvest.

[0201] The proportion of Treg cells at harvest time of the test sample was detected using the same amplification method as in Example 1.

[0202] The proportion of Treg cells at the time of TILs harvesting is shown in Figure 15 . Among them, A is the tumor sample from subject LC001; B is the tumor sample from subject LC004; C is the tumor sample from subject LC005; D is the tumor sample from subject LC008. The results show that in different test samples, the proportion of Treg cells at the time of harvesting has no significant difference between the present application and the FOST method.

[0203] Example 16: The present application method has no significant effect on the proportion of TILs stemness regulatory transcription factor TCF-1 +

[0204] The proportion of TILs stemness regulatory transcription factor TCF-1 + was detected in the test samples using the same amplification method as in Example 1.

[0205] The proportion of TILs stemness regulatory transcription factor TCF-1 + is shown in Figure 16 . Among them, A is the tumor sample from subject LC003; B is the tumor sample from subject LC006; C is the tumor sample from subject LC008. The results show that in different test samples, the proportion of TILs stemness regulatory transcription factor TCF-1 + has no significant difference between the present application and the FOST method.

[0206] Example 17: The present application method has no significant effect on the proportion of transcription factor BCL6 regulating TILs early exhaustion +

[0207] The proportion of transcription factor BCL6 regulating TILs early exhaustion + was detected in the test samples using the same amplification method as in Example 1.

[0208] The proportion of transcription factor BCL6 regulating TILs early exhaustion + is shown in Figure 17 . Among them, A is the tumor sample from subject LC002; B is the tumor sample from subject LC006; C is the tumor sample from subject LC009. The results show that in different test samples, the proportion of transcription factor BCL6 regulating TILs early exhaustion + has no significant difference between the present application and the FOST method.

[0209] Example 18: The present application method has no significant effect on the proportion of TILs terminal exhaustion marker TIM-3 + BLIMP1 + ​​the proportion of TIM-3

[0210] The proportion of TIM-3 + BLIMP1 + was detected.

[0211] The proportion of TIM-3 + BLIMP1 + was detected. Figure 18 The detection results of the proportion of TIM-3 + BLIMP1 + are shown in Table 1. In Table 1, A represents the tumor sample from subject LC003; B represents the tumor sample from subject LC006; and C represents the tumor sample from subject LC009. The results show that there is no significant difference between the proportion of TIM-3 + BLIMP1 + in the different test samples and the proportion of TIM-3 + BLIMP1 + determined by the FOST method.

[0212] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. The illustrative description of the above terms in the specification does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0213] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and purposes of the present application within the scope of the present application.

Claims

1. A method of expanding tumor infiltrating lymphocytes, characterized in that, The method comprises: culturing a tumor sample obtained from a subject in a culture medium containing a first factor and a second factor, thereby obtaining an expanded tumor infiltrating lymphocyte population, and the culture medium does not comprise feeder cells; wherein the first factor comprises: a CD3 agonist, a 4-1BB agonist, and IL-2; and the second factor comprises: at least one of IL-7, IL-15, TGFβ, IL-27, and CD25-bias IL-2.

2. The method of claim 1, wherein, The CD3 agonist comprises an anti-CD3 antibody and / or an antigen-binding fragment thereof, optionally a humanized anti-CD3 antibody and / or an antigen-binding fragment thereof; Preferably, the CD3 agonist is OKT3. Optionally, the final concentration of the CD3 agonist is 10 ng / mL-30 ng / mL.

3. The method of claim 1, wherein, The 4-1BB agonist comprises a ligand-competitive 4-1BB agonistic antibody and / or an antigen-binding fragment thereof, or a ligand-non-competitive 4-1BB agonistic antibody and / or an antigen-binding fragment thereof; Preferably, the 4-1BB agonist is selected from Utomilumab or Urelumab. Optionally, the final concentration of the 4-1BB agonist is 1 µg / mL-10 µg / mL.

4. The method of claim 1, wherein, The final concentration of IL-2 in the culture medium is 1000 IU / mL-3000 IU / mL.

5. The method according to any one of claims 1 to 4, characterized in that, The second factor comprises: IL-7 and IL-15; Optionally, the second factor comprises: IL-27 and CD25-bias IL-2. Optionally, the second factor comprises: CD25-bias IL-2. Optionally, the second factor comprises: TGFβ.

6. The method of claim 5, wherein, The final concentration of IL-7 in the culture medium is 5 ng / mL-15 ng / mL; Preferably, the final concentration of IL-7 in the culture medium is 10 ng / mL.

7. The method of claim 5, wherein, The final concentration of IL-15 in the culture medium is 5 ng / mL-15 ng / mL; Preferably, the final concentration of IL-15 in the culture medium is 10 ng / mL.

8. The method of claim 5, wherein, The final concentration of IL-27 in the culture medium is 20 ng / mL-30 ng / mL; Preferably, the final concentration of IL-15 in the culture medium is 25 ng / mL.

9. The method of claim 5, wherein, The final concentration of CD25-bias IL-2 in the culture medium is 500 IU / mL-1500 IU / mL; Preferably, the final concentration of CD25-bias IL-2 in the culture medium is 1000 IU / mL.

10. The method of claim 5, wherein, The final concentration of TGFβ in the culture medium is 1 ng / mL-5 ng / mL; Preferably, the final concentration of TGFβ in the culture medium is 2 ng / mL.

11. The method of claim 1, wherein, The medium exchange is performed every 2 to 5 days; Optionally, the medium exchange comprises adding fresh medium to the culture, wherein the fresh medium comprises IL-2 and does not comprise the CD3 agonist and the 4-1BB agonist.

12. The method of claim 1, wherein, The tumor infiltrating lymphocyte culture lasts for 14-26 days; Optionally, the tumor infiltrating lymphocyte culture lasts for at least 14 days.

13. A population of tumor infiltrating lymphocytes, characterized in that, The tumor infiltrating lymphocyte population is obtained by the method of any one of claims 1-12.

14. A pharmaceutical composition, characterized by, Comprises: The population of tumor infiltrating lymphocytes of claim 13, and optionally a pharmaceutically acceptable carrier.

15. Use of a population of tumor infiltrating lymphocytes obtained according to the method of any one of claims 1-12 or of a population of tumor infiltrating lymphocytes of claim 13 or of a pharmaceutical composition of claim 14 for the manufacture of a medicament for the prevention and / or treatment of a tumor.

16. The use of claim 15, wherein the tumor is a solid tumor; Optionally, 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, gastric cancer, colorectal cancer, and renal cancer.

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