Expanded tumor-reactive t cell composition

Abstract

The present invention relates to a tumor-reactive T cell composition obtained by expansion from a patient sample, wherein upon stimulation with tumor cell antigens, the proportion of CD8+CD137+ cells among CD45+ cells in the composition is at least greater than 25%, and / or the proportion of CD45RA+CD62L+ cells in the composition is at least greater than 30%. The present invention further relates to a method for obtaining a tumor-reactive T cell composition by expansion from a patient sample. Tumor-reactive T cells in the composition of the present invention have a high total count, a long lifespan, a high viability rate, and a potent tumor-killing capacity, thereby providing a basis for the use of the composition in tumor therapy.

Description

An expanded tumor-reactive T cell composition TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an expanded tumor-reactive T cell composition and a method for expanding the same. BACKGROUND

[0002] Tumor-Infiltrating Lymphocytes (TIL) refers to a cell population derived from a patient's body, which is originally obtained as a white blood cell, and has migrated out of the blood circulation and invaded the inside of a tumor tissue at least once before being obtained. TIL includes but is not limited to CD8+ T cells, CD4+ T cells, natural killer (NK) cells, B cells, dendritic cells, macrophages, and the like. TIL includes primary TIL and secondary TIL. Primary TIL refers to cells directly obtained from a tumor tissue sample of a patient, and secondary TIL refers to a TIL composition further expanded or proliferated on the basis of primary TIL, including but not limited to cells obtained by rapid expansion culture.

[0003] Treatment of bulky, refractory cancers with adoptively transferred tumor infiltrating lymphocytes represents an effective approach to treat patients with poor prognosis (Gattinoni et al., Nat. Rev. Immunol., 2006, 6, 383-393). A large number of TILs are required for successful immunotherapy and a robust and reliable expansion method is needed for commercial applications. Cell expansion technology faces multiple challenges in practical applications, including technical bottlenecks, logistical management, and regulatory requirements. Due to its speed and efficiency, IL-2-based TIL expansion followed by a "rapid expansion process (REP)" has become the preferred method for TIL expansion. Dudley et al., Science, 2002, 298, 850-54; Dudley et al., J. Clin. Oncol., 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol., 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. Immunother., 2003, 26, 332-42. REP can expand TILs by 1000-fold within 14 days, although it typically requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs) from multiple donors as feeder cells, and anti-CD3 antibody (OKT3) and high doses of IL-2. Dudley et al., J. Immunother., 2003, 26, 332-42. TILs that undergo the REP procedure have produced successful adoptive cell therapy in melanoma patients after receiving lymphodepleting conditioning.

[0004] With the in-depth study in recent years, it is found that the main killer in tumor infiltrating lymphocytes is T cells, and the main killer in tumor infiltrating T cells is tumor-reactive T cells. Tumor-reactive T cells refer to a specific cell population or composition in tumor infiltrating tissues or non-tumor infiltrating tissues (such as para-carcinoma tissues, tumor-draining lymph nodes, pleural effusion, PBMC, etc.), which can be activated after being stimulated by corresponding tumor cell antigens, and produce enhanced tumor reactivity and / or specific killing ability.

[0005] Despite some progress in the treatment of solid tumors with tumor-reactive T cells, there are still some challenges. For example, the preparation process of a tumor-reactive T cell composition is complex and time-consuming, which not only requires extracting a sample from a patient, but also requires multiple steps such as cell separation, screening, maintenance, and expansion. The expansion fold of the tumor-reactive T cell composition obtained by expansion in the prior art is generally only a few thousand (Creelan et al., Nat Med 27(8): 1410-1418 (2021); Chiffelle et al., Immunity 10:S1074-7613(24)00413-8 (2024)), the proportion of tumor-reactive T cells in the composition after stimulation by tumor cell antigens, represented by the proportion of CD8+CD137+ cells among CD45+ cells, is low, which is less than 10%, and the proportion of young cells in the composition, represented by the proportion of CD45RA+CD62L+ cells, is also low, which is less than 20% (Vodnala et al., Science 363(6434): eaau0135 (2019); Onimus et al., Identity 40:20 (2022); Fennell et al., J Thorac Oncol 18(11):S381-S382 (2023)), which seriously limits the application range of the tumor-reactive T cell composition. The above documents are incorporated by reference in their entirety into the present application.

[0006] Therefore, there is an urgent need in the art for an expanded tumor-reactive T cell composition with higher total number of T cells, higher proportion of tumor-reactive T cells, and / or higher proportion of young cells, and a method for expanding the same. If the total number of T cells, the proportion of viable T cells, and the proportion of tumor-reactive T cells are all improved, and the tumor-reactive T cells are younger and have a longer lifespan, a stronger and more persistent tumor-killing ability can be provided. To this end, the present application has been made. SUMMARY

[0007] To this end, the present application provides an expanded tumor-reactive T cell composition and a method for expanding the same, wherein the composition is significantly improved in the following aspects compared to the prior art:

[0008] i) the proportion of CD8+CD137+ cells among CD45+ cells after stimulation by tumor cell antigens;

[0009] ii) the proportion of CD45RA+CD62L+ cells in the composition;

[0010] iii) an increase in the amount of expression of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1 and KLF7 relative to before expansion and / or a decrease in the amount of expression of one or more gene markers selected from the group consisting of TBX1, ID2 and HOPX relative to before expansion.

[0011] In particular, the present application solves the technical problems existing in the prior art by the following technical solutions:

[0012] 1. An expanded tumor reactive T cell composition, wherein:

[0013] (a) the composition has a proportion of CD8+CD137+ cells to CD45+ cells greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55% or greater than 60% after being stimulated by tumor cell antigens; and / or

[0014] (b) the composition has a proportion of CD45RA+CD62L+ cells greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, greater than 60% or greater than 70%; and / or

[0015] (c) the composition has an increase of more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 58% or more than 60% in the amount of expression of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1 and KLF7 relative to before expansion and / or a decrease of more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 58% or more than 60% in the amount of expression of one or more gene markers selected from the group consisting of TBX1, ID2 and HOPX relative to before expansion.

[0016] 2. The composition of item 1, wherein the composition is obtained by one or more rounds of expansion of a sample from a patient.

[0017] 3. The composition of item 2, wherein the sample is from a tumor, a lymph node, a pleural effusion, an abdominal effusion, peripheral blood or cerebrospinal fluid of a patient, preferably the tumor is a sample selected from the group consisting of a surgically resected tumor sample, a punch biopsy tumor sample, a core biopsy tumor sample and a small biopsy tumor sample.

[0018] 4. The composition of any one of items 1-3, wherein the tumor is a solid tumor, preferably selected from the group consisting of mesothelioma, lung cancer and glioma, more preferably a non-small cell lung cancer.

[0019] 5. The composition of any one of items 1-4, wherein the proportion of CD45+ cells in the composition is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

[0020] 6. The composition of any one of items 1-5, wherein the proportion of CD45+ CD3+ cells in the composition is greater than 90%, greater than 92%, greater than 94%, greater than 96%, or greater than 98%.

[0021] 7. The composition of any one of items 1-6, wherein the composition is obtained within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, or within 15 days after the start of expansion.

[0022] 8. The composition of any one of items 1-7, wherein the total number of tumor reactive T cells in the composition is at least 10,000-fold, at least 20,000-fold, at least 50,000-fold, at least 80,000-fold, or at least 100,000-fold greater than before expansion, or the total number of tumor reactive T cells per milliliter of the composition is at least 1 x 10 10 , at least 2 x 10 10 , at least 4 x 10 10 , at least 8 x 10 10 , at least 1 x 10 11 , at least 2 x 10 11 , at least 3 x 10 11 , at least 4 x 10 11 , or at least 5 x 10 11 .

[0023] 9. The composition of any one of items 1-8, wherein the percentage of viable tumor reactive T cells in the composition is 80% or more, 85% or more, or 90% or more.

[0024] 10. The composition of any one of items 1-9, wherein the composition is capable of producing 500 pg / mL or more, 1000 pg / mL or more, 2000 pg / mL or more, 3000 pg / mL or more, 4000 pg / mL or more, or 4500 pg / mL or more of interferon gamma (IFN-γ) after being stimulated with tumor cell antigens.

[0025] 11. The composition of any one of items 1-10, wherein the composition has any one or more of the following properties:

[0026] (i) the proportion of CD45+ cells from the composition in the subject is higher than 20%, higher than 25%, higher than 30% or higher than 35% of the total CD45+ in the subject 7-14 days after administration to the subject, preferably the proportion of CD45+ cells from the composition in the subject is higher than 10%, higher than 15% or higher than 20% of the total CD45+ in the subject 28 days after administration to the subject;

[0027] (ii) the proportion of CD3+ cells from the composition in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50% or higher than 60% of the total CD3+ in the subject 7-14 days after administration to the subject, preferably the proportion of CD3+ cells from the composition in the subject is higher than 15%, higher than 20%, higher than 30%, higher than 40%, higher than 50% or higher than 60% of the total CD3+ in the subject 28-63 days after administration to the subject;

[0028] (iii) the proportion of TCR species from the composition in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50% or higher than 60% of the total TCR species in the subject 7-14 days after administration to the subject, preferably the proportion of TCR species from the composition in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50% or higher than 60% of the total TCR species in the subject 28-56 days after administration to the subject;

[0029] (iv) the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 1000 cells / μL, higher than 2000 cells / μL, higher than 4000 cells / μL, higher than 6000 cells / μL, higher than 8000 cells / μL or higher than 10000 cells / μL 7-14 days after administration to the subject, preferably the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 500 cells / μL, higher than 1000 cells / μL, higher than 1500 cells / μL, higher than 2000 cells / μL, higher than 2500 cells / μL, higher than 3000 cells / μL, higher than 3500 cells / μL or higher than 4000 cells / μL 28-56 days after administration to the subject.

[0030] 12. The composition of any one of items 1-11, wherein the composition is capable of at least 6, at least 7, at least 8, at least 9 or at least 10 consecutive killings of tumor cells.

[0031] 13. The composition of any one of clauses 1-12, wherein the composition comprises or does not comprise feeder layer cells.

[0032] 14. A method of making a tumor reactive T cell composition by expansion, comprising:

[0033] (1) providing a sample containing tumor reactive T cells;

[0034] (2) pre-treating the sample;

[0035] (3) subjecting the pre-treated sample to a first stage of expansion culture in a first culture medium comprising IL-2, OKT3, and / or a SMO inhibitor;

[0036] (4) subjecting the product of the first stage of expansion culture to a second stage of expansion culture in a second culture medium comprising IL-2, IL-7, IL-15, OKT3, and / or a SMO inhibitor; and

[0037] (5) optionally harvesting the composition.

[0038] 15. The method of clause 14, wherein the sample containing tumor reactive T cells is a sample from a patient, preferably the sample is from a tumor, lymph node, pleural effusion, peritoneal effusion, peripheral blood, or cerebrospinal fluid of a patient, more preferably the sample from a tumor of a patient is a sample selected from the group consisting of a surgically resected tumor sample, a punch biopsy tumor sample, a core biopsy tumor sample, and a small biopsy tumor sample.

[0039] 16. The method of clause 15, wherein the tumor is a solid tumor, preferably selected from the group consisting of mesothelioma, lung cancer, and glioma, more preferably a non-small cell lung cancer.

[0040] 17. The method of any one of clauses 14-16, wherein the SMO inhibitor is selected from one or more of the group of compounds consisting of BMS-833923 (XL-139), SAG, Cyclopamine, Purmorphamine, Glasdegib, Sonidegib, Taladegib, and Halcinonide.

[0041] 18. The method of any one of clauses 14-17, wherein the pre-treatment comprises one or more procedures selected from the group consisting of removing tissue, fragmentation, washing, red blood cell lysis, resuspension, and cryopreservation.

[0042] 19. The method of clause 18, wherein the fragmentation is performed by:

[0043] (1) grinding the sample; and / or

[0044] (2) mincing the sample.

[0045] 20. The method of item 18 or 19, wherein the washing is performed by centrifugation in 1-50 times the sample volume of wash solution.

[0046] 21. The method of item 20, wherein the wash solution comprises: an antibiotic and potassium ions.

[0047] 22. The method of item 20 or 21, wherein the centrifugation is performed by centrifugation at 1200-1800 rpm for 2-5 minutes.

[0048] 23. The method of any one of items 18-22, wherein the red blood cell lysis is performed by adding 1-50 times the sample volume of red blood cell lysis solution and mixing, inverting every 1-2 minutes for a total of no more than 5 minutes.

[0049] 24. The method of item 23, wherein the red blood cell lysis solution comprises metal ions, preferably potassium ions, sodium ions, and / or ammonium ions.

[0050] 25. The method of any one of items 18-24, wherein the resuspension is resuspension of the sample in the first culture medium.

[0051] 26. The method of any one of items 14-25, wherein the cryopreservation is performed by centrifugation of the sample followed by addition of a cryopreservation solution and cryopreservation using a programable cryogenic freezer.

[0052] 27. The method of item 26, wherein the cryopreservation solution comprises:

[0053] (a) CS10;

[0054] (b) human blood albumin; and

[0055] (c) a compound electrolyte injection solution.

[0056] 28. The method of any one of items 14-27, wherein the first stage expansion culture is performed in a culture bag for 4-8 days, preferably the culture bag is placed in a 37°C, 5% CO2 incubator.

[0057] 29. The method of any one of items 14-28, wherein the first culture medium is supplemented to the culture on days 1-3 of the first stage expansion culture.

[0058] 30. The method of any one of clauses 14-29, wherein the total number of tumor reactive T cells in the resulting composition after the first phase expansion culture is at least 20-fold, at least 40-fold, at least 50-fold, at least 80-fold, or at least 100-fold greater than prior to expansion, or the total number of tumor reactive T cells per milliliter of the composition is at least 5 x 10 8 , at least 6 x 10 8 , at least 7 x 10 8 , at least 8 x 10 8 , at least 9 x 10 8 , or at least 1 x 10 9 .

[0059] 31. The method of any one of clauses 14-30, wherein the second phase expansion culture is performed in a culture bag for 7-10 days, preferably the culture bag is placed in a 37°C, 5% CO2 incubator.

[0060] 32. The method of any one of clauses 14-31, wherein the second culture medium is supplemented to the culture on days 2-3, 4-5, and / or 6-7 of the second phase expansion culture.

[0061] 33. The method of any one of clauses 14-32, wherein the total number of tumor reactive T cells in the resulting composition after the second phase expansion culture is at least 10,000-fold, at least 20,000-fold, at least 50,000-fold, at least 80,000-fold, or at least 100,000-fold greater than prior to expansion, or the total number of tumor reactive T cells per milliliter of the composition is at least 1 x 10 10 , at least 2 x 10 10 , at least 4 x 10 10 , at least 8 x 10 10 , at least 1 x 10 11 , at least 2 x 10 11 , at least 3 x 10 11 , at least 4 x 10 11 , or at least 5 x 10 11 .

[0062] 34. The method of any one of clauses 14-33, wherein harvesting the composition is performed by centrifuging to collect the cells after the second phase expansion culture.

[0063] 35. The method of any one of clauses 14-34, wherein harvesting the composition further optionally comprises washing the composition with a wash solution and / or cryopreserving the composition with a cryopreservation solution.

[0064] 36. The method of any one of clauses 14-35, wherein:

[0065] (a) the proportion of CD8+CD137+ cells out of CD45+ cells after stimulation with tumor cell antigens is greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, or greater than 60%; and / or

[0066] (b) the proportion of CD45RA+CD62L+ cells in the composition is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, greater than 60%, or greater than 70%; and / or

[0067] (c) the expression level of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1, and KLF7 is increased by more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 58%, or more than 60% in the composition compared to before expansion and / or the expression level of one or more gene markers selected from the group consisting of TBX1, ID2, and HOPX is decreased by more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 58%, or more than 60% in the composition compared to before expansion.

[0068] 37. The method of any of clauses 14-36, wherein the proportion of CD45+ cells in the composition is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

[0069] 38. The method of any of clauses 14-37, wherein the proportion of CD45+CD3+ cells in the composition is greater than 90%, greater than 92%, greater than 94%, greater than 96%, or greater than 98%.

[0070] 39. The method of any of clauses 14-38, wherein the composition is obtained within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, or within 15 days after the start of expansion.

[0071] 40. The method of any of clauses 14-39, wherein the total number of tumor reactive T cells in the composition is at least 10,000-fold, at least 20,000-fold, at least 50,000-fold, at least 80,000-fold, or at least 100,000-fold higher than before expansion, or the total number of tumor reactive T cells per milliliter of the composition is at least 1 x 10 10 , at least 2 x 10 10 , at least 4 x 10 10 , at least 8 x 10 10 , at least 1 x 10 11at least 2 x 10 11 at least 3 x 10 11 at least 4 x 10 11 at least 5 x 10 11 .

[0072] 41. The method of any one of clauses 14-40, wherein the percentage of viable cells of tumor reactive T cells in the composition is 80% or more, 85% or more, or 90% or more.

[0073] 42. The method of any one of clauses 14-41, wherein the composition is capable of producing 500 pg / mL or more, 1000 pg / mL or more, 2000 pg / mL or more, 3000 pg / mL or more, 4000 pg / mL or more, or 4500 pg / mL or more of interferon gamma (IFN-γ) upon stimulation with tumor cell antigens.

[0074] 43. The method of any one of clauses 14-42, wherein the composition has any one or more of the following properties:

[0075] (i) the proportion of CD45+ cells from the composition to total CD45+ in the subject is higher than 20%, higher than 25%, higher than 30%, or higher than 35% in the subject 7-14 days after administration to the subject, preferably the proportion of CD45+ cells from the composition to total CD45+ in the subject is higher than 10%, higher than 15%, or higher than 20% in the subject 28 days after administration to the subject;

[0076] (ii) the proportion of CD3+ cells from the composition to total CD3+ in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% in the subject 7-14 days after administration to the subject, preferably the proportion of CD3+ cells from the composition to total CD3+ in the subject is higher than 15%, higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% in the subject 28-63 days after administration to the subject;

[0077] (iii) the proportion of TCR species from the composition to total TCR species in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% in the subject 7-14 days after administration to the subject, preferably the proportion of TCR species from the composition to total TCR species in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% in the subject 28-56 days after administration to the subject;

[0078] (iv) the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 1000 cells per pL, higher than 2000 cells per pL, higher than 4000 cells per pL, higher than 6000 cells per pL, higher than 8000 cells per pL, or higher than 10000 cells per pL after 7-14 days of administration to the subject; preferably the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 500 cells per pL, higher than 1000 cells per pL, higher than 1500 cells per pL, higher than 2000 cells per pL, higher than 2500 cells per pL, higher than 3000 cells per pL, higher than 3500 cells per pL, or higher than 4000 cells per pL after 28-56 days of administration to the subject.

[0079] 44. The method of any one of items 14-43, wherein the composition is capable of at least 6, at least 8, at least 10 consecutive killing of tumor cells.

[0080] 45. The method of any one of items 14-44, wherein the composition comprises or does not comprise feeder cells.

[0081] 46. A tumor reactive T cell composition prepared by the method of any one of items 14-45.

[0082] In order to make the technical solutions of the present application clearer, the present application will be further described in detail in combination with the specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figures 1A and IB show the culture of the tumor reactive T cell composition of the present application. Figure 1A shows that the total number of T cells in the tumor reactive T cell composition of the present application obtained on day 20 of expansion is about 136500 times of that before expansion (test group); while the total number of T cells in the composition of the prior art (i.e., see T cell composition prepared in methods 2A of examples 5-10 and table 19 of US10894063B2) obtained under the same expansion condition is less than 40 times of that before expansion (control group). Figure IB shows that the proportion of viable cells in the tumor reactive T cell composition of the present application obtained on day 14 of expansion is about 85% (test group); while the proportion of viable cells in the composition of the prior art obtained under the same expansion condition is about 66.5% (control group).

[0084] Figures 2A-2E show that the proportion of CD45RA+CD62L+ cells in the expanded tumor reactive T cell compositions of the present application (test group) is significantly higher than the proportion of CD45RA+CD62L+ cells in the prior art compositions (control group) for multiple batches from different sample sources.

[0085] Figure 3 shows the proportion of CD45+ cells and / or CD45+CD3+ cells in the expanded tumor reactive T cell compositions of the present application for multiple batches from different sample sources.

[0086] Figures 4A-4E show that the proportion of CD8+CD137+ cells out of CD45+ cells after stimulation with tumor cell antigen in the expanded tumor reactive T cell compositions of the present application (test group) is significantly higher than the proportion of CD8+CD137+ cells out of CD45+ cells after stimulation with tumor cell antigen in the prior art compositions (control group) for multiple batches from different sample sources.

[0087] Figure 5A shows the tumor killing profile of the expanded tumor reactive T cell compositions of the present application and the prior art compositions in terms of IFN-γ concentration (pg / mL) at day 18; Figure 5B shows the tumor killing profile of the expanded tumor reactive T cell compositions of the present application for multiple batches under different culture conditions in terms of IFN-γ concentration (pg / mL).

[0088] Figure 6A shows that the proportion of CD45+ out of total CD45+ from the expanded tumor reactive T cell compositions of the present application is about 60% at 7 days after administration to a subject, about 40% at 14 days after administration to a subject, and still higher than 20% at 28 days after administration to a subject (test group); while the proportion of CD45+ out of total CD45+ from the prior art compositions is less than 10% at 14 days after administration to a subject, and about 0% at 28 days after administration to a subject (control group); Figure 6B shows that the proportion of CD3+ out of total CD3+ from the expanded tumor reactive T cell compositions of the present application is higher than 90% at 14 days after administration to a subject, and still higher than 40% at 28 days after administration to a subject (test group).

[0089] Figure 7 shows the PK effect of the expanded tumor reactive T cell compositions of the present application in humans (in terms of absolute counts of CD3+ or CD3+CD8+ in peripheral blood). As shown in Figure 7, the expanded tumor reactive T cell compositions of the present application have significantly enhanced expansion ability and persistence in humans after administration to human subjects.

[0090] Figure 8 shows that the proportion of TCR species from the expanded tumor- reactive T cell composition of the present application (Testing+Testing_QLQ) to the total TCR species in the subject is higher than 70% at 7 days, 14 days, 28 days and 56 days after administration to the subject.

[0091] Figure 9 shows that the expanded tumor-reactive T cell composition of the present application maintains more than 80% target lysis after 10 consecutive rounds of killing in vitro (test group), while the composition of the prior art has a target lysis close to 0 after 6 consecutive rounds of killing (control group).

[0092] DETAILED DESCRIPTION

[0093] T cell therapy is expected to be a relatively safe and well-tolerated adoptive T cell product. However, the expansion of the T cell composition expanded in the prior art has a limited expansion fold, and the proportion of tumor cell antigen-reactive cells and the proportion of young T cells are also low. Therefore, the present application provides an expanded tumor-reactive T cell composition, in which the total number of T cells is high, the proportion of viable cells is high, the proportion of tumor-reactive T cells is high, and the life span is long, thus having stronger and / or more persistent killing ability on tumors, which can provide a basis for the application of the composition in tumor treatment.

[0094] 1. Definitions

[0095] In order to facilitate the understanding of the present application, some terms and phrases are defined below.

[0096] As used herein, the term "tumor-reactive T cell" refers to a specific T cell contained in tumor infiltrating lymphocytes (TIL) which, after being stimulated by tumor cell antigens, can produce enhanced tumor reactivity and / or enhanced specific killing ability compared to other T cells (J. Chiffelle et al., Immunity 10: S1074-7613 (24) 00413-8 (2024)). The enhancement of tumor reactivity is generally characterized in the art by the proportion of CD8+CD137+cells to CD45+cells. For example, the proportion of CD8+CD137+cells to CD45+cells increases from less than 3% to 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, but it is difficult to achieve further increase in the proportion of CD8+CD137+cells to CD45+cells in the prior art.

[0097] As used herein, the term "tumor cell antigen", also known as tumor antigen, refers to an antigenic substance that appears or is overexpressed during the process of tumorigenesis and development, and its production is related to factors such as gene mutation, abnormal gene activation, ectopic expression of embryonic antigens, and viral gene intervention. Tumor antigen should be understood to refer to those antigens presented on tumor cells, which can be generally divided into tumor-specific antigens and tumor-associated antigens according to the expression abundance of the antigens in normal cells. Among them, tumor-specific antigens usually refer to a class of antigens that are presented by tumor cells and cannot be presented by normal cells. While tumor-associated antigens are a class of antigens that can be presented by both tumor cells and normal cells, but the expression amount in tumor cells is significantly higher than that in normal cells. Tumor cell antigens can be used to activate tumor-reactive T cells, and the T cell receptor on the surface of the activated tumor-reactive T cells usually has specific complementary relationship with the MHC I-antigen peptide complex. In particular to the present disclosure, in the preparation of tumor-reactive T cell composition, appropriate tumor cell antigens can be selected for T cell stimulation in the amplification process according to the target indication, so as to obtain tumor-reactive T cell composition with high tumor killing effect.

[0098] As used herein, the term "CD (Cluster of Differentiation)" refers to a cluster of differentiation. As used herein, the term "CD8+" refers to a cell that is positive for CD8, i.e., a cell that expresses the CD8 gene, or a cell that has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD8 antibody), and a cell that can be seen as a distinct population. As used herein, the term "CD137+" refers to a cell that is positive for CD137, i.e., a cell that expresses the CD137 gene, or a cell that has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD137 antibody), and a cell that can be seen as a distinct population. CD137 is also known as 4-1BB, TNFRSF9, etc. As used herein, the term "CD8+CD137+" refers to a cell that is double positive for CD8 and CD137, i.e., a cell that expresses both CD8 and CD137 genes, or a cell that has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD8 and CD137 antibodies), and a cell that can be seen as a distinct population. As used herein, the term "CD45+" refers to a cell that is positive for CD45, i.e., a cell that expresses the CD45 gene, or a cell that has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD45 antibody), and a cell that can be seen as a distinct population. As used herein, the term "CD3+" refers to a cell that is positive for CD3, i.e., a cell that expresses the CD3 gene, or a cell that has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD3 antibody), and a cell that can be seen as a distinct population. As used herein, the term "CD45+CD3+" refers to a cell that is double positive for CD45 and CD3, i.e., a cell that expresses both CD45 and CD3 genes, or a cell that has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD45 and CD3 antibodies), and a cell that can be seen as a distinct population.

[0099] As used herein, the term "young T cell" refers to a T cell that is low in differentiation, or expresses markers with high concordance with an initial phenotype. Young T cells are capable of maintaining their killing activity in a subject for a long duration, and are capable of killing for a longer time / or more times compared to non-young T cells, and thus are useful for more effective and long-lasting control of tumors (see Lynn, R.C. et al., Nature 576(7786): 293-300 (2019); and Gattinoni, L. et al., Nat Med 17(10): 1290-1297 (2011). One or more of the following are commonly used in the art to characterize the degree of youthfulness of a cell: (i) high expression of CD45RA and CD62L (the proportion of CD45RA+CD62L+ cells in the composition); (ii) high expression of one or more selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1, and KLF7 (the percentage of increase in expression compared to before expansion); (iii) low expression of one or more selected from the group consisting of TBX1, ID2, and HOPX (the percentage of decrease in expression compared to before expansion); (iv) any of (i)-(iii) in combination with high expression of one or more selected from the group consisting of CCR7, CD27, CD28, BACH2, LEF1, TCF7; (v) any of (i)-(iv) in combination with low expression of one or more selected from the group consisting of TBX1, ID2, and HOPX.

[0100] As used herein, the term "CD45RA+" refers to a cell that is positive for CD45RA, i.e., a cell that expresses the CD45RA gene, or has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD45RA antibody), and a cell that can be seen as a distinct population. As used herein, the term "CD62L+" refers to a cell that is positive for CD62L, i.e., a cell that expresses the CD62L gene, or has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD62L antibody), and a cell that can be seen as a distinct population. CD62L is also known as SELL, LNHR, LSEL, LECAM1, etc. As used herein, the term "CD45RA+CD62L+" refers to a cell that is positive for both CD45RA and CD62L, i.e., a cell that expresses both the CD45RA and CD62L genes, or has a higher value of corresponding fluorescence intensity compared to a negative control (a cell that is not stained with CD45RA and CD62L antibodies), and a cell that can be seen as a distinct population.

[0101] As used herein, the term“engineered” refers to targeted modification of a cell. In some aspects, cell engineering includes viral genetic engineering, non-viral genetic engineering, introduction of a receptor that allows for tumor-specific targeting (e.g., TCR, TCRm, and / or CAR), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve T cell function, or any combination thereof.

[0102] As used herein, the term“expand” or“expansion” refers to a process of stimulating or activating cells and culturing the cells. Following stimulation or activation and culturing of the cells, the expansion process can result in an increase in the proportion or total number of desired cells in the cultured cell population, e.g., an increase in the total number and / or proportion of tumor-reactive T cells. Expansion does not require that the number of all cell types in the cultured cell population increase. Rather, in some aspects, only the number of a subset of cells in the cultured cell population increases during the expansion process, while the number of other cell types can not change or can decrease.

[0103] As used herein, the term“expanded tumor-reactive T cell composition” does not include formulated tumor-reactive T cell compositions (i.e., compositions resulting from combining purified specific T cells in a particular ratio (e.g., a ratio defined by the present disclosure)), and the term“expanded tumor-reactive T cell composition” does not include engineered T cell populations or compositions (i.e., compositions resulting from targeted modification from a source cell).

[0104] As used herein, the term“OKT3” refers to a monoclonal antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, and including commercially available forms, such as OKT-3 (30 ng / mL, MACS GMP CD3 Pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned ATCC Accession Number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalog Number 86022706.

[0105] As used herein, the term "SMO inhibitor" refers to a class of small molecule targeted therapies that are used to specifically inhibit the activity of the Smoothened (SMO) protein. "SMO inhibitors" of the present application include, but are not limited to, BMS-833923 (XL-139), SAG, Cyclopamine, Purmorphamine, Glasdegib, Sonidegib, Taladegib, and Halcinonide.

[0106] 2. A tumor reactive T cell composition of the present application

[0107] In a first aspect, the present application provides a tumor reactive T cell composition. In one embodiment, the tumor reactive T cell composition of the present application is obtained by expansion. In one embodiment, the tumor reactive T cell composition of the present application is obtained by one or more rounds of expansion. In one embodiment, the tumor reactive T cell composition of the present application is obtained by two rounds of expansion. In one embodiment, the tumor reactive T cell composition of the present application is obtained by one or more (preferably two) rounds of expansion from a sample from a patient of tumor, lymph node, pleural effusion, peritoneal effusion, peripheral blood, or cerebrospinal fluid. In one embodiment, the tumor reactive T cell composition of the present application is obtained by one or more (preferably two) rounds of expansion from a sample from a patient of tumor. In one embodiment, the tumor is a sample selected from the group consisting of a surgically resected tumor sample, a punch biopsy tumor sample, a core biopsy tumor sample, and a small biopsy tumor sample. In one embodiment, the tumor is a solid tumor. In one embodiment, the tumor is selected from the group consisting of mesothelioma, lung cancer, and glioma. In one embodiment, the tumor is non-small cell lung cancer.

[0108] In one embodiment, the tumor reactive T cell compositions of the present application have a higher tumor reactivity compared to prior art T cell compositions. In one embodiment, the tumor reactivity is characterized by the proportion of CD8+CD137+ cells out of CD45+ cells (CD8+CD137+ / CD45+) after stimulation of the composition with tumor cell antigens, as is commonly employed in the art. In one embodiment, the tumor reactive T cell compositions of the present application comprise a high proportion of tumor reactive T cells. In one embodiment, the tumor reactive T cell compositions of the present application have a proportion of CD8+CD137+ cells out of CD45+ cells (CD8+CD137+ / CD45+) after stimulation with tumor cell antigens that is greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, or greater than 70%.

[0109] In one embodiment, the tumor reactive T cell compositions of the application have a higher degree of naivety than prior art T cell compositions. In one embodiment, the degree of naivety is characterized by the proportion of CD45RA+CD62L+ cells in the composition as is commonly employed in the art. In one embodiment, the tumor reactive T cell compositions of the application comprise a high proportion of naive T cells. In one embodiment, the proportion of CD45RA+CD62L+ cells in the tumor reactive T cell compositions of the application is greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79%, or greater than 80%. In one embodiment, the degree of naivety is characterized by the proportion of cells with one or more of the following selected from the group: STAB1+, HES1+, RBPJ+, PMEPA1+, KLF7+, TBX1-, ID2-, and HOPX- in the composition as is commonly employed in the art. In one embodiment, the tumor reactive T cell compositions of the application comprise a high proportion of naive T cells.In one embodiment, the proportion of cells in the tumor reactive T cell composition of the application that are selected from one or more of the group consisting of STAB1+, HES1+, RBPJ+, PMEPA1+, KLF7+, TBX1-, ID2- and HOPX- is greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79% or greater than 80%. In one embodiment, the degree of youthfulness of the tumor reactive T cell composition of the application is characterized by the percentage increase in the expression level of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1 and KLF7 as compared to before expansion, as is commonly employed in the art. In one embodiment, the tumor reactive T cell composition of the application has an increase of greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79% or greater than 80% in the expression level of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1 and KLF7 as compared to before expansion.In one embodiment, the degree of youthful state is characterized by the percentage decrease in the amount of expression of one or more gene markers selected from the group consisting of the following in the composition compared to before amplification: TBX1, ID2, and HOPX, as commonly employed in the art. In one embodiment, the tumor reactive T cell composition of the application has a decrease of more than 20%, more than 21%, more than 22%, more than 23%, more than 24%, more than 25%, more than 26%, more than 27%, more than 28%, more than 29%, more than 30%, more than 31%, more than 32%, more than 33%, more than 34%, more than 35%, more than 36%, more than 37%, more than 38%, more than 39%, more than 40%, more than 41%, more than 42%, more than 43%, more than 44%, more than 45%, more than 46%, more than 47%, more than 48%, more than 49%, more than 50%, more than 51%, more than 52%, more than 53%, more than 54%, more than 55%, more than 56%, more than 57%, more than 58%, more than 59%, more than 60%, more than 61%, more than 62%, more than 63%, more than 64%, more than 65%, more than 66%, more than 67%, more than 68%, more than 69%, more than 70%, more than 71%, more than 72%, more than 73%, more than 74%, more than 75%, more than 76%, more than 77%, more than 78%, more than 79%, or more than 80% in the amount of expression of one or more gene markers selected from the group consisting of the following: TBX1, ID2, and HOPX.

[0110] In one embodiment, the proportion of CD45+ cells in a tumor reactive T cell composition of the application is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 95.5%, greater than 96%, greater than 96.5%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, or greater than 99.8%. In one embodiment, the proportion of CD3+ cells in a tumor reactive T cell composition of the application is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 95.5%, greater than 96%, greater than 96.5%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, or greater than 99.8%. In one embodiment, the proportion of CD45+CD3+ cells in a tumor reactive T cell composition of the application is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 95.5%, greater than 96%, greater than 96.5%, greater than 97%, greater than 97.5%, greater than 97.6%, greater than 97.7%, greater than 97.8%, greater than 97.9%, or greater than 98%.

[0111] In one embodiment, a tumor reactive T cell composition of the application contains a higher total number of T cells than a composition of the prior art. In one embodiment, the total number of T cells in a tumor reactive T cell composition of the application is at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least 10000-fold, at least 12000-fold, at least 15000-fold, at least 20000-fold, at least 25000-fold, at least 30000-fold, at least 35000-fold, at least 40000-fold, at least 45000-fold, at least 50000-fold, at least 55000-fold, at least 60000-fold, at least 65000-fold, at least 70000-fold, at least 75000-fold, at least 80000-fold, at least 85000-fold, at least 90000-fold, at least 95000-fold, at least 100000-fold, or more, than the total number of T cells prior to expansion. In one embodiment, the total number of tumor reactive T cells per milliliter of a tumor reactive T cell composition of the application is at least 1 x 10 10 10 10 In one embodiment, a tumor reactive T cell composition of the application contains a higher total number of T cells than a composition of the prior art. In one embodiment, the total number of T cells in a tumor reactive T cell composition of the application is at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least 10000-fold, at least 12000-fold, at least 15000-fold, at least 20000-fold, at least 25000-fold, at least 30000-fold, at least 35000-fold, at least 40000-fold, at least 45000-fold, at least 50000-fold, at least 55000-fold, at least 60000-fold, at least 65000-fold, at least 70000-fold, at least 75000-fold, at least 80000-fold, at least 85000-fold, at least 90000-fold, at least 95000-fold, at least 100000-fold, or more, than the total number of T cells prior to expansion. In one embodiment, the total number of tumor reactive T cells per milliliter of a tumor reactive T cell composition of the application is at least 1 x 10​​10 one, 5 x 10 10 one, 6 x 10 10 one, 7 x 10 10 one, 8 x 10 10 one, 9 x 10 10 one, 1 x 10 11 one, 2 x 10 11 one, 3 x 10 11 one, 4 x 10 11 one, 5 x 10 11 one or higher.

[0112] In one embodiment, the tumor reactive T cell compositions of the present application expand more quickly and / or more efficiently than prior art compositions. In one embodiment, the tumor reactive T cell compositions of the present application (e.g., having higher tumor reactivity, higher degree of youthfulness, and / or containing higher total number of T cells) are obtained within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, within 15 days, or within 14 days of the start of expansion. In one embodiment, the one or more (e.g., two) rounds of expansion are completed within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, within 15 days, or within 14 days.

[0113] In one embodiment, the tumor reactive T cell compositions of the present application have a higher percentage of viable T cells than prior art compositions. In one embodiment, the percentage of viable T cells in the tumor reactive T cell compositions of the present application (e.g., having higher tumor reactivity, higher degree of youthfulness, and / or containing higher total number of T cells) is 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0114] In one embodiment, the tumor reactive T cell compositions of the present application have greater tumor cell killing capacity than prior art compositions. In one embodiment, the killing capacity is characterized by the concentration of gamma-interferon (IFN-g) produced after stimulation with tumor cell antigens, as is commonly employed in the art. In one embodiment, the tumor reactive T cell compositions of the present application are capable of producing greater than 500 pg / mL, greater than 600 pg / mL, greater than 700 pg / mL, greater than 800 pg / mL, greater than 900 pg / mL, greater than 1000 pg / mL, greater than 1100 pg / mL, greater than 1200 pg / mL, greater than 1300 pg / mL, greater than 1400 pg / mL, greater than 1500 pg / mL, greater than 1600 pg / mL, greater than 1700 pg / mL, greater than 1800 pg / mL, greater than 1900 pg / mL, greater than 2000 pg / mL, greater than 2100 pg / mL, greater than 2200 pg / mL, greater than 2300 pg / mL, greater than 2400 pg / mL, greater than 2500 pg / mL, greater than 2600 pg / mL, greater than 2700 pg / mL, greater than 2800 pg / mL, greater than 2900 pg / mL, greater than 3000 pg / mL, greater than 3100 pg / mL, greater than 3200 pg / mL, greater than 3300 pg / mL, greater than 3400 pg / mL, greater than 3500 pg / mL, greater than 3600 pg / mL, greater than 3700 pg / mL, greater than 3800 pg / mL, greater than 3900 pg / mL, greater than 4000 pg / mL, greater than 4100 pg / mL, greater than 4200 pg / mL, greater than 4300 pg / mL, greater than 4400 pg / mL, or greater than 4500 pg / mL of gamma-interferon (IFN-g) after stimulation with tumor cell antigens.

[0115] In one embodiment, the tumor reactive T cell compositions of the present application have greater in vivo expansion capacity and / or longer in vivo survival time of tumor reactive T cells after administration to a subject (e.g., a human) than prior art compositions. In one embodiment, the in vivo expansion capacity and / or in vivo survival time is characterized by parameters commonly employed in the art selected from the group consisting of the proportion of CD45+ cells from the composition to total CD45+ in the subject, the proportion of CD3+ cells from the composition to total CD3+ in the subject, TCR abundance (i.e., the proportion of TCR species from the composition to the total number of TCR species in the subject), and / or the absolute count of CD3+ cells or CD3+CD8+ cells in the subject (e.g., peripheral blood).

[0116] In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD45+ cells from the composition that is greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, or greater than about 60% of the total CD45+ in the subject 7 days after administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD45+ cells from the composition that is greater than 15%, greater than 20%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, or greater than about 40% of the total CD45+ in the subject 14 days after administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD45+ cells from the composition that is greater than 15%, greater than 20%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, or greater than 35% of the total CD45+ in the subject 21 days after administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD45+ cells from the composition that is greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 16%, greater than 17%, greater than 18%, greater than 19%, greater than 20% of the total CD45+ in the subject 28-56 days after administration to the subject.

[0117] In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD3+ cells from the composition that is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79%, or greater than 80% of the total CD3+ in the subject 7 days after administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD3+ cells from the composition that is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, or greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95% of the total CD3+ in the subject 14 days after administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD3+ cells from the composition that is greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45% of the total CD3+ in the subject 28 days after administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD3+ cells from the composition that is greater than 20%, greater than 25%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, or greater than 40% of the total CD3+ in the subject 42 days after administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of CD3+ cells from the composition that is greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15% of the total CD3+ in the subject 63 days after administration to the subject.

[0118] In one embodiment, the tumor reactive T cell composition of the application has a proportion of TCR species from the composition to the total number of TCR species in the subject that is higher than 20%, higher than 21%, higher than 22%, higher than 23%, higher than 24%, higher than 25%, higher than 26%, higher than 27%, higher than 28%, higher than 29%, higher than 30%, higher than 31%, higher than 32%, higher than 33%, higher than 34%, higher than 35%, higher than 36%, higher than 37%, higher than 38%, higher than 39%, higher than 40%, higher than 41%, higher than 42%, higher than 43%, higher than 44%, higher than 45%, higher than 46%, higher than 47%, higher than 48%, higher than 49%, higher than 50%, higher than 51%, higher than 52%, higher than 53%, higher than 54%, higher than 55%, higher than 56%, higher than 57%, higher than 58%, higher than 59%, higher than 60% after 7-14 days of administration to the subject. In one embodiment, the tumor reactive T cell composition of the application has a proportion of TCR species from the composition to the total number of TCR species in the subject that is higher than 20%, higher than 21%, higher than 22%, higher than 23%, higher than 24%, higher than 25%, higher than 26%, higher than 27%, higher than 28%, higher than 29%, higher than 30%, higher than 31%, higher than 32%, higher than 33%, higher than 34%, higher than 35%, higher than 36%, higher than 37%, higher than 38%, higher than 39%, higher than 40%, higher than 41%, higher than 42%, higher than 43%, higher than 44%, higher than 45%, higher than 46%, higher than 47%, higher than 48%, higher than 49%, higher than 50%, higher than 51%, higher than 52%, higher than 53%, higher than 54%, higher than 55%, higher than 56%, higher than 57%, higher than 58%, higher than 59%, higher than 60% after 28-56 days of administration to the subject.

[0119] In one embodiment, the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject is higher than 1000 cells per μL, higher than 2000 cells per μL, higher than 3000 cells per μL, higher than 4000 cells per μL, higher than 5000 cells per μL, higher than 6000 cells per μL, higher than 7000 cells per μL, higher than 8000 cells per μL, higher than 9000 cells per μL, or higher than 10000 cells per μL after 7-14 days of administration of the tumor reactive T cell composition of the application to the subject. In one embodiment, the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject is higher than 500 cells per μL, higher than 800 cells per μL, higher than 1000 cells per μL, higher than 1200 cells per μL, higher than 1500 cells per μL, higher than 1800 cells per μL, higher than 2000 cells per μL, higher than 2200 cells per μL, higher than 2500 cells per μL, higher than 2800 cells per μL, higher than 3000 cells per μL, higher than 3200 cells per μL, higher than 3500 cells per μL, higher than 3800 cells per μL, or higher than 4000 cells per μL after 28-56 days of administration of the tumor reactive T cell composition of the application to the subject.

[0120] In one embodiment, the tumor reactive T cell composition of the application has a stronger continuous killing ability against tumor cells compared to the compositions of the prior art. In one embodiment, the tumor reactive T cell composition of the application is capable of continuous killing of tumor cells for at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times. In one embodiment, the tumor reactive T cell composition of the application retains at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% of target lysis (%) after 6 times, 7 times, 8 times, 9 times, 10 times of continuous killing of tumor cells.

[0121] In one embodiment, the tumor reactive T cell composition of the application can or can not comprise feeder cells / stromal cells.

[0122] In one embodiment, the tumor reactive T cell composition of the application is the composition obtained by the method of the application for preparing the tumor reactive T cell composition of the application.

[0123] 3. Method for preparing a tumor reactive T cell composition

[0124] In a second aspect, the present application provides a method of making a tumor- reactive T cell composition. In one embodiment, the method is by expansion to make the tumor- reactive T cell composition. In one embodiment, the method is by one or more rounds of expansion to make the tumor-reactive T cell composition. In one embodiment, the method is by two rounds of expansion to make the tumor-reactive T cell composition. In one embodiment, the method comprises: (1) providing a sample containing tumor-reactive T cells; (2) pre-treating the sample; (3) performing a first-stage expansion culture of the pre-treated sample in a first medium comprising IL-2, OKT3, and / or a SMO inhibitor; (4) performing a second-stage expansion culture of the product of the first-stage expansion culture in a second medium comprising IL-2, IL-7, IL-15, OKT3, and / or a SMO inhibitor; and (5) optionally harvesting the composition.

[0125] In one embodiment, the sample containing tumor-reactive T cells is a sample from a patient. In one embodiment, the sample is a tumor, lymph node, pleural effusion, peritoneal effusion, peripheral blood, or cerebrospinal fluid from a patient. In one embodiment, the tumor is a sample selected from the group consisting of a surgically resected tumor sample, a punch biopsy tumor sample, a core biopsy tumor sample, and a small biopsy tumor sample. In one embodiment, wherein the tumor is a solid tumor. In one embodiment, wherein the tumor is selected from the group consisting of mesothelioma, lung cancer, and glioma. In one embodiment, wherein the tumor is non-small cell lung cancer.

[0126] In one embodiment, the first medium comprises IL-2. In one embodiment, the first medium comprises OKT3. In one embodiment, the first medium comprises a SMO inhibitor. In one embodiment, the first medium comprises IL-2 and OKT3. In one embodiment, the first medium comprises IL-2 and a SMO inhibitor. In one embodiment, the first medium comprises OKT3 and a SMO inhibitor. In one embodiment, the first medium comprises IL-2, OKT3, and a SMO inhibitor. In one embodiment, the second medium comprises IL-2. In one embodiment, the second medium comprises IL-7. In one embodiment, the second medium comprises IL-15. In one embodiment, the second medium comprises OKT3. In one embodiment, the second medium comprises a SMO inhibitor. In one embodiment, the second medium comprises IL-2 and IL-7. In one embodiment, the second medium comprises IL-2 and IL-15. In one embodiment, the second medium comprises IL-2 and OKT3. In one embodiment, the second medium comprises IL-2 and a SMO inhibitor. In one embodiment, the second medium comprises IL-7 and IL-15. In one embodiment, the second medium comprises IL-7 and OKT3. In one embodiment, the second medium comprises IL-7 and a SMO inhibitor. In one embodiment, the second medium comprises IL-15 and OKT3. In one embodiment, the second medium comprises IL-15 and a SMO inhibitor. In one embodiment, the second medium comprises IL-2, IL-7, and IL-15. In one embodiment, the second medium comprises IL-2, IL-7, and OKT3. In one embodiment, the second medium comprises IL-2, IL-7, and a SMO inhibitor. In one embodiment, the second medium comprises IL-2, IL-15, and OKT3. In one embodiment, the second medium comprises IL-2, IL-15, and a SMO inhibitor. In one embodiment, the second medium comprises IL-2, OKT3, and a SMO inhibitor. In one embodiment, the second medium comprises IL-2, IL-7, IL-15, and OKT3. In one embodiment, the second medium comprises IL-2, IL-7, IL-15, and a SMO inhibitor. In one embodiment, the second medium comprises IL-2, IL-7, OKT3, and a SMO inhibitor. In one embodiment, the second medium comprises IL-2, IL-15, OKT3, and a SMO inhibitor. In one embodiment, the second medium comprises IL-7, IL-15, OKT3, and a SMO inhibitor. In one embodiment, the second medium comprises IL-2, IL-7, IL-15, OKT3, and a SMO inhibitor.

[0127] In one embodiment, the SMO inhibitor is selected from the group consisting of BMS-833923 (XL-139), SAG, Cyclopamine, Purmorphamine, Glasdegib, Sonidegib, Taladegib, and Halcinonide. In one embodiment, the SMO inhibitor comprises BMS-833923 (XL-139).

[0128] In one embodiment, the pre-treatment comprises one or more operations selected from the group consisting of removing tissue, fragmenting, washing, lysing red blood cells, resuspension, and cryopreservation. In one embodiment, the removing tissue comprises removing necrotic tissue and / or removing adipose tissue. In one embodiment, whether to perform removing tissue is determined according to the condition of the tissue. In one embodiment, the removing tissue comprises transferring the tissue into a culture dish and then removing necrotic tissue and adipose tissue in the culture dish. In one embodiment, the fragmenting is performed by grinding the tissue or mincing the tissue. In one embodiment, the fragmenting is performed by grinding the sample on a cell strainer. In one embodiment, the grinding the tissue on a cell strainer is suitable for softer tissue. In one embodiment, the grinding is performed by placing the strainer on a centrifuge tube with hemostatic forceps, transferring the softer tissue onto the strainer with forceps, grinding the tissue with the end of a syringe plunger handle, and then washing with a wash solution. In one embodiment, the fragmenting is performed by mincing the sample. In one embodiment, the mincing is performed by transferring the sample into a centrifuge tube with forceps, keeping it moist, and mincing to about 1-3 mm pieces (e.g., with ophthalmic scissors). 3and centrifuging the minced sample in a centrifuge tube. In one embodiment, the washing is performed by centrifugation in 1-50 times the sample volume of wash solution. In one embodiment, the wash solution comprises potassium ions. In one embodiment, the wash solution comprises: an antibiotic and potassium ions. In one embodiment, the centrifugation is performed by centrifugation at 1000-2000 rpm for 1-5 minutes. In one embodiment, the centrifugation is performed by centrifugation at 1200-1800 rpm for 2-3 minutes. In one embodiment, the red blood cell lysis is suitable for a tissue with hypererythrosis. In one embodiment, the red blood cell lysis is performed by adding 1-50 times the sample volume of red blood cell lysis solution and mixing. In one embodiment, the mixing is performed by pipetting and / or inverting mixing. In one embodiment, the red blood cell lysis is no more than 8 minutes or no more than 5 minutes. In one embodiment, the mixing is performed by inverting mixing every 1-2 minutes for a total of no more than 5 minutes. In one embodiment, the lysis is terminated by adding an equal volume of wash solution and inverting mixing. In one embodiment, the sample after red blood cell lysis is optionally washed again. In one embodiment, the red blood cell lysis solution comprises ions, preferably metal ions. In one embodiment, the red blood cell lysis solution comprises potassium ions, sodium ions, and / or ammonium ions. In one embodiment, the red blood cell lysis solution comprises potassium ions, sodium ions, and ammonium ions. In one embodiment, the resuspension is resuspending the sample in the first culture medium again. In one embodiment, the wherein the cryopreservation is cryopreservation after centrifugation of the sample by adding a cryopreservation solution and using a programable cryopreservation instrument. In one embodiment, the cryopreservation solution comprises human serum albumin. In one embodiment, the cryopreservation solution comprises: (a) CS10; (b) human serum albumin; and (c) Compound Electrolyte Injection.

[0129] In one embodiment, the first culture medium can be the same as or different from the second culture medium.

[0130] In one embodiment, the first-stage expansion culture is performed in a culture bag placed in a 37°C, 5% CO2 incubator for 4-8 days, preferably 4-5 days. In one embodiment, the culture is optionally supplemented with the first culture medium on days 1-3 of the first-stage expansion culture. In one embodiment, after the first-stage expansion culture, the total number of tumor-reactive T cells in the resulting composition is at least 20-fold, at least 40-fold, at least 50-fold, at least 80-fold, or at least 100-fold greater than before expansion. In one embodiment, after the first-stage expansion culture, the total number of cells per milliliter of the resulting composition is at least 5 x 10 8 cells, 6 x 108 7×10 8 8×10 8 9×10 8 One or 1×10 9 indivual.

[0131] In one embodiment, the second-stage expansion culture is carried out in a culture bag placed in a 37°C, 5% CO2 incubator for 7-13 days, preferably 7-10 days. In one embodiment, optionally, a second culture medium is added to the culture on days 2-3, 4-5, and / or 6-7 of the second-stage expansion culture. In one embodiment, after the second-stage expansion culture, the total number of tumor-reactive T cells in the resulting composition is at least 10,000 times, at least 20,000 times, at least 50,000 times, at least 80,000 times, or at least 100,000 times that before expansion. In one embodiment, after the second-stage expansion culture, the total number of T cells per milliliter of the resulting composition is at least 1 × 10⁻⁶. 10 1.5 × 10 10 1, 2×10 10 1, 3×10 10 1, 4×10 10 5×10 10 6×10 10 7×10 10 8×10 10 9×10 10 1×10 11 1, 2×10 11 1, 3×10 11 1, 4×10 11 5×10 11 One or more.

[0132] In one embodiment, harvesting of the composition is performed by centrifuging to collect cells after the second stage of expansion culture. In one embodiment, harvesting the composition optionally further includes washing the composition with a washing buffer and / or freezing the composition with a cryopreservation solution. In one embodiment, the washing buffer contains potassium ions. In one embodiment, the washing buffer contains: an antibiotic and potassium ions. In one embodiment, the cryopreservation solution contains human serum albumin. In one embodiment, the cryopreservation solution contains: (a) CS10; (b) human serum albumin; and (c) compound electrolyte injection.

[0133] In one embodiment, the tumor reactive T cell composition obtained by the methods of the application is obtained in multiple batches under different culture conditions. In one embodiment, the different culture conditions include, but are not limited to, different sample sources, different pre-treatment conditions, different first media compositions and concentrations, different second media compositions and concentrations, and / or different cryopreservation conditions, etc.

[0134] In one embodiment, the tumor reactive T cell composition obtained by the methods of the application has a higher tumor reactivity compared to prior art T cell compositions. In one embodiment, the tumor reactivity is characterized by the proportion of CD8+CD137+cells out of CD45+cells (CD137+CD8+ / CD45+) of the composition after stimulation with tumor cell antigens, in a manner commonly employed in the art. In one embodiment, the tumor reactive T cell composition obtained by the methods of the application comprises a high proportion of tumor reactive T cells. In one embodiment, the tumor reactive T cell composition obtained by the methods of the application has a proportion of CD8+CD137+cells out of CD45+cells (CD8+CD137+ / CD45+) of greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, or greater than 70% after stimulation with tumor cell antigens. In one embodiment, the tumor cell antigens are obtained from the patient to be treated or from a different patient having the same indication.

[0135] In one embodiment, the tumor reactive T cell compositions obtained by the methods of the application have a higher degree of naivety than prior art T cell compositions. In one embodiment, the degree of naivety is characterized by means commonly employed in the art, e.g., the proportion of CD45RA+CD62L+cells in the composition. In one embodiment, the tumor reactive T cell compositions obtained by the methods of the application comprise a high proportion of naive T cells. In one embodiment, the proportion of CD45RA+CD62L+cells in the tumor reactive T cell compositions obtained by the methods of the application is greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79%, or greater than 80%. In one embodiment, the degree of naivety is characterized by means commonly employed in the art, e.g., the proportion of cells in the composition having one or more selected from the group consisting of STAB1+, HES1+, RBPJ+, PMEPA1+, KLF7+, TBX1-, ID2-, and HOPX-. In one embodiment, the tumor reactive T cell compositions obtained by the methods of the application comprise a high proportion of naive T cells.In one embodiment, the proportion of cells in the tumor reactive T cell composition obtained by the method of the application that are one or more selected from the group consisting of: STAB1+, HES1+, RBPJ+, PMEPA1+, KLF7+, TBX1-, ID2- and HOPX- is greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79% or greater than 80%. In one embodiment, the degree of youthfulness is characterized by the percentage increase in the amount of expression of one or more gene markers selected from the group consisting of: STAB1, HES1, RBPJ, PMEPA1 and KLF7 in the composition compared to before expansion by means commonly employed in the art.In one embodiment, the expression level of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1, and KLF7 is increased by more than 20%, more than 21%, more than 22%, more than 23%, more than 24%, more than 25%, more than 26%, more than 27%, more than 28%, more than 29%, more than 30%, more than 31%, more than 32%, more than 33%, more than 34%, more than 35%, more than 36%, more than 37%, more than 38%, more than 39%, more than 40%, more than 41%, more than 42%, more than 43%, more than 44%, more than 45%, more than 46%, more than 47%, more than 48%, more than 49%, more than 50%, more than 51%, more than 52%, more than 53%, more than 54%, more than 55%, more than 56%, more than 57%, more than 58%, more than 59%, more than 60%, more than 61%, more than 62%, more than 63%, more than 64%, more than 65%, more than 66%, more than 67%, more than 68%, more than 69%, more than 70%, more than 71%, more than 72%, more than 73%, more than 74%, more than 75%, more than 76%, more than 77%, more than 78%, more than 79%, or more than 80% in the tumor reactive T cell composition obtained by the methods of the present application as compared to the pre-expansion level. In one embodiment, the degree of youthfulness is characterized by the percentage decrease in the expression level of one or more gene markers selected from the group consisting of TBX1, ID2, and HOPX in the composition as compared to the pre-expansion level by means commonly employed in the art (e.g., the percentage decrease in the expression level of one or more gene markers selected from the group consisting of TBX1, ID2, and HOPX in the composition). In one embodiment, the expression level of one or more gene markers selected from the group consisting of TBX1, ID2, and HOPX is decreased by more than 20%, more than 21%, more than 22%, more than 23%, more than 24%, more than 25%, more than 26%, more than 27%, more than 28%, more than 29%, more than 30%, more than 31%, more than 32%, more than 33%, more than 34%, more than 35%, more than 36%, more than 37%, more than 38%, more than 39%, more than 40%, more than 41%, more than 42%, more than 43%, more than 44%, more than 45%, more than 46%, more than 47%, more than 48%, more than 49%, more than 50%, more than 51%, more than 52%, more than 53%, more than 54%, more than 55%, more than 56%, more than 57%, more than 58%, more than 59%, more than 60%, more than 61%, more than 62%, more than 63%, more than 64%, more than 65%, more than 66%, more than 67%, more than 68%, more than 69%, more than 70%, more than 71%, more than 72%, more than 73%, more than 74%, more than 75%, more than 76%, more than 77%, more than 78%, more than 79%, or more than 80% in the tumor reactive T cell composition obtained by the methods of the present application as compared to the pre-expansion level.

[0136] In one embodiment, the proportion of CD45+ cells in the tumor-reactive T-cell composition obtained by the method of the present invention is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 95.5%, greater than 96%, greater than 96.5%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, or greater than 99.8%. In one embodiment, the proportion of CD3+ cells in the tumor-reactive T-cell composition obtained by the method of the present invention is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 95.5%, greater than 96%, greater than 96.5%, greater than 97%, greater than 97.5%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, or greater than 99.8%. In one embodiment, the tumor-reactive T-cell composition obtained by the method of the present invention has a CD45+CD3+ cell ratio greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 95.5%, greater than 96%, greater than 96.5%, greater than 97%, greater than 97.5%, greater than 97.6%, greater than 97.7%, greater than 97.8%, greater than 97.9%, or greater than 98%.

[0137] In one embodiment, the tumor-reactive T-cell composition obtained by the method of the present invention contains a higher total number of T cells compared with compositions of the prior art. In one embodiment, the total number of T cells in the tumor-reactive T cell composition obtained by the method of the present invention is at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, at least 7000 times, at least 8000 times, at least 9000 times, at least 10000 times, at least 12000 times, at least 15000 times, at least 20000 times, at least 25000 times, at least 30000 times, at least 35000 times, at least 40000 times, at least 45000 times, at least 50000 times, at least 55000 times, at least 60000 times, at least 65000 times, at least 70000 times, at least 75000 times, at least 80000 times, at least 85000 times, at least 90000 times, at least 95000 times, at least 100000 times or higher than before expansion. In one embodiment, the total number of tumor-reactive T cells in each milliliter of the tumor-reactive T cell composition obtained by the method of the present invention is at least 1 × 10⁻⁶. 10 At least 2×1010 at least 4 x 10 10 at least 8 x 10 10 at least 1 x 10 11 at least 2 x 10 11 at least 3 x 10 11 at least 4 x 10 11 at least 5 x 10 11 at least 6 x 10

[0138] In one embodiment, the tumor reactive T cell composition obtained by the methods of the present application expands more rapidly than prior art compositions. In one embodiment, the tumor reactive T cell composition obtained by the methods of the present application (e.g., a tumor reactive T cell composition having greater tumor reactivity, a greater degree of youthfulness, and / or containing a greater total number of T cells) is obtained within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, or within 15 days of the start of expansion. In one embodiment, the one or more (e.g., two) rounds of expansion are completed within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, or within 15 days.

[0139] In one embodiment, the tumor reactive T cell composition obtained by the methods of the present application has a greater percentage of viable T cells than prior art compositions. In one embodiment, the tumor reactive T cell composition obtained by the methods of the present application (e.g., a tumor reactive T cell composition having greater tumor reactivity, a greater degree of youthfulness, and / or containing a greater total number of T cells) has a percentage of viable T cells of 80% or greater, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater.

[0140] In one embodiment, the tumor reactive T cell compositions resulting from the methods of the application have greater tumor cell killing capacity than prior art compositions. In one embodiment, the killing capacity is characterized by means commonly employed in the art, such as concentration of gamma-interferon (IFN-g) produced upon stimulation with tumor cell antigens. In one embodiment, the tumor reactive T cell compositions resulting from the methods of the application are capable of producing 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 800 pg / mL or more, 900 pg / mL or more, 1000 pg / mL or more, 1100 pg / mL or more, 1200 pg / mL or more, 1300 pg / mL or more, 1400 pg / mL or more, 1500 pg / mL or more, 1600 pg / mL or more, 1700 pg / mL or more, 1800 pg / mL or more, 1900 pg / mL or more, 2000 pg / mL or more, 2100 pg / mL or more, 2200 pg / mL or more, 2300 pg / mL or more, 2400 pg / mL or more, 2500 pg / mL or more, 2600 pg / mL or more, 2700 pg / mL or more, 2800 pg / mL or more, 2900 pg / mL or more, 3000 pg / mL or more, 3100 pg / mL or more, 3200 pg / mL or more, 3300 pg / mL or more, 3400 pg / mL or more, 3500 pg / mL or more, 3600 pg / mL or more, 3700 pg / mL or more, 3800 pg / mL or more, 3900 pg / mL or more, 4000 pg / mL or more, 4100 pg / mL or more, 4200 pg / mL or more, 4300 pg / mL or more, 4400 pg / mL or more, or 4500 pg / mL or more of gamma-interferon (IFN-g) upon stimulation with tumor cell antigens.

[0141] In one embodiment, the tumor reactive T cell compositions resulting from the methods of the application have greater in vivo expansion capacity and / or longer in vivo survival time of tumor reactive T cells after administration to a subject (e.g., a human) than prior art compositions. In one embodiment, the in vivo expansion capacity and / or in vivo survival time is characterized by parameters commonly employed in the art selected from the group consisting of the proportion of CD45+ cells from the composition to total CD45+ in the subject, the proportion of CD3+ cells from the composition to total CD3+ in the subject, TCR abundance (e.g., the proportion of TCR species from the composition to the total number of TCR species in the subject), and absolute counts of CD3+ cells or CD3+CD8+ cells in the subject (e.g., peripheral blood).

[0142] In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD45+ cells from the composition that is greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, or greater than about 60% of the total CD45+ in the subject 7 days after administration to the subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD45+ cells from the composition that is greater than 15%, greater than 20%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, or greater than about 40% of the total CD45+ in the subject 14 days after administration to the subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD45+ cells from the composition that is greater than 15%, greater than 20%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, or greater than 35% of the total CD45+ in the subject 21 days after administration to the subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD45+ cells from the composition that is greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 16%, greater than 17%, greater than 18%, greater than 19%, greater than 20% of the total CD45+ in the subject 28-56 days after administration to the subject.

[0143] In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD3+ cells from the composition that is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79%, or greater than 80% of the total CD3+ in the subject 7 days after administration to the subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD3+ cells from the composition that is greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, or greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95% of the total CD3+ in the subject 14 days after administration to the subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD3+ cells from the composition that is greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45% of the total CD3+ in the subject 28 days after administration to the subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD3+ cells from the composition that is greater than 20%, greater than 25%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, or greater than 40% of the total CD3+ in the subject 42 days after administration to the subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of CD3+ cells from the composition that is greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15% of the total CD3+ in the subject 63 days after administration to the subject.

[0144] In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of TCR species from the composition that is greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60% of the total number of TCR species after 7-14 days of administration to a subject. In one embodiment, the tumor reactive T cell composition resulting from the methods of the application has a proportion of TCR species from the composition that is greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, greater than 30%, greater than 31%, greater than 32%, greater than 33%, greater than 34%, greater than 35%, greater than 36%, greater than 37%, greater than 38%, greater than 39%, greater than 40%, greater than 41%, greater than 42%, greater than 43%, greater than 44%, greater than 45%, greater than 46%, greater than 47%, greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60% of the total number of TCR species after 28-56 days of administration to a subject.

[0145] In one embodiment, the tumor reactive T cell composition obtained by the methods of the application has an absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject that is greater than 1000 cells per μL, greater than 2000 cells per μL, greater than 3000 cells per μL, greater than 4000 cells per μL, greater than 5000 cells per μL, greater than 6000 cells per μL, greater than 7000 cells per μL, greater than 8000 cells per μL, greater than 9000 cells per μL, or greater than 10000 cells per μL 7-14 days after administration to the subject. In one embodiment, the tumor reactive T cell composition obtained by the methods of the application has an absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject that is greater than 500 cells per μL, greater than 800 cells per μL, greater than 1000 cells per μL, greater than 1200 cells per μL, greater than 1500 cells per μL, greater than 1800 cells per μL, greater than 2000 cells per μL, greater than 2200 cells per μL, greater than 2500 cells per μL, greater than 2800 cells per μL, greater than 3000 cells per μL, greater than 3200 cells per μL, greater than 3500 cells per μL, greater than 3800 cells per μL, or greater than 4000 cells per μL 28-56 days after administration to the subject. In one embodiment, the tumor reactive T cell composition obtained by the methods of the application has an absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject that is greater than 1000 cells per μL, greater than 2000 cells per μL, greater than 3000 cells per μL, greater than 4000 cells per μL, greater than 5000 cells per μL, greater than 6000 cells per μL, greater than 7000 cells per μL, greater than 8000 cells per μL, greater than 9000 cells per μL, or greater than 10000 cells per μL 7-14 days after administration to the subject. In one embodiment, the tumor reactive T cell composition obtained by the methods of the application has an absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject that is greater than 500 cells per μL, greater than 800 cells per μL, greater than 1000 cells per μL, greater than 1200 cells per μL, greater than 1500 cells per μL, greater than 1800 cells per μL, greater than 2000 cells per μL, greater than 2200 cells per μL, greater than 2500 cells per μL, greater than 2800 cells per μL, greater than 3000 cells per μL, greater than 3200 cells per μL, greater than 3500 cells per μL, greater than 3800 cells per μL, or greater than 4000 cells per μL 28-56 days after administration to the subject.

[0146] In one embodiment, the tumor reactive T cell composition obtained by the method of the present application has a stronger serial killing ability against tumor cells than prior art compositions. In one embodiment, the tumor reactive T cell composition obtained by the method of the present application is capable of serial killing of tumor cells for at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times. In one embodiment, the tumor reactive T cell composition obtained by the method of the present application retains at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% of target lysis (%) after 6, 7, 8, 9, 10 serial killing of tumor cells.

[0147] In one embodiment, the tumor reactive T cell composition obtained by the method of the present application can or can not comprise feeder cells / stromal cells.

[0148] 4. The tumor reactive T cell composition of the present application

[0149] In a third aspect, the present application provides a tumor reactive T cell composition. In one embodiment, the tumor reactive T cell composition of the present application is prepared by the method of the present application for preparing a tumor reactive T cell composition.

[0150] 5. Advantages of the present application

[0151] Compared with prior art expanded T cell compositions and expansion methods, the composition and expansion method of the present application has at least the following advantages:

[0152] (1) The tumor reactive T cell composition of the present application has at least 25% of CD8+CD137+ cells among CD45+ cells after stimulation with tumor cell antigens, which indicates that the composition of the present application has stronger killing ability, which is also evidenced by the high IFN-γ concentration produced by the composition after stimulation with tumor cell antigens;

[0153] (2) The tumor reactive T cell composition of the present application has at least 30% of CD45RA+CD62L+ cells, which indicates that the composition of the present application has a higher degree of naivety, which can be used for sustained and effective killing of tumor (e.g. can kill more times and / or reduce the volume of tumor cells or make tumor cells disappear for a longer period of time);

[0154] (3) The tumor reactive T cell composition of the present application has a higher total number of T cells and / or a higher number of viable T cells (higher viability);

[0155] (4) The tumor reactive T cell composition of the present application has stronger in vivo expansion ability and / or longer in vivo survival time of tumor reactive T cells after administration to a subject (e.g., a human) compared to the compositions of the prior art, and thus has stronger continuous killing ability to tumor cells.

[0156] The combination of the advantages of (1) to (4) described above results in a higher total number of young, tumor reactive T cells per unit volume, and thus more stable, powerful and persistent killing of tumor cells. DETAILED DESCRIPTION

[0157] The present application will be further described with reference to the following specific examples, but these specific examples should not be construed as limiting the scope of the present application. Those skilled in the art can make various changes or modifications to the specific examples without departing from the scope of the technical solutions of the present application, and the changed and modified embodiments still fall within the scope of the present application.

[0158] All chemicals or products used in the examples are commercially available.

[0159] Example 1. Expansion preparation of the tumor reactive T cell composition of the present application

[0160] This example describes a novel simplified procedure for generating a composition comprising clinically relevant numbers of tumor specific T cells from tumor tissue resected from a patient (e.g., mesothelioma, glioma, non-small cell lung cancer, and other solid tumor patients) in a culture bag and cryopreserving the final product.

[0161] Example 1.1 Expansion preparation of the tumor reactive T cell composition of the present application from non-small cell lung cancer tumor tissue

[0162] I. Definitions / Abbreviations

[0163] BSC: Biological Safety Cabinet

[0164] °C: degrees Celsius

[0165] CO2: carbon dioxide

[0166] CM1: first culture medium

[0167] CM2: second culture medium

[0168] CRF: Control Rate Freezer

[0169] EtOH: ethanol

[0170] GMP: Good Manufacturing Practice

[0171] IL-2, rIL-2: interleukin-2, recombinant human interleukin-2

[0172] IL-7: interleukin-7

[0173] IL-15: interleukin-15

[0174] IU: international unit

[0175] L / mL / μL: liter / milliliter / microliter

[0176] LN2: liquid nitrogen

[0177] mM / μM: millimolar / micromolar

[0178] μm: micrometer

[0179] NA: not applicable

[0180] PPE: personal protective equipment

[0181] TIL: tumor infiltrating lymphocytes

[0182] TIWB: TIL isolation wash buffer

[0183] SOP: standard operating procedure

[0184] Prior art composition (control): T cell composition prepared according to methods 2A in examples 5-10 and table 19 in US 10894063 B2 (this document is incorporated herein by reference) using samples from the same tumor patients as in the examples of the present application.

[0185] II. Preparation procedure

[0186] 1 Preparation in advance: day 0 (up to 36 hours in advance)

[0187] 1.1 Preparation of CM1: the CM1 comprises 2000-7000 UI / mL of IL-2 and 0.01-10 μΜ of BMS-833923 (XL-139) on top of a regular medium in the art. The regular medium includes, but is not limited to: MEM, DMEM, IMDM, RPMI-1640, Xpander TM , X-VIVO and KBM581. The CM1 is stored at 4°C for up to 96 hours. Before use, it is warmed at 37°C for at least 1 hour.

[0188] 2 Reception of non-small cell lung cancer tumor tissue (biopsy sample)

[0189] 2.1 Retain all documentation received with the tumor tissue (including patient's informed consent) and take a photograph of the shipping container and tumor tissue.

[0190] 2.2 If shipping documentation is provided, print and retain the relevant documents; save as PDF.

[0191] 2.3 Remove the tumor sample and secondary container (zip-locked bag) from the shipper and store at 4°C until ready for processing.

[0192] 2.4 Unused tumor is shipped in HypoThermasol, which is commercially available from BioLife Solutions, Inc.

[0193] 3 Tumor processing for TILs, Day 0

[0194] 3.1 Sterilely transfer the following materials (i.e., ophthalmic scissors, ophthalmic forceps) to the BSC as needed to prepare sterile surgical instruments 3 ophthalmic scissors, 3 ophthalmic forceps per sample.

[0195] 3.2 Prepare wash solution (e.g., 50 mL PBS with 187.5 μΐ gentamicin sulfate injection and 9.8 μΐ vancomycin hydrochloride for injection to make a potassium ion concentration of 35 mM) and red blood cell lysis solution (e.g., 45 mL purified water with 5 mL of commercial red blood cell lysis solution (available from Biolegend, RBC Lysis Buffer (10X)) to make a potassium ion, sodium ion, and / or ammonium ion concentration of 35 mM, filter through a 0.22 μιη filter into a new 50 mL centrifuge tube).

[0196] 3.3 Spray the surface of the tissue sample tube with alcohol, transfer the sample into the safety cabinet, and further check the sample.

[0197] 3.4 Remove necrotic tissue and adipose tissue: determine if this is to be performed based on the tissue (if necrotic tissue and adipose tissue are present); transfer the tissue into a 60 mm petri dish and remove the necrotic tissue and adipose tissue.

[0198] 3.5 Grind the tissue, grind the tissue on a cell strainer (for soft tissue): place a 40 μιη strainer on a 50 mL centrifuge tube using hemostatic forceps, transfer the soft tissue to the strainer using sterile ophthalmic forceps, grind the tissue using the end of a syringe plunger handle, and drop the ground tissue into the centrifuge tube.

[0199] 3.6 Chop the tissue: transfer the sample to a 1.5 mL centrifuge tube using sterile ophthalmic forceps, keep moist, and chop to approximately 1-3 mm using ophthalmic scissors. 3 .

[0200] 3.7 Wash the ground or minced tissue: Add 10 volumes of wash solution, centrifuge at 1500 rpm for 3 minutes at room temperature, and discard the supernatant.

[0201] 3.8 Red cell lysis (for tissues with excessive red blood cells): Add 10 volumes of red cell lysis solution to the cell pellet, mix by pipetting up and down, and invert the tube every 1-2 minutes for no more than 5 minutes. Add an equal volume of wash solution, invert the tube to stop the lysis. Centrifuge at 1500 rpm for 3 minutes at room temperature, and discard the supernatant. Add 10 volumes of wash solution, centrifuge at 1500 rpm for 3 minutes at room temperature, and discard the supernatant.

[0202] 3.9 Resuspend the sample: Add 10 volumes of CM1 to resuspend the minced tissue. Take a portion of the sample directly for inoculation, and use the rest for QC testing. The rest of the sample is frozen;

[0203] 3.10 Freeze the rest of the sample: Centrifuge the sample to be frozen at room temperature at 1500 rpm for 3 minutes, and discard the supernatant. Add freezing solution (e.g., the freezing solution can contain the following components: CS10, human serum albumin, and Compound Electrolyte Injection), and use a programable freezer to freeze the sample.

[0204] 4 Inoculate 0.3 L bags

[0205] 4.1 Sterilely transfer the 0.3 L bags and CM1 to the BSC as needed.

[0206] 4.2 Place 100 mL of pre-warmed CM1 in each of the desired 0.3 L bags.

[0207] 4.3 Use a pipette to transfer an appropriate amount of tumor pieces to each 0.3 L bag.

[0208] 4.4 Record the volume of tissue added to each bag.

[0209] 4.5 Place each bag in a 37°C, 5% CO2 incubator.

[0210] 5 First stage cell expansion, days 4-8

[0211] 5.1 Prepare 2 L of CM1. Warm at 37°C for 1 hour before use.

[0212] 5.2 Remove the bags from the CO2 incubator and transfer to the BSC. Transfer CM1 to the BSC, and add an appropriate amount (e.g., about 0.2 L) of CM1 to the bags using a 50 mL syringe as needed. Return the bags to the incubator after the procedure is complete.

[0213] 5.3 The volume of media in a 0.3L bag should not exceed 300mL, if it does, the cell culture should be transferred to a 2L bag, and the volume of media in a 2L bag should not exceed 1800mL.

[0214] 5.4 T cells are counted and when the cell number reaches the range of 5E8 to 1E9, the second phase of cell expansion can begin.

[0215] 6 Second phase of cell expansion, days 8-14

[0216] 6.1 Day 8: Prepare 0.6L of CM2. The CM2 contains 2000-7000 UI / mL of IL-15 and 0.01-10 μΜ of BMS-833923 (XL-139) on top of a conventional media used in the art. The conventional media includes but is not limited to: MEM, DMEM, IMDM, RPMI-1640, Xpander TM , X-VIVO, and KBM581. The CM2 is warmed at 37°C for 1 hour before use.

[0217] 6.2 Day 8: Replace media: remove the bag containing the cell culture from the CO2 incubator, use a sterile tubing machine to connect the bag to the Corning® FlexPro consumables, use the Corning® FlexPro device to centrifuge the bag to collect the cells, remove the CM1, collect the remaining sample into one bag, in a biological safety cabinet, dispense the remaining sample into 6-12 2L bags, and add 0.6L of CM2 containing IL-15 to each bag, return the bags to the incubator for continued culture.

[0218] 6.3 Day 10: Add CM2, remove the bag from the CO2 incubator and transfer to a BSC, transfer the CM2 to the BSC, add an amount (e.g., about 0.6L) of CM2 containing IL-15 to the bag as needed, and return the bag to the incubator for continued culture after the procedure is complete.

[0219] 6.4 Day 12: Add CM2, remove the bag from the CO2 incubator and transfer to a BSC, transfer the CM2 to the BSC, add an amount (e.g., about 0.6L) of CM2 containing IL-15 to the bag as needed, and return the bag to the incubator for continued culture after the procedure is complete.

[0220] 6.5 Day 14: Pre-harvest count, remove the bag from the CO2 incubator and transfer to a BSC, mix the bag, remove 2mL of the culture, and count the cells using a cell counting instrument. Harvest the cells when the cell count is greater than 2E10.

[0221] 7 Cell harvest and cryopreservation, days 14-21

[0222] 7.1 Pre-harvest preparation: Prepare harvest wash solution (e.g., 100 mL wash solution is prepared by mixing 3 mL of commercially purchased human serum albumin and 47 mL of commercially purchased compound electrolyte injection solution, and the potassium ion concentration is 35 mM) and cryopreservation solution (e.g., the cryopreservation solution can contain the following components: CryoStor CS10, human serum albumin, and compound electrolyte injection solution).

[0223] 7.2 Take out the culture bag containing the cell culture from the CO2 incubator, and connect the culture bag and the CytoSorb FlexPro cell harvest kit through a sterile connector;

[0224] 7.3 Use the CytoSorb FlexPro cell harvest automation device to centrifugally collect the cells in the culture bag, and use the CytoSorb FlexPro to wash the cell culture with the wash solution.

[0225] 7.4 Mix the cryopreservation solution and the cells well through the CytoSorb FlexPro device, and sub-pack into cryopreservation bags (100-150 mL per bag for subject infusion, and 10-20 mL per bag for QC detection samples).

[0226] 7.5 Label each cryopreservation bag with product description, name, and date, volume, cell count, and viability.

[0227] 7.6 Place each cryopreservation bag into a pre-cooled aluminum freezing can.

[0228] 7.7 Use the program cooling refrigerator (CRF) to freeze the resulting composition.

[0229] 7.8 Follow the standard procedure of the program cooling refrigerator.

[0230] 7.9 After using the CRF, store the cryopreservation bag in liquid nitrogen (LN2).

[0231] 8 Determine the expected results and detection acceptance criteria.

[0232] The total number of T cells and the percentage of viable cells were monitored during the culturing process and compared with the relevant parameters of the prior art compositions described above. As shown in Figures 1A and 1B, the number of cells obtained and the viability of the cells of the test group were significantly better than the control group. Figure 1A shows that the total number of T cells in the tumor-reactive T cell composition of the present application obtained on day 20 of expansion was about 136,500 times the number of T cells before expansion (test group); whereas the total number of T cells obtained from the prior art composition (i.e., see T cell compositions prepared in Examples 5-10 of US 10894063 B2 and Table 19 using method 2A) under the same expansion conditions was less than 40 times the number of T cells before expansion (control group). Figure 1B shows that the percentage of viable cells in the tumor-reactive T cell composition of the present application obtained on day 14 of expansion was about 85% (test group); whereas the percentage of viable cells obtained from the prior art composition under the same expansion conditions was about 66.5% (control group).

[0233] Example 1.2 Expansion of tumor-reactive T cell compositions of the present application from different types of samples from non-small cell lung cancer patients

[0234] Different types (e.g., pleural effusion, peritoneal effusion, and peripheral blood) of non-small cell lung cancer samples were obtained and the tumor-reactive T cell compositions of the present application were prepared by expansion in multiple batches according to the procedure of Example 1.1 with the initial number of cells inoculated, the total number of cells at the end of the culture, the expansion fold (= total number of cells at the end of the culture / initial number of cells inoculated), and the percentage of viable cells as shown in Table 1 below:

[0235] Table 1. Tumor-reactive T cell compositions of the present application obtained from multiple batches under different culture conditions

[0236] Example 1.3 Expansion of tumor-reactive T cell compositions of the present application from other solid tumor tissues

[0237] Different types (e.g., pleural effusion, peritoneal effusion, and peripheral blood) of non-small cell lung cancer samples were obtained and the tumor-reactive T cell compositions of the present application were prepared by expansion in multiple batches according to the procedure of Example 1.1 with the initial number of cells inoculated, the total number of cells at the end of the culture, the expansion fold (= total number of cells at the end of the culture / initial number of cells inoculated), and the percentage of viable cells as shown in Table 1 below:

[0238] Table 2. Tumor-reactive T cell compositions of the present application obtained from multiple batches under different culture conditions

[0239] Example 2. Cell phenotyping of tumor-reactive T cell compositions of the present application

[0240] In this example, flow cytometry was used to detect and identify the phenotype of the tumor reactive T cell compositions of the application (e.g., the tumor reactive T cell compositions obtained from the multiple lots of Example 1.1 and 1.3).

[0241] 1. Prepare wash solution: PBS containing 0.5% (v / v) human blood albumin.

[0242] 2. Prepare antibody mixture: ① Prepare Zombie NIR™ staining solution according to the number of staining. 0.1 μL of Zombie NIR™ staining solution was added to 100 μL of PBS. Each sample required 100 μL of Zombie NIR™ staining solution. ② Calculate the total volume of antibody mixture required for each sample. Dilute each antibody and fluorescent dye corresponding to the staining group with wash solution at a ratio of 1:1000.

[0243] 3. Cell fluorescent staining

[0244] 3.1 Sampling: Transfer the stained samples and negative control group to a 96-well plate, centrifuge (1500 rpm, 3 minutes), and aspirate the supernatant. Add 100 μL / well of wash solution without antibody or dye to the negative control group to resuspend the cells.

[0245] 3.2 Zombie staining: Add 100 μL / well of prepared Zombie NIR™ staining solution to the cell pellet, mix well by blowing and sucking, and incubate at room temperature for 10-20 minutes in the dark. Add 100 μL / well of PBS to the incubated cells, mix well, centrifuge (1500 rpm, 3 minutes), and aspirate all the supernatant.

[0246] 3.3 Incubation: Add 100 μL / well of prepared antibody mixture to the cell pellet, mix well by blowing and sucking, and incubate at room temperature for 20 minutes in the dark.

[0247] 3.4 Washing: Add 100 μL / well of wash solution to the incubated cells, mix well, centrifuge (1500 rpm, 3 minutes), and aspirate 150 μL / well of supernatant. Resuspend, add 150 μL / well of wash solution, mix well by blowing and sucking, and seal the film.

[0248] 4. Prepare for machine detection. If immediate detection is not possible, store at 4°C in the dark for 0-24 hours before machine detection.

[0249] The results of detecting the proportion of CD45RA+CD62L+ cells in the tumor-reactive T-cell compositions of the present invention (e.g., multiple batches of tumor-reactive T-cell compositions obtained in Examples 1.1 and 1.3) are shown in Figures 2A-2E. As shown in Figures 2A-2C, the proportion of CD45RA+CD62L+ cells in the test groups of multiple batches of Example 1.1 was significantly higher than that in the control group (approximately 2.0-3.7 times that of the control group). As shown in Figures 2D-2E, the proportion of CD45RA+CD62L+ cells in the test group of Example 1.3 was higher than 35% or 40%. Furthermore, as shown in Figure 3, the average proportions of CD45+ cells and CD45+CD3+ cells in the tumor-reactive T-cell compositions of the present invention obtained in multiple batches of Examples 1.1 and 1.3 exceeded 99.5% and 97.8%, respectively.

[0250] Example 3. Characterization of the tumor-responsive T cell composition of the present invention by stimulation test

[0251] In this embodiment, a stimulation test was performed on the tumor-reactive T-cell composition of the present invention (e.g., multiple batches of the tumor-reactive T-cell composition obtained in Examples 1.1 and 1.3). The stimulation test included resuscitating tumor cells, washing cells, collecting samples, and flow cytometry analysis. The conventional culture medium used in this embodiment was derived from one or more of the following: MEM, DMEM, IMDM, RPMI-1640, etc. Xpander, X-VIVO, and KBM581. The T-cell stimulation culture medium of the present invention may contain 0.01-10 ng / mL OKT3 in addition to conventional culture media in the art.

[0252] 1. MG gel preparation: 1640 basal medium + MG (from Corning, catalog number 354277) (volume ratio 50:1). Lay the MG gel in 96-well plates according to the plate laying instructions, 40 μL / well. After laying, transfer to 4°C, then transfer to a 37°C incubator 30 minutes before use.

[0253] 2. Resuscitate tumor cells.

[0254] Before inoculation, centrifuge the required number of cells (1500 rpm, 3 minutes) and then use ImmunoCult. TM Resuspend XF T Cell Expansion Medium and prepare for inoculation. After incubating the MG gel at 37°C for 30 minutes, discard all liquid in the 96-well plate. Inoculate the T+TUM, T+TUM+HLA, TUM, and TUM+HLA groups with 1x10 tumor cells according to the plate placement requirements. 4Cells: 5 x 10 cells / 100 μL / well, HLA concentration: 40 μg / mL. Among them: T represents the tumor-reactive T cell composition of the application; TUM represents the primary tumor cells; HLA represents the human leukocyte antigen (HLA) antibody (Biolegend, item number 311447 or 361702).

[0255] 3. T+HLA group and T group only added corresponding 1640 basic culture solution + HLA (40 μg / mL) / 1640 basic culture solution 100 μL / well. Finally transferred to the incubator, and after 4 hours, T cells were added to start killing.

[0256] 4. Killing T cells, according to the T cell count results on the 14th day, the required cell suspension volume of each culture condition was calculated (according to 10% error), and the cells were collected into centrifuge tubes according to the calculation results, and centrifuged (1500 rpm, 3 minutes).

[0257] 5. Discard all supernatant, resuspend in 1640 killing culture solution, centrifuge again (1500 rpm, 3 minutes), discard all supernatant, resuspend in 1640 killing culture solution to 5 x 10 4 cells / 100 μL. According to the plate diagram, add T cells to the corresponding position of the 96-well plate inoculated with tumor cells, 5 x 10 4 cells / 100 μL, and the final volume is 200 μL / well. Only tum, tum+HLA group supplemented with 100 μL killing culture solution. Return to the incubator and record the killing start time.

[0258] 6. Killing for about 24 hours, and killing sample collection.

[0259] 6.1 Blow and suck the cells to mix and aspirate all, take part of the cells for multi-tag sequencing under each condition, and the remaining cells are subjected to flow cytometry staining.

[0260] 6.2 Flow cytometry staining of cells after killing

[0261] 6.2.1 Preparation of washing solution: PBS containing 0.5% (v / v) human blood albumin.

[0262] 6.2.2 Preparation of antibody mixture: ① Zombie NIR TM dyeing solution: configure Zombie NIR TM dyeing solution according to the number of staining, 0.1 μL added to 100 μL PBS, 100 μL Zombie NIR TM dyeing solution required for each sample. ② Calculate the total volume of antibody mixture (including CD3, CD4, CD8, CD137 antibodies) required for each sample. Dilute each staining group corresponding antibody and fluorescent dye with washing solution at 1:1000.

[0263] 6.2.3 Cell fluorescence staining

[0264] (1) Sampling: Transfer the stained sample and negative control group to a 96-well plate, centrifuge (1500 rpm, 3 minutes), and discard the supernatant. Add 100 μL / well of the washing solution without antibody or dye to the negative control group to resuspend the cells.

[0265] (2) Zombie staining: Add 100 μL / well of the prepared Zombie NIR TM dye solution to the cell precipitate, mix by blowing and sucking, incubate at room temperature for 10-20 minutes in the dark, add 100 μL / well of PBS to the incubated cells, mix, centrifuge (1500 rpm, 3 minutes), and discard all the supernatant.

[0266] (3) Incubation: Add 100 μL / well of the prepared antibody mixture to the cell precipitate, mix by blowing and sucking, and incubate at room temperature for 20 minutes in the dark.

[0267] (4) Washing: Add 100 μL / well of the washing solution to the incubated cells, mix, centrifuge (1500 rpm, 3 minutes), and discard 150 μL / well of the supernatant. Resuspend, add 150 μL / well of the washing solution, mix by blowing and sucking, and seal the film.

[0268] 6.2.4 Prepare for detection on the machine. If immediate detection is not possible, store at 4°C in the dark for 0-24 hours before detection on the machine.

[0269] The proportion of CD8+CD137+cells to CD45+cells in the tumor-reactive T cell composition of the present application (e.g., the tumor-reactive T cell composition obtained in Example 1.1 and Example 1.3) was determined by flow cytometry, and the results are shown in FIGS. 4A-4E. As shown in FIGS. 4A-4D, the proportion of CD8+CD137+cells to CD45+cells in the test group obtained in Example 1.1 was significantly higher than that in the control group (about 10.4-15.9 times that of the control group) after stimulation by tumor cell antigens. As shown in FIG. 4E, the proportion of CD8+CD137+cells to CD45+cells in the test group obtained in Example 1.3 (mesothelioma) was higher than 35% after stimulation by tumor cell antigens.

[0270] Example 4. Killing test of the tumor-reactive T cell composition of the present application

[0271] In this example, a killing test was performed on the tumor-reactive T cell composition of the present application (e.g., the tumor-reactive T cell composition obtained in Example 1.1), which included ELISA, sample collection, and flow detection.

[0272] 1. MG gel configuration: 1640 base medium + MG (volume ratio 50:1). 96-well plate MG gel laying according to the layout requirements, 40 μL / well, after laying, transfer into 4℃, 30 minutes before use, transfer into 37℃ incubator for standby.

[0273] 2. Resuscitate tumor cells, after resuscitation, culture with 1640 base medium for 0-2 days, then inoculate.

[0274] Before inoculation, centrifuge the required cells (1500 rpm, 3 minutes), resuspend with ImmunoCult TM XF T Cell Expansion Medium, prepare for inoculation. After placing the MG gel plate in the 37℃ incubator for 30 minutes, discard all the liquid in the 96-well plate, inoculate tumor cells 1x10 4 cells / 100 μL / well into the T+TUM, T+TUM+HLA, TUM, TUM+HLA groups according to the layout requirements. The HLA concentration is 40 μg / mL. Among them: T represents the tumor-reactive T cell composition of the application; TUM represents primary tumor cells; HLA represents HLA antibody.

[0275] 3. Add corresponding 1640 base medium + HLA (40 μg / mL) / 1640 base medium 100 μL / well to the T+HLA group and the T only group, then transfer into the incubator for incubation for 4 hours.

[0276] 4. Add the tumor-reactive T cell composition obtained in Example 1.1 for killing, according to the T cell count results on the 14th day, calculate the required cell suspension volume for each culture condition (according to 10% error amount), collect the cells into centrifuge tubes according to the calculation results, centrifuge (1500 rpm, 3 minutes).

[0277] 5. Discard all supernatant, resuspend in 1640 killing medium, centrifuge again (1500 rpm, 3 minutes), discard all supernatant, resuspend in 1640 killing medium to 5x10 4 cells / 100 μL. According to the layout diagram, add T cells to the corresponding positions of the 96-well plate inoculated with tumor cells, 5x10 4 cells / 100 μL, the final volume is 200 μL / well. Add 100 μL of killing medium to the tum and tum+HLA groups. Return to the incubator and record the killing start time.

[0278] 6. Killing sampling and detection

[0279] 6.1 Collect 80 μL of supernatant for ELISA detection after about 24 hours of killing. Blow and suck the cells to mix and suck them all out, take part of the cells for multi-tag sequencing for each condition, and the remaining for flow cytometry staining.

[0280] 6.2 Flow cytometry staining after killing

[0281] 6.2.1 Prepare wash solution: PBS containing 0.5% (v / v) human blood albumin.

[0282] 6.2.2 Prepare antibody mixture: ① Zombie NIR™ staining solution: prepare Zombie NIR™ staining solution according to the number of staining, 0.1 μL to 100 μL PBS, each sample needs 100 μL Zombie NIR™ staining solution. ② Calculate the total volume of human leukocyte antigen (HLA) antibody (Biolegend, item number 311447 or 361702) mixture required for each sample. Dilute each corresponding antibody and fluorescent dye in the staining group with wash solution at 1:1000.

[0283] 6.2.3 Cell fluorescent staining

[0284] (1) Sampling: transfer the staining samples and negative control group to a 96-well plate, centrifuge (1500 rpm, 3 minutes), aspirate the supernatant, and resuspend the cells by adding 100 μL / well of wash solution without antibody or dye to the negative control group.

[0285] (2) Zombie staining: add 100 μL / well of prepared Zombie NIR™ staining solution to the cell pellet, mix well by blowing and sucking, incubate at room temperature for 10-20 minutes in the dark, add 100 μL / well of PBS to the incubated cells, mix well, centrifuge (1500 rpm, 3 minutes), and aspirate all the supernatant.

[0286] (3) Incubation: add 100 μL / well of prepared antibody mixture to the cell pellet, mix well by blowing and sucking, and incubate at room temperature for 20 minutes in the dark.

[0287] (4) Washing: add 100 μL / well of wash solution to the incubated cells, mix well, centrifuge (1500 rpm, 3 minutes), and aspirate 150 μL / well of supernatant. Resuspend, add 150 μL / well of wash solution, mix well by blowing and sucking, and seal the film.

[0288] 6.2.4 Prepare for machine detection. If immediate detection is not possible, store at 4°C in the dark for 0-24 hours before machine detection.

[0289] The tumor killing profiles of the tumor reactive T cell compositions of the present application (e.g., tumor reactive T cell compositions obtained in Example 1.1) and prior art compositions at day 18 are shown in FIG. 5A as concentration of IFN-γ (pg / mL). The tumor killing profiles of the tumor reactive T cell compositions of the present application obtained from different batches are shown in FIG. 5B as concentration of IFN-γ (pg / mL). As shown in FIGS. 5A and 5B, the concentration of IFN-γ of the test groups is significantly higher than that of the control groups, and the concentration of IFN-γ of individual batches of the test groups can reach 4500 pg / mL.

[0290] Example 5. PK test in mice

[0291] In this example, experimental data of PK of the tumor reactive T cell compositions of the present application (e.g., tumor reactive T cell compositions obtained in Example 1.1) in mice were measured to observe the metabolism of the tumor reactive T cell compositions in vivo. The tumor reactive T cell compositions (indicated by CD3+ cells and CD45+ cells) were measured at different time points to determine the proportion of the total CD3+ cells and total CD45+ cells in the mice (subjects), respectively, and the specific steps are as follows:

[0292] 1. Prepare a cell suspension of the tumor reactive T cell composition: Take an appropriate amount of T cells from the tumor reactive T cell composition obtained after culture in the manner described in Example 1.1 into a new 50 ml centrifuge tube, centrifuge at 1500 rpm at room temperature for 3 minutes, and discard the supernatant. Then add 10 ml of washing solution (normal saline + 0.5% human blood albumin), mix the cells by blowing and sucking, centrifuge at 1500 rpm at room temperature for 3 minutes, discard the supernatant, and after adding resuspension solution (normal saline + 0.5% human blood albumin), move the resuspended cells to a new 1.5 ml centrifuge tube, and label it. Store at 2-8°C temporarily;

[0293] 2. Mouse injection: Use 6-8 week old female mice, inject the resuspended cell composition obtained in the first step and the control group (prior art composition or solvent control in Example 1) through the tail vein injection method, and give human IL2 injection intraperitoneally every day for 16 consecutive days, once a day;

[0294] 3. Sampling: After the completion of step 2 above, sample the tail tip blood of the mice at the corresponding time points (3, 4, 7, 14, 28, 35, 42, and 63 days);

[0295] 4. Fluorescent antibody staining and flow cytometry: After the completion of step 3 above, fluorescent antibody staining was performed on each sample collected, and then flow cytometry was performed. The specific steps of fluorescent antibody staining and flow cytometry were as follows: about 5 times the volume of fresh 1x red blood cell lysis solution was added to the sample collected in step 3, and the sample was allowed to stand at room temperature for about 5-10 minutes. After the red blood cells were lysed, an equal volume of PBS was added to terminate the lysis. The sample was centrifuged to discard the supernatant, and an antibody mixture was added to the sample and mixed well. The sample was incubated at room temperature for about 20 minutes in the dark, and then PBS was added and mixed well. The sample was centrifuged to discard the supernatant. PBS was added to resuspend the cells, and flow cytometry (Cytoflex, Beckman) was used to detect the cells. The sample was placed in the sample injector, and the record or automatic record was started. The proportion of CD45+ cells from the composition to the total CD45+ cells in the mouse (as shown in FIG. 6A) and the proportion of CD3+ cells from the composition to the total CD3+ cells in the mouse (as shown in FIG. 6B) were analyzed. As shown in FIG. 6A, the proportion of the tumor-reactive T cell composition obtained by the method of the present application in the body was at least 2 times the proportion of the control group from the 4th day. This indicates that the amount and lifespan of the tumor-reactive T cell composition of the present application in the body are significantly higher than those of the control group.

[0296] Example 6. PK test in humans

[0297] In this example, the PK data in humans was measured to observe the persistence of the tumor-reactive T cell composition of the present application (e.g., the tumor-reactive T cell composition obtained in Example 1.1) in the human body.

[0298] (1) Determining the absolute count of CD3+ or CD3+CD8+ in the peripheral blood of a human subject

[0299] 1. Calculate and record the number of cells of the tumor-reactive T cell composition required, and transfer to a new 50 ml centrifuge tube. Centrifuge at 1500 rpm and room temperature for 3 minutes, and discard the supernatant.

[0300] 2. Add 10 ml of washing solution (normal saline + 0.5% human blood albumin), gently blow and suck to mix the cells, centrifuge at 1500 rpm and room temperature for 3 minutes, and discard the supernatant.

[0301] 3. Repeat the washing once.

[0302] 4. Calculate and record the volume of resuspension solution required for injection into the human body, and add the resuspension solution (normal saline + 0.5% human blood albumin). Resuspend the washed tumor-reactive T cell composition, transfer the resuspended cells to a new 1.5 ml centrifuge tube, and label it. Store at 2-8°C.

[0303] 5. Inject the cells into the human subject.

[0304] 6. Blood is collected from the human subject and subjected to flow cytometry experiments.

[0305] The absolute counts of CD3+ or CD3+CD8+ in the peripheral blood of the human subject after 14 days of administration of the tumor-reactive T cell composition of the present application (e.g., the tumor-reactive T cell composition obtained in Example 1.1) are shown in Figure 7. As can be seen from the figure, the expansion ability and persistence of the tumor-reactive T cell composition obtained by expansion after administration to the human subject are significantly enhanced.

[0306] (2) Determining the proportion of the number of TCR species from the composition in the human subject to the total number of TCR species in the subject

[0307] 1. Sampling: tumor tissue / thoracic fluid samples are collected from human patients, and tumor-reactive T cell compositions are obtained after culture in the manner described in Example 1.1 (testing group). After the patient is cleared of lymph (cyclophosphamide combined with fludarabine), the tumor-reactive T cell compositions are reinfused into the patient, and the patient is tracked and sampled at the corresponding time points. The sampling time points are: QLQ (the day before the tumor-reactive T cell compositions are reinfused, the baseline point), after the tumor-reactive T cell compositions are reinfused (7th, 14th, 28th, and 56th days);

[0308] 2. The samples extracted at each sampling point need to be subjected to lysis-RNA extraction-library preparation-machine sequencing to obtain TCR-seq data. The TCR sequencing process can be performed according to the manual of any commercially available TCR product;

[0309] 3. Analysis process of TCR-seq data:

[0310] a) Check the TCR-seq data and perform quality control: use FastQC (version 0.11.9) to check the quality of the sequencing data, and use Trimmomatic software (version 0.39) to remove the adapter sequences of the sequencing data;

[0311] b) Use MiXCR software (version 3.0.13) to obtain TCR data information in each sample from the quality-controlled data;

[0312] c) Read the TCR data information using R package immunarch software (version 0.9.0), and divide into 4 parts according to whether all TCRs exist in the testing group or the benchmark point (QLQ) group: testing_QLQ (TCR species contained in both the testing group and the benchmark point group), QLQ (TCR species contained only in the benchmark point group), testing (TCR species contained only in the testing group), and other (TCR species from sources other than testing, testing_QLQ). Calculate the TCR clone ratio of each part and visualize the results by ggplot2 software (Elegant Graphics for Data Analysis, version 3.4.3).

[0313] The determination results are shown in Figure 8. The proportion of TCR species (testing + testing_QLQ) from the tumor-reactive T cell composition of the method of the application (for example, Example 1.1) to the total number of TCR species in the subject is higher than 70% after 7 days, 14 days, 28 days and 56 days of administration to a human subject, respectively.

[0314] Example 7. Continuous killing test

[0315] In this example, the tumor-reactive T cell composition of the application is used to perform multiple continuous killing of advanced solid tumors (for example, mesothelioma) in vitro, and the proportion of tumor cells killed each time is observed.

[0316] 1. Mesothelioma tumor cells (referred to as "tumor cells" in this example) are inoculated into a 96-well plate, 1e4 tumor cells per well;

[0317] 2. After 24 hours of tumor cell inoculation, the tumor cell culture solution is removed, and the tumor-reactive T cell composition of the application (for example, the tumor-reactive T cell composition derived from the mesothelioma tumor sample in Example 1.3) is inoculated into the tumor cell culture well, 5e4 cells per well, and co-cultured with the tumor cells. The control group is the composition of the prior art (see the related description in Example 1);

[0318] 3. After co-culturing with tumor cells every 24 hours, the tumor-reactive T cell composition is gently aspirated into a newly inoculated tumor cell culture well, and continuous killing experiments are continued.

[0319] 4. After removing the co-cultured tumor-reactive T cell composition, the remaining tumor cells are lysed for live cell detection. Compared with the tumor cell control group without T cells, the proportion of tumor cells killed in co-culture (tumor cell lysis %) is calculated.

[0320] The amplified tumor reactive T cell composition of the present application was subjected to a serial killing test in mice, and the results are shown in Figure 9. The amplified tumor reactive T cell composition of the present application maintained more than 80% tumor cell lysis after 10 serial killings of tumor cells in vitro (test group); while the prior art composition had almost 0% tumor cell lysis after 6 serial killings of tumor cells (control group).

[0321] The above description is only preferred embodiments of the present application, and does not limit the present application in any form. Although the specific embodiments of the present application are described above, those skilled in the art should understand that this is only an illustration, and the protection scope of the present application is defined by the appended claims. Those skilled in the art can make various changes or modifications to the disclosed technology without departing from the scope of the technical solutions of the present application, and these changes and modifications all fall within the protection scope of the present application.

Claims

1. An expanded tumor reactive T cell composition, wherein: (a) the composition has a proportion of CD8+CD137+ cells to CD45+ cells greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, or greater than 60% after stimulation with tumor cell antigens; and / or (b) the composition has a proportion of CD45RA+CD62L+ cells greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, greater than 60%, or greater than 70%; and / or (c) the composition has an increase in expression of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1, and KLF7 greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, or greater than 60% compared to before expansion and / or a decrease in expression of one or more gene markers selected from the group consisting of TBX1, ID2, and HOPX greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, or greater than 60% compared to before expansion.

2. The composition of claim 1, wherein the composition is obtained by expanding a sample from a patient by one or more rounds of expansion.

3. The composition of claim 2, wherein the sample is from a tumor, a lymph node, a pleural effusion, a peritoneal effusion, peripheral blood, or cerebrospinal fluid of a patient, preferably the tumor is a sample selected from the group consisting of a surgically resected tumor sample, a punch biopsy tumor sample, a core biopsy tumor sample, and a small biopsy tumor sample.

4. The composition of any one of claims 1-3, wherein the tumor is a solid tumor, preferably selected from the group consisting of mesothelioma, lung cancer, and glioma, more preferably non-small cell lung cancer.

5. The composition of any one of claims 1-4, wherein the proportion of CD45+ cells in the composition is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

6. The composition of any one of claims 1-5, wherein the proportion of CD45+CD3+ cells in the composition is greater than 90%, greater than 92%, greater than 94%, greater than 96%, or greater than 98%.

7. The composition of any one of claims 1-6, wherein the composition is obtained within 20 days, 19 days, 18 days, 17 days, 16 days, or 15 days after the start of expansion.

8. The composition of any one of claims 1-7, wherein the total number of cells of tumor reactive T cells in the composition is at least 10,000-fold, at least 20,000-fold, at least 50,000-fold, at least 80,000-fold, or at least 100,000-fold greater than prior to expansion, or the total number of cells of tumor reactive T cells per milliliter of the composition is at least 1 x 10 10 , at least 2 x 10 10 , at least 4 x 10 10 , at least 8 x 10 10 , at least 1 x 10 11 , at least 2 x 10 11 , at least 3 x 10 11 , at least 4 x 10 11 , or at least 5 x 10 11 .

9. The composition of any one of claims 1-8, wherein the composition has a percentage of viable tumor reactive T cells greater than 80%, greater than 85%, or greater than 90%.

10. The composition of any one of claims 1-9, wherein the composition is capable of producing 500 pg / mL or more, 1000 pg / mL or more, 2000 pg / mL or more, 3000 pg / mL or more, 4000 pg / mL or more, or 4500 pg / mL or more of interferon-gamma (IFN-γ) upon stimulation with tumor cell antigens.

11. The composition of any one of claims 1-10, wherein the composition has any one or more of the following properties: (i) the proportion of CD45+ cells from the composition to total CD45+ in a subject is higher than 20%, higher than 25%, higher than 30%, or higher than 35% after 7-14 days of administration to the subject, preferably the proportion of CD45+ cells from the composition to total CD45+ in a subject is higher than 10%, higher than 15%, or higher than 20% after 28 days of administration to the subject; (ii) the proportion of CD3+ cells from the composition to total CD3+ in a subject is higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% after 7-14 days of administration to the subject, preferably the proportion of CD3+ cells from the composition to total CD3+ in a subject is higher than 15%, higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% after 28-63 days of administration to the subject; (iii) the proportion of TCR species from the composition to total TCR species in a subject is higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% after 7-14 days of administration to the subject, preferably the proportion of TCR species from the composition to total TCR species in a subject is higher than 20%, higher than 30%, higher than 40%, higher than 50%, or higher than 60% after 28-56 days of administration to the subject; (iv) the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of a subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 1000 cells / µL, higher than 2000 cells / µL, higher than 4000 cells / µL, higher than 6000 cells / µL, higher than 8000 cells / µL, or higher than 10000 cells / µL after 7-14 days of administration to the subject, preferably the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of a subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 500 cells / µL, higher than 1000 cells / µL, higher than 1500 cells / µL, higher than 2000 cells / µL, higher than 2500 cells / µL, higher than 3000 cells / µL, higher than 3500 cells / µL, or higher than 4000 cells / µL after 28-56 days of administration to the subject.

12. The composition of any one of claims 1-11, wherein the composition is capable of at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive rounds of killing tumor cells.

13. The composition of any one of claims 1-12, wherein the composition comprises or does not comprise feeder cells.

14. A method of preparing a tumor reactive T cell composition by expansion, comprising: (1) providing a sample containing tumor reactive T cells; (2) pre-treating the sample; (3) subjecting the pre-treated sample to a first stage of expansion culture in a first medium comprising IL-2, OKT3, and / or a SMO inhibitor; (4) subjecting the product of the first stage of expansion culture to a second stage of expansion culture in a second medium comprising IL-2, IL-7, IL-15, OKT3, and / or a SMO inhibitor; and (5) optionally harvesting the composition.

15. The method of claim 14, wherein the sample containing tumor reactive T cells is a sample from a patient, preferably the sample is from a tumor, lymph node, pleural effusion, peritoneal effusion, peripheral blood, or cerebrospinal fluid of a patient, more preferably the sample from a tumor of a patient is a sample selected from the group consisting of a surgically resected tumor sample, a punch biopsy tumor sample, a core biopsy tumor sample, and a small biopsy tumor sample.

16. The method of claim 15, wherein the tumor is a solid tumor, preferably selected from the group consisting of mesothelioma, lung cancer, and glioma, more preferably non-small cell lung cancer.

17. The method of any one of claims 14-16, wherein the SMO inhibitor is selected from one or more of the group of compounds consisting of BMS-833923 (XL-139), SAG, Cyclopamine, Purmorphamine, Glasdegib, Sonidegib, Taladegib, and Halcinonide.

18. The method of any one of claims 14-17, wherein the pre-treatment comprises one or more manipulations selected from the group consisting of removing tissue, fragmentation, washing, red blood cell lysis, resuspension, and cryopreservation.

19. The method of claim 18, wherein the fragmentation is performed by: (1) grinding the sample; and / or (2) mincing the sample.

20. The method of claim 18 or 19, wherein the washing is performed by centrifugation in 1-50 times the sample volume of a wash solution.

21. The method of claim 20, wherein the wash solution comprises an antibiotic and potassium ions.

22. The method of claim 20 or 21, wherein the centrifugation is performed by centrifugation at 1200-1800 rpm for 2-5 minutes.

23. The method of any one of claims 18-22, wherein the red blood cell lysis is performed by adding 1-50 times the volume of the sample of a red blood cell lysis solution and mixing, inverting every 1-2 minutes for a total of no more than 5 minutes.

24. The method of claim 23, wherein the red blood cell lysis solution comprises a metal ion, preferably a potassium ion, a sodium ion, and / or an ammonium ion.

25. The method of any one of claims 18-24, wherein the resuspension is resuspension of the sample in the first medium.

26. The method of any one of claims 14-25, wherein the cryopreservation is performed after centrifugation of the sample by adding a cryopreservation solution and using a programable cryopreservation instrument.

27. The method of claim 26, wherein the cryopreservation solution comprises: (a) CS10; (b) human blood albumin; and (c) a compound electrolyte injection solution.

28. The method of any one of claims 14-27, wherein the first-stage expansion culture is performed in a culture bag for 4-8 days, preferably the culture bag is placed in a 37 °C, 5% CO2 incubator.

29. The method of any one of claims 14-28, wherein the first medium is supplemented to the culture on days 1-3 of the first-stage expansion culture.

30. The method of any of claims 14-29, wherein following the first-stage expansion culture, the total number of tumor-reactive T cells in the resulting composition is at least 20-fold, at least 40-fold, at least 50-fold, at least 80-fold, or at least 100-fold greater than prior to expansion, or the total number of cells per milliliter of the composition that are tumor-reactive T cells is at least 5 x 10 8 , at least 6 x 10 8 , at least 7 x 10 8 , at least 8 x 10 8 , at least 9 x 10 8 , or at least 1 x 10 9 .

31. The method of any one of claims 14-30, wherein the second-stage expansion culture is performed in a culture bag for 7-10 days, preferably the culture bag is placed in a 37 °C, 5% CO2 incubator.

32. The method of any one of claims 14-31, wherein the second medium is supplemented to the culture on days 2-3, 4-5, and / or 6-7 of the second-stage expansion culture.

33. The method of any of claims 14-32, wherein following the second phase expansion culture, the total number of tumor reactive T cells in the resulting composition is at least 10,000-fold, at least 20,000-fold, at least 50,000-fold, at least 80,000-fold, or at least 100,000-fold greater than prior to expansion, or the total number of cells per milliliter of the composition that are tumor reactive T cells is at least 1 x 10 10 , at least 2 x 10 10 , at least 4 x 10 10 , at least 8 x 10 10 , at least 1 x 10 11 , at least 2 x 10 11 , at least 3 x 10 11 , at least 4 x 10 11 , or at least 5 x 10 11 .

34. The method of any one of claims 14-33, wherein the harvesting of the composition is performed by centrifugation to collect the cells after the second-stage expansion culture.

35. The method of any one of claims 14-34, wherein the harvesting of the composition further optionally comprises washing the composition with a wash solution and / or cryopreserving the composition with a cryopreservation solution.

36. The method of claim 14-35, wherein: (a) the composition has a proportion of CD8+CD137+ cells to CD45+ cells that is greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, or greater than 60% after stimulation with a tumor cell antigen; and / or (b) the composition has a proportion of CD45RA+CD62L+ cells that is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, greater than 60%, or greater than 70%; and / or (c) the amount of expression of one or more gene markers selected from the group consisting of STAB1, HES1, RBPJ, PMEPA1, and KLF7 is increased by greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, or greater than 60% in the composition compared to prior to expansion and / or the amount of expression of one or more gene markers selected from the group consisting of TBX1, ID2, and HOPX is decreased by greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 58%, or greater than 60% in the composition compared to prior to expansion.

37. The method of any one of claims 14-36, wherein the proportion of CD45+ cells in the composition is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

38. The method of any one of claims 14-37, wherein the proportion of CD45+CD3+ cells in the composition is greater than 90%, greater than 92%, greater than 94%, greater than 96%, or greater than 98%.

39. The method of any one of claims 14-38, wherein the composition is obtained within 20 days, within 19 days, within 18 days, within 17 days, within 16 days, or within 15 days after the start of expansion.

40. The method of any of claims 14-39, wherein the total number of cells of tumor reactive T cells in the composition is at least 10,000-fold, at least 20,000-fold, at least 50,000-fold, at least 80,000-fold, or at least 100,000-fold greater than prior to expansion, or the total number of cells of tumor reactive T cells per milliliter of the composition is at least 1 x 10 10 , at least 2 x 10 10 , at least 4 x 10 10 , at least 8 x 10 10 , at least 1 x 10 11 , at least 2 x 10 11 , at least 3 x 10 11 , at least 4 x 10 11 , or at least 5 x 10 11 .

41. The method of any one of claims 14-40, wherein the percentage of viable cells of tumor reactive T cells in the composition is greater than 80%, greater than 85%, or greater than 90%.

42. The method of any one of claims 14-41, wherein the composition is capable of producing greater than 500 pg / mL, greater than 1000 pg / mL, greater than 2000 pg / mL, greater than 3000 pg / mL, greater than 4000 pg / mL, or greater than 4500 pg / mL of interferon gamma (IFN-γ) after stimulation with tumor cell antigens.

43. The method of any one of claims 14-42, wherein the composition has any one or more of the following properties: (i) the proportion of CD45+ cells from the composition in a subject that are total CD45+ in the subject is greater than 20%, greater than 25%, greater than 30%, or greater than 35% after 7-14 days of administration to the subject, preferably the proportion of CD45+ cells from the composition in a subject that are total CD45+ in the subject is greater than 10%, greater than 15%, or greater than 20% after 28 days of administration to the subject; (ii) the proportion of CD3+ cells from the composition in a subject that are total CD3+ in the subject is greater than 20%, greater than 30%, greater than 40%, greater than 50%, or greater than 60% after 7-14 days of administration to the subject, preferably the proportion of CD3+ cells from the composition in a subject that are total CD3+ in the subject is greater than 15%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, or greater than 60% after 28-63 days of administration to the subject; (iii) the proportion of TCR species from the composition to the total number of TCR species in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50% or higher than 60% in the subject after 7-14 days of administration to the subject, preferably the proportion of TCR species from the composition to the total number of TCR species in the subject is higher than 20%, higher than 30%, higher than 40%, higher than 50% or higher than 60% in the subject after 28-56 days of administration to the subject; (iv) the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 1000 cells / μL, higher than 2000 cells / μL, higher than 4000 cells / μL, higher than 6000 cells / μL, higher than 8000 cells / μL or higher than 10000 cells / μL in the subject after 7-14 days of administration to the subject; preferably the absolute count of CD3+ cells or CD3+CD8+ cells in the peripheral blood of the subject or the absolute count of CD3+ cells or CD3+CD8+ cells from the composition is higher than 500 cells / μL, higher than 1000 cells / μL, higher than 1500 cells / μL, higher than 2000 cells / μL, higher than 2500 cells / μL, higher than 3000 cells / μL, higher than 3500 cells / μL or higher than 4000 cells / μL in the subject after 28-56 days of administration to the subject.

44. The method of any one of claims 14-43, wherein the composition is capable of at least 6, at least 8, at least 10 consecutive killing of tumor cells.

45. The method of any one of claims 14-44, wherein the composition comprises or does not comprise feeder cells.

46. A tumor reactive T cell composition prepared by the method of any one of claims 14-45.

Images