A method of preparing tumor antigen-specific t cells

Abstract

The application discloses a method for preparing tumor antigen specific T cells. A tumor antigen peptide is also disclosed, and the amino acid sequence of the tumor antigen peptide is shown as SEQ ID NO:1. Specifically, the method comprises the following steps: co-culturing primary TILs cells with DC cells loaded with antigen peptides; then sorting tumor antigen specific T cells using an antigen specific activation marker to obtain marker positive cells; and expanding the marker positive cells. The method provided by the application can provide a stronger co-stimulatory signal to activate tumor antigen specific T cells in TILs, avoid losing effective cells with anti-tumor functions, and be suitable for various tumor specific antigens, and thus has a wide application range. The end product obtained by using the method provided by the application has stronger tumor killing ability.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing tumor antigen-specific T cells. Background Technology

[0002] Adoptive cell therapy (ACT) refers to the treatment of tumors by infusing cancer patients with immune cells that have anti-tumor activity, which directly kill or stimulate the body's immune response to kill tumor cells.

[0003] Tumor tissue often contains tumor-infiltrating lymphocytes (TILs) surrounding and within the tumor, which include a large number of tumor antigen-specific T cells. Rosenberg's team was the first to use TILs for ACT therapy, and their research showed that under the influence of IL-2, TILs in tumor tissue can proliferate efficiently while maintaining their ability to kill autologous tumors.

[0004] The cellular composition and phenotype of tumor-responsive ILs (TILs) products are significantly correlated with clinical treatment efficacy. TILs obtained directly from tumor tissue have a complex cell composition, containing not only tumor-responsive T cells but also a large number of bystander cells that lack anti-tumor activity. Enriching and culturing tumor-responsive TILs can enhance their anti-tumor function and reduce the amount of cells required for reinfusion.

[0005] Some techniques utilize the phenomenon that tumor-responsive T cells are more abundant in T cell populations positive for molecular markers such as PD-1, LAG-3, and TIM-3 than in negative populations, to sort and culture these marker-positive TILs. However, the antigen-responsive T cell content in the PD-1-positive T cell population obtained by this method is only relatively increased; the tumor-responsive T cell content remains unclear.

[0006] Tumor antigens that can serve as targets for immunotherapy can include tumor-associated virus-derived antigens (TAVs), tumor-associated antigens (TAAs), cancer-testis antigens (CTAs), and tumor neoantigens (TNFs). One study reported a patient who did not respond to bulk TILs (unenriched TILs). Subsequent enrichment culture of ERBB2IP gene-mutant responsive CD4 cells from the TILs yielded a final product with a mutation-responsive T cell content as high as 95%. After reinfusion of the enriched TILs, rapid shrinkage of liver and lung metastases was observed. This study demonstrates that enriching and culturing tumor antigen-responsive TILs can effectively enhance the anti-tumor ability of TILs. Therefore, isolating and enriching tumor antigen-specific T cells from TILs is beneficial for obtaining cell products with stronger anti-tumor functions.

[0007] Common strategies for isolating tumor antigen-specific T cells can be divided into two categories based on whether prior stimulation of antigen-specific T cells is required: (1) direct isolation without stimulation, which mainly uses peptide-MHC polymeric complexes to sort and enrich antigen-specific T cells; (2) specific stimulation using antigens, followed by capture of specific T cell populations using activation markers regulated on the cell surface. However, the preparation of peptide-MHC polymeric complexes is time-consuming and costly, and only specific MHC subtypes have commercially available reagents, which is not conducive to large-scale application. Furthermore, numerous studies have shown that tumor antigen-specific CD4 cells in TILs also play an important role in anti-tumor therapy. The preparation of peptide-MHC polymers of MHC class II molecules is relatively difficult, making it challenging to perform tumor antigen-specific CD4 cell sorting using this method (MRParkhurst et al. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers, CancerDiscov, 9(2019)1022-1035. http: / / doi.org / 10.1158 / 2159-8290.CD-18-1494).

[0008] Following antigen stimulation, specific cell populations are isolated and enriched using surface activation markers. This method has a wider range of applications; however, the choice of stimulant, the stimulation time, and the further amplification and culture of the sorted products all significantly affect the effectiveness of the final product. Dendritic cells (DCs) are currently the most potent antigen-presenting cells known and can effectively activate antigen-presenting cells. CD137 molecules respond to antigen stimulation and are expressed on the surface of CD8 and CD4 cells, making them an effective cell surface marker for antigen-specific CD8 and CD4 expression. Based on research, the following relatively mature preparation processes exist:

[0009] Option 1: Jianjian Jin et al. (J. Jin et al. Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment, J Immunother, 35(2012)283-292. http: / / doi.org / 10.1097 / CJI.0b013e31824e801f) published a method for preparing TILs. This method directly uses unsorted TILs obtained from preliminary culture from tumor tissue to expand in large quantities to obtain the final product. The resulting final product contains a large number of non-tumor responsive T cells, and may even lack tumor-specific T cells, making the content of its effective components uncontrollable.

[0010] Option 2: Patent publication number CN104946589A discloses a method for isolating and culturing tumor-specific TILs. This method utilizes the phenomenon that the number of tumor-responsive T cells in T cell populations positive for molecular markers such as PD-1, LAG-3, and TIM-3 is higher than that in negative populations, and sorts and cultures these marker-positive TILs. However, the antigen-responsive T cell content in the PD-1 positive T cell population obtained by this method is only relatively increased; the tumor-responsive T cell content remains unclear.

[0011] Option 3: Mark E. Dudley et al. (MEDudley et al. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients, J Immunother, 26(2003)332-342. http: / / doi.org / 10.1097 / 00002371-200307000-00005) used a method for selecting tumor-specific TILs for expansion culture. This method utilizes autologous tumor cells co-cultured with various TIL cultures, selecting positive reaction wells for further expansion. The positive wells selected in this method only contain tumor-responsive T cells, the specific proportion of which is unknown, and the success rate of autologous tumor extracellular culture is uncontrollable, with high limiting factors for different cancer types.

[0012] Option 4: Patent CN113106062A discloses a method for co-culturing tumor antigen-specific tumor-infiltrating lymphocytes. This method involves whole-exome sequencing of normal and tumor tissues, comparing the two sets of sequencing data to determine amino acid mutations, and then synthesizing five tumor-specific antigen peptides N1-N5. The tumor tissue is then digested, and the lymphocyte (TILs) layer and tumor cell layer are collected using density gradient centrifugation. This method has two main problems: firstly, the method directly uses antigen peptides to activate specific cells in TILs, resulting in limited activation efficiency; furthermore, the activated TILs are prone to apoptosis and terminal differentiation due to prolonged antigen contact; secondly, while neoantigen-specific T cells proliferate relatively predominantly, other non-specific T cells also proliferate significantly, resulting in a limited content of neoantigen-specific TILs in the final product.

[0013] Option 5: Patent publication number CN113272419A discloses a method for preparing therapeutic T lymphocytes. This method uses a tandem gene expressing tumor neoantigens (TMG) to transfect antigen-presenting cells (APCs). The transfected antigen-presenting cells stimulate TILs, and T cell surface activation markers are used to isolate neoantigen-specific TILs. Rapid expansion is then performed to obtain the final product. However, the construction of the TMG gene and the APC cell gene transfection process are complex and time-consuming, making large-scale production difficult.

[0014] Option 6: Patent CN109988748A discloses a method for screening tumor-specific T cells from TILs. This method uses MHC-peptide complexes as a method for isolating and enriching tumor neoantigens from TILs. However, the success rate of MHC-peptide complex preparation is highly correlated with the affinity of the peptides, making it more suitable for fixed peptides. In actual clinical applications, tumor neoantigens vary greatly among individuals and are often unique to each patient, making it difficult to prepare them in advance and ensure their effectiveness. Currently, only a few MHC I molecule subtypes have commercially available kits for preparing tetramers, limiting the applicable population. Furthermore, the preparation of MHC-peptide complexes for MHC II subtypes is technically difficult and rarely successful. Therefore, MHC-peptide complexes can only achieve the isolation and enrichment of CD8 cells in individuals with specific MHC subtypes, and cannot achieve the isolation and enrichment of tumor neoantigen-specific CD4 cells. Therefore, the large-scale clinical application of the MHC-peptide complex multimer method is significantly limited. Summary of the Invention

[0015] To address the shortcomings of the aforementioned technical processes, where the selection of stimulants, the duration of stimulant application, and further amplification and culture of sorted products can significantly impact the effectiveness of the final product, this invention provides a method for preparing GMP-grade tumor antigen-specific TILs (tumor-infiltrating lymphocytes). The method of this invention significantly improves the yield and proportion of tumor antigen-specific T cells in the final T cell product, and simultaneously obtains tumor antigen-specific CD4 and CD8 cells, maximizing the diversity of tumor-responsive T cells. Furthermore, the overall culture time of this invention does not exceed 5 weeks, the amplification process uses a low dose of IL-2, and the obtained TILs are young TILs, exhibiting better proliferative capacity and longer persistence in patients, thus enhancing their anti-tumor capabilities.

[0016] This invention provides a highly efficient protocol for isolating and expanding tumor antigen-specific TILs applicable to both CD8 and CD4 cells, to obtain TILs therapeutic products with a high proportion of anti-tumor active T cells. The protocol first co-cultures dendritic cells (DCs) with synthetically produced tumor antigen peptides, loading the DCs with these peptides to prepare activated, mature DCs. These activated mature DCs are then co-cultured with pre-expanded TILs cells. Tumor antigen-specific T cells are isolated using an activated surface marker (4-1BB / CD137), and then the T cells are rapidly expanded using feeder cells, an anti-CD3 monoclonal antibody, and IL-2.

[0017] The key technical point of this invention lies in stimulating initially cultured TILs cells with DC cells loaded with tumor antigen peptides, followed by sorting tumor antigen-specific TILs using CD137, an antigen-specific activation marker, with a culture time not exceeding 5 weeks. Using a combination of cytokines to amplify young TILs, this method significantly increases the number and proportion of tumor antigen-specific T cells in the final TIL product, enabling the production of therapeutically viable young TILs within 5 weeks. This makes them more suitable for clinical use, exhibiting strong in vivo proliferation and specific anti-tumor effects. The culture process is simple, widely applicable, and easy to scale up for production.

[0018] To address the problems of the prior art, the first aspect of the present invention provides a tumor antigen peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0019] A second aspect of the present invention provides a method for preparing tumor antigen-specific T cells, the method comprising the following steps:

[0020] A) Co-cultured cells were obtained by co-culturing DC cells loaded with tumor antigen peptides with primary TILs cells;

[0021] B) Subsequently, tumor antigen-specific T cells were sorted using antigen-specific activation markers to obtain marker-positive cells; and

[0022] C) Amplify the marker-positive cells.

[0023] In some embodiments, the marker is CD137, CD107a, or CD134, for example, CD137.

[0024] In some embodiments, the preparation process of the DC cells includes the following steps:

[0025] (1) After filtering and centrifuging the suspension of peripheral blood mononuclear cells, CD14 magnetic beads were added and incubated. After sorting, CD14 positive cells were obtained.

[0026] (2) The CD14 positive cells were resuspended in DC cell culture medium, and then differentiated by adding directed differentiation factor and maturation factor.

[0027] (3) Add the tumor antigen peptide as described in the first aspect of the present invention for co-incubation to obtain DC cells loaded with tumor antigen peptide.

[0028] In some embodiments, in step (1), the peripheral blood mononuclear cells are derived from blood cells of a tumor patient; and / or,

[0029] The filter size is 35-45 μm, for example, 40 μm; and / or,

[0030] The centrifugation conditions are 300-500g for 5-10 minutes, for example, 500g for 10 minutes; and / or,

[0031] The incubation temperature is 2-4℃, for example, 4℃;

[0032] In step (2), the DC cell culture medium is a DC cell culture medium containing autologous serum or HSA (human serum albumin); and / or,

[0033] The directed differentiation factor is one or more of rhGM-CSF (granulocyte-macrophage colony-stimulating factor) and rhIL-4; and / or,

[0034] The maturation factors are one or more of rhGM-CSF, rhIL-4, rhIL-1β, rhIL-6, rhTNF-α, PGE-2, and Poly I:C; and / or,

[0035] The differentiation process lasts 3-5 days; and / or,

[0036] The culture period is 1-6 days;

[0037] In step (3), the co-incubation period is 6 days. In this article, "rh" represents "recombinant human", for example, rhGM-CSF is recombinant human granulocyte-macrophage colony-stimulating factor.

[0038] In some embodiments, the preparation process of the primary TILs cells includes:

[0039] (a) Digesting tumor tissue to obtain a single-cell suspension; the tumor tissue, for example, is derived from cervical cancer;

[0040] (b) The single-cell suspension was mixed with HIPP medium A containing 1-5% autologous plasma or serum substitute and IL-2 at a concentration of 500-6000 IU / mL;

[0041] (c) Change the medium using the HIPP medium A, and then culture to obtain primary TILs cells.

[0042] The digestion can be performed using the gentleMACS Dissociator and a tumor tissue dissociation kit. The aforementioned tumor tissue dissociation kit can be any tumor tissue dissociation kit commonly used in the art.

[0043] The gentleMACS Dissociator and tumor tissue dissociation kit described above can digest different types of tumor tissue into single cells according to standardized operations and procedures.

[0044] In some embodiments, in step C), the HIPP medium A is a HIPP medium containing 500 IU / mL IL-2 and 2% autologous plasma or serum substitute; and / or,

[0045] The fluid replacement is a half-volume replacement; and / or,

[0046] The fluid change frequency is once every 2-3 days; and / or,

[0047] The total duration of the re-culturing is 1-3 weeks, for example, 2 weeks.

[0048] In some embodiments, the method includes the following steps:

[0049] In step A), the DC cells and the primary TILs cells are mixed at a ratio of 1:4 to 1:20, resuspended in HIPP medium B containing 1-5% HSA, and co-cultured with tumor antigen peptides and rhIL-21 as described in the first aspect of the present invention, and the co-cultured cells are collected.

[0050] In step B), tumor antigen-specific T cells are separated using a CD137-positive cell sorting reagent to obtain marker-positive cells.

[0051] In some embodiments, in step A), the ratio is 1:10; and / or,

[0052] The HIPP medium B is a HIPP medium containing 2.5% HSA; and / or,

[0053] The final concentration of the tumor antigen peptide is 1-20 μg / mL, preferably 10 μg / mL; and / or,

[0054] The final concentration of rhIL-21 is 30 ng / mL; and / or,

[0055] The co-culture duration is 12-48 hours, preferably 18 hours.

[0056] In some embodiments, step C) includes one or more of the following conditions:

[0057] (i) Irradiated allogeneic PBMC cells and TILs cells were mixed at a ratio of 1:20 to 1:100 to obtain mixed cells;

[0058] (ii) The mixed cells were resuspended in HIPP medium 1 containing 1-5% autologous plasma or serum substitutes and cultured with one or more of anti-CD3 antibody, anti-CD28 antibody, rhIL-2, rhIL-7 and rhIL-15.

[0059] (iii) Change the medium using HIPP medium 2 containing 1-5% autologous plasma or serum substitutes, for example, by half-volume replacement;

[0060] (iv) Continue culturing at a rate of 0.5-2×10⁻⁶. 6 Cells were resuspended at a density of 1-5% autologous plasma or serum substitutes in HIPP medium and cytokines were added.

[0061] Preferably, the amplification step further includes the step of:

[0062] (v) Supplement the cytokines every 2-3 days, repeating 2-5 times.

[0063] In some embodiments, step C) includes one or more of the following conditions:

[0064] In step (i), the radiation is X-ray radiation, preferably with an irradiation dose of 200 Gy, and / or,

[0065] The ratio is 1:100;

[0066] In step (ii), the HIPP medium 1 is a HIPP medium containing 2.5% autologous plasma or serum substitute.

[0067] The concentration of the anti-CD3 antibody is 30-600 ng / mL, for example, 600 ng / mL.

[0068] The concentration of the anti-CD28 antibody is 30-600 ng / mL, for example, 600 ng / mL.

[0069] The concentration of rhIL-2 is 100-1000 IU / mL, for example, 200 IU / mL.

[0070] The concentration of rhIL-7 is 10-100 ng / mL, for example, 10 ng / mL.

[0071] The concentration of rhIL-15 is 10-100 ng / mL, for example, 10 ng / mL, and / or,

[0072] The culture period is 4-6 days;

[0073] In step (iii), the HIPP culture medium 2 contains 100-1000 IU / mL (e.g., 200 IU / mL) of rhIL-2, 10-100 ng / mL (e.g., 10 ng / mL) of rhIL-7, and 10-100 ng / mL (e.g., 10 ng / mL) of rhIL-15, and contains 2% autologous plasma or serum substitutes, and / or,

[0074] The duration of the continued culture is 2-5 days, preferably 2 days;

[0075] The HIPP-T009 lymphocyte serum-free culture medium is a HIPP-T009 lymphocyte serum-free culture medium containing 2% autologous plasma, and / or,

[0076] The cytokines are 100-1000 IU / mL, for example, 200 IU / mL rhIL-2, 10-100 ng / mL, for example, 10 ng / mL rhIL-7, and 10-100 ng / mL, for example, 10 ng / mL rhIL-15.

[0077] The third aspect of the present invention provides the application of the tumor antigen peptide described in the first aspect of the present invention in the preparation of tumor antigen-specific T cells and a detection kit containing the same.

[0078] The technical solution adopted in this invention is: a method for preparing GMP-grade tumor antigen-specific TILs, characterized in that it is prepared according to the following steps:

[0079] 1. Tumor antigen prediction and antigenic peptide synthesis

[0080] Peripheral blood and tumor tissue from cancer patients were sequenced. Whole-exome sequencing was performed on peripheral blood, while whole-exome and transcriptome sequencing were performed on tumor tissue. By comparing the exome sequencing data from peripheral blood and tumor tissue, tumor cell-specific variations and differentially expressed proteins were analyzed. Based on this, combined with tumor tissue transcriptome sequencing data and patient HLA typing data, tumor-specific antigens (including tumor neoantigens, tumor-associated antigens, cancer-testis antigens, and tumor-associated viral-derived antigen peptides) that the patient might produce were predicted. Peptides that could be synthesized based on the predicted tumor-specific antigen peptide sequences that could be combined with MHC I and / or MHC II were used in subsequent DC loading experiments.

[0081] 2. Obtain primary TILs (pre-TILs) from tumor tissue through in vitro culture;

[0082] 2.1 Obtain tumor tissue and cut it into pieces of 1-4 mm. 3 Size of tissue blocks;

[0083] The tumor tissue used in this invention can be human tumors, mouse tumors, or brain tumors (including glioblastoma, neuroblastoma, medulloblastoma), such as human solid tumors (including breast cancer, lung cancer, colon cancer, melanoma).

[0084] 2.2 Tumor tissue was digested using the gentleMACS Dissociator and a tumor tissue dissociation kit (Mitteni, Germany, catalog number 130-095-929) to obtain a single-cell suspension;

[0085] 2.3 1×10 7 -4×10 7 Single-cell suspensions were added to G-rex bottles and cultured in pre-TILs medium (HIPP medium containing 1-5% autologous plasma or serum substitutes) containing 200-6000 IU / mL IL-2.

[0086] 2.4 After day 5, half of the medium was replaced using pre-TILs medium containing 200-6000 IU / mL IL-2;

[0087] After 2.5, change half the solution every 2-3 days;

[0088] 2.6 The total culture time should not exceed 3 weeks, until the pre-TILs cell count reaches greater than 1×10⁻⁶. 8 One is used for tumor antigen-specific T cell isolation.

[0089] 3. Subject's autologous DC culture

[0090] 3.1 Mix peripheral blood or apheresis blood thoroughly and centrifuge to collect the lower layer of blood cells. Dilute the blood cells with DPBS, add them to Ficoll (sucrose) separation solution, and centrifuge. Collect the cells in the middle white membrane layer, which are peripheral blood mononuclear cells (PBMCs).

[0091] 3.2 Pass the PBMC suspension through a 40μm sieve, centrifuge at 500g for 10 min, add CD14 magnetic beads (Miltenyi Biotec) and incubate for 30 min at 4℃. After incubation, centrifuge at 300g for 10 min and resuspend the cell pellet. Add the cell suspension to the center of the LS sorting column. Then, push the stopcock of the sorting column sharply to transfer the cells from the tube to the collection tube to collect CD14 positive cells.

[0092] 3.3 The sorted CD14-positive cells were resuspended in DC cell culture medium containing autologous serum or human serum albumin (HSA), and rhGM-CSF and rhIL-4 were added for directed differentiation. On days 3-5, maturation factors rhGM-CSF, rhIL-4, rhIL-1β, rhIL-6, rhTNF-α, PGE-2, and Poly I:C were added, and culture continued. On days 6-9, tumor antigen peptides were co-incubated with DC cells, and mature DC cells loaded with antigen peptides were collected.

[0093] 4. Isolation of tumor antigen peptide-specific TILs cells

[0094] Mature dendritic cells loaded with the above-mentioned antigenic peptides were co-cultured with the above-mentioned primary TILs cells to stimulate upregulation of CD137 expression on the surface of tumor antigen-specific T cells. The corresponding T cells were then isolated using CD137 antibody. The specific steps are as follows:

[0095] 4.1 Mature DC cells loaded with antigenic peptides were mixed with T cells at a ratio of 1:4 to 1:20, resuspended in HIPP medium containing 2.5% HSA, and the peptide fragments of tumor antigen peptides were added to a final concentration of 1-20 μg / mL, and rhIL-21 was added to 30 ng / mL.

[0096] 4.2 After co-culturing for 12-48 hours, collect the cells that have been co-cultured overnight;

[0097] 4.3 Tumor antigen-specific T cells were isolated using CD137-positive cell sorting reagent (Miltenyi Biotec).

[0098] 5. Rapid and massive expansion of CD137-positive cells

[0099] 5.1 Allogeneic PBMCs were irradiated with X-rays and used as helper cells. The CD137 positive cells were mixed with the helper cells at a ratio of 1:20 to 1:100.

[0100] 5.2 Resuspend the cells in 40 mL of HIPP medium containing 1-5% autologous plasma or serum substitute, add them to a GREX culture flask, and add 30-600 ng / mL anti-CD3 antibody, 30-600 ng / mL anti-CD28 antibody, 100-1000 IU / mL rhIL-2, 10-100 ng / mL rhIL-7 and 10-100 ng / mL rhIL-15;

[0101] 5.3 After 4-6 days of culture, half of the medium was replaced with HIPP medium containing 1-5% autologous plasma or serum substitutes, supplemented with 100-1000 IU / mL rhIL-2, 10-100 ng / mL rhIL-7 and 10-100 ng / mL rhIL-15.

[0102] 5.4 After continuing culture for 2 days, expand the culture system, with each flask of cells at a rate of 0.5-2 × 10⁻⁶. 6 Resuspend cells / mL in HIPP medium containing 1-5% autologous plasma to a final volume of 200-400 mL, place in a GREX culture flask, and add cytokines.

[0103] After 5.5, add cytokines every 2-3 days, and collect the finished product after culturing for 9-14 days.

[0104] The technical solution used in this invention is to culture pre-TILs using a single-cell suspension obtained from tumor tissue digestion, then pre-stimulate TILs cells with DCs loaded with tumor antigen peptides, and then use CD137 as a T cell activation marker to enrich and expand tumor antigen-specific TILs cells.

[0105] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0106] The reagents and raw materials used in this invention are all commercially available.

[0107] The positive and progressive effects of this invention are as follows:

[0108] 1) This invention uses autologous DCs as antigen-presenting cells, which can provide stronger co-stimulatory signals and activate tumor antigen-specific T cells in TILs.

[0109] 2) The CD137 activation marker can be used to simultaneously isolate tumor antigen-specific CD4 and CD8 TILs cells, avoiding the loss of effective anti-tumor cells.

[0110] 3) The tumor antigen-specific T cell isolation strategy in this invention is not limited by the patient's HLA typing and has a wide range of applications.

[0111] 4) The tumor antigen-specific TILs cells in the final product obtained by this invention are greatly improved compared with the existing methods, and the final product has a stronger tumor killing ability.

[0112] 5) This invention is applicable to a variety of tumor-specific antigens, such as tumor-associated virus-derived antigens, tumor-associated antigens, cancer testis antigens, tumor neoantigens, etc.

[0113] 6) The overall culture time of this invention is about 5 weeks, and the expansion process uses a low dose of rhIL-2. The obtained TILs cells are young TILs cells, which are more suitable for continuous existence and killing function in the in vivo environment.

[0114] 7) The present invention is simple to conduct large-scale culture and is more suitable for preparing immunotherapy products that meet the GMP requirements for clinical application. Attached Figure Description

[0115] Figure 1 The graph shows the ELISPOT detection results of the immune response of T2 cells to tumor antigen peptide-loaded TILs in Examples 2 and 3.

[0116] Figure 2 The results of T2 cells loaded with cytotoxic peptides in Examples 2 and 3 are shown in the figure.

[0117] Figure 3 The results of inhibiting tumor growth in mice in Examples 2 and 3 are shown in the figure. Detailed Implementation

[0118] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0119] Example 1: Tumor Antigen Prediction and Peptide Synthesis

[0120] Peripheral blood and tumor tissue from cancer patients were sequenced. Whole-exome sequencing was performed on peripheral blood, while whole-exome and transcriptome sequencing were performed on tumor tissue. Tumor cell-specific variations were analyzed by comparing the exome sequencing data from peripheral blood and tumor tissue. The expression of tumor-specific or differentially expressed proteins, or tumor-associated viral proteins, was analyzed using tumor tissue transcriptome sequencing data. Based on this, and combined with the patient's HLA typing data, tumor-specific antigens that the patient may produce were predicted, including tumor-associated viral antigens, tumor-associated antigens, cancer-testis antigens, and tumor neoantigens.

[0121] Table 1. Predicted tumor-specific antigen peptide sequences

[0122] Tumor antigen peptides REFIPLPWL 1 B40:01 Wild-type peptides REFIPLPRL 2

[0123] Example 2: Bulk TILs Cell Culture

[0124] 1. Primary TILs cell culture (pre-TILs)

[0125] Obtain cervical cancer tumor tissue and cut the tumor tissue into 2-4mm pieces. 3 The tissue blocks were then digested using the gentleMACS Dissociator and a tumor tissue dissociation kit (Mitteni, Germany, catalog number 130-095-929) to obtain a single-cell suspension; 1×10 7 The single-cell suspension was added to a Grex-10 flask, and 30 mL of HIPP medium (Bei'anji, FG0103801) containing 500 IU / mL rhIL-2 and 2% autologous plasma or serum substitute was added for culture. After day 5, half of the medium was replaced with pre-TILs medium containing 500 IU / mL rhIL-2. Thereafter, half of the medium was replaced every 2-3 days. After 3 weeks of culture, the pre-TILs cells were harvested.

[0126] 2. Rapid amplification of unsorted pre-TILs to prepare unsorted TILs

[0127] Allogeneic PBMCs were irradiated and used as feeder cells. Unsorted pre-TILs cells were mixed with feeder cells at a ratio of 1:100. The cells were then resuspended in 40 mL of HIPP medium containing 2% autologous plasma or serum substitutes and added to GREX culture flasks. 600 ng / mL anti-CD3 antibody, 600 ng / mL anti-CD28 antibody, 200 IU / mL IL-2, 10 ng / mL rhIL-7, and 10 ng / mL rhIL-15 were added. After 4-6 days, half the medium was replaced with HIPP medium containing 2% autologous plasma or serum substitutes, supplemented with 200 IU / mL IL-2, 10 ng / mL rhIL-7, and 10 ng / mL rhIL-15. After culturing for another 2 days, the culture system was expanded, with each flask containing 0.5-2 × 10⁶ cells. 6 Cells were resuspended at a density of 200-400 mL in HIPP-T009 lymphocyte serum-free medium (Bio-Enzyme) containing 2% autologous plasma or serum substitute. The cells were then placed in GREX culture flasks and cytokines were added. Cytokines were then added every 2-3 days, and cells were harvested after 10-14 days of culture. Cell yield is shown in Table 2.

[0128] Example 3: Isolation and rapid expansion of tumor antigen peptide-specific TILs cells

[0129] 1. Subject's autologous DC culture

[0130] 100 mL of peripheral blood was collected from the patient, and PBMCs were isolated from the blood using Ficoll separation solution. CD14-positive cells in the PBMCs were isolated using CliniMACS CD14 Reagent (Miltenyi Biotec). Specifically, the PBMC suspension was passed through a 40 μm sieve, centrifuged at 500 g for 10 min, and then incubated with CD14 magnetic beads (Miltenyi Biotec) for 30 min at 4°C. After incubation, the cells were centrifuged at 300 g for 10 min, and the cell pellet was resuspended. The cell suspension was added to the center of an LS sorting column, and then the column stopcock was pushed sharply to transfer the cells from the tube to the collection tube to collect the CD14-positive cells.

[0131] The sorted CD14+ cells were resuspended in DC cell culture medium (CellGenix, 20801-0500) containing autologous serum or human serum albumin (HSA), and rh GM-CSF was added to a final concentration of 50 ng / ml. rh IL-4 was added to a final concentration of 100 ng / ml for directed differentiation. From day 3 to 5, the following concentrations were added: rhGM-CSF (final concentration 100 ng / ml), rhIL-4 (final concentration 100 ng / ml), rhIL-1β (final concentration 10 ng / ml), rhIL-6 (final concentration 10 ng / ml), rhTNF-α (final concentration 10 ng / ml), PGE-2 (final concentration 100 ng / ml), and Poly I:C (final concentration 5 μg / ml), and cultured further. On day 6, tumor antigen peptides were co-incubated with the DC cells, and the mature DC cells loaded with the antigen peptides were collected.

[0132] 2. Primary TILs cell culture (pre-TILs)

[0133] Obtain cervical cancer tumor tissue and cut the tumor tissue into 2mm pieces. 3 The tissue blocks were then digested using the gentleMACS Dissociator and a tumor tissue dissociation kit (Mitteni, Germany, catalog number 130-095-929) to obtain a single-cell suspension; 1×10 7 The single-cell suspension was added to a Grex-10 flask, and 30 mL of HIPP medium containing 500 IU / mL rhIL-2 and 2% autologous plasma or serum substitute was added for culture. After day 5, half of the medium was replaced with HIPP medium containing 500 IU / mL rhIL-2. Thereafter, half of the medium was replaced every 2-3 days. Primary TILs cells were harvested after 2 weeks of culture.

[0134] 3. Isolation of tumor antigen peptide-specific T cells using CD137 labeling

[0135] The DCs loaded with tumor antigen peptides were mixed with the primary TILs at a ratio of 1:10 and resuspended in HIPP medium containing 2.5% HSA. The tumor antigen peptides were added to a final concentration of 10 μg / mL, and rhIL-21 was added to a final concentration of 30 ng / mL. After co-culturing DCs and T cells for 18 h, the cells co-cultured overnight were collected, and CD137 positive cells in the co-culture were sorted using CD137 Antibody, Anti-human, Biotin (Miltenyi Biotec) and Anti-Biotin MicroBeads UltraPure (Miltenyi Biotec).

[0136] 4. Rapid expansion of CD137-positive cells to prepare tumor antigen-specific TILs cells

[0137] Allogeneic PBMCs were irradiated with X-rays (200 Gy) and used as helper cells. The aforementioned CD137-positive cells were mixed with the helper cells at a 1:100 ratio. Next, the cells were resuspended in 40 mL of HIPP medium containing 2.5% autologous plasma and added to GREX culture flasks. 600 ng / mL anti-CD3 antibody, 600 ng / mL anti-CD28 antibody, 200 IU / mL IL-2, 10 ng / mL rhIL-7, and 10 ng / mL rhIL-15 were added. After 4-6 days, half the medium was replaced with HIPP medium containing the same concentrations of cytokines and 2% autologous plasma or serum substitutes. After culturing for another 2 days, the culture system was expanded, with each flask containing 0.5-2 × 10⁶ cells. 6 Cells were resuspended at a density of 2% autologous plasma or serum substitute in serum-free HIPP-T009 lymphocyte culture medium (200-400 mL) and placed in GREX culture flasks with added cytokines. Cytokines were then added every 2-3 days, and cells were harvested after 10-14 days of culture. Cell yield is shown in Table 2.

[0138] Table 2 TILs cell yield

[0139] Example 2 <![CDATA[1×10 7 ]]> <![CDATA[5.28×10 10 ]]> Example 3 <![CDATA[1×10 7 ]]> <![CDATA[4.69×10 10 ]]>

[0140] Example 4: Flow cytometry analysis of the proportion of PD-1 (CD279) positive cells in cells from Examples 2 and 3.

[0141] 1. Take 6 × 10 5 The cells obtained in Example 2 or Example 3 were resuspended in 300 μL of DPBS containing 2% FBS, centrifuged at 400g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 200 μL of DPBS containing 2% FBS.

[0142] 2. Take 50 μL of cell suspension and place it into two 1.5 mL centrifuge tubes, one as the control group and the other as the experimental group. The control group is untreated, while the experimental group is treated with 2 μL of APC-CD279 antibody, 2 μL of APC-Cy7-CD3 antibody, and 2 μL of AlexaFlour@700-CD8 antibody. After mixing the solutions, incubate at room temperature in the dark for 20 min.

[0143] 4. After staining, add DPBS to the centrifuge tube, mix well, centrifuge at 400g for 5 min, and discard the supernatant. Disperse the cell pellet, resuspend the cells in 200μL of DPBS.

[0144] 5. Add 5 μL of 7-ADD (Biolegend) staining solution to each tube, incubate at room temperature in the dark for 5 min, and then perform the analysis using a flow cytometer (Beckman).

[0145] 6. First, select the 7-ADD negative cell population, and then select the CD3+CD8 double positive population from the 7-ADD negative cell population. Analyze the PD-1 expression in this population.

[0146] The test results are shown in Table 3.

[0147] Table 3. Results of detection of tumor antigen peptides and specific T cell peptides in pre-TILs and enriched TILs after culture.

[0148]

[0149] Example 5: IFN-γELISPOTs detection of the immune response of TILs cells to tumor antigen peptide-loaded T2 cells in Examples 2 and 3.

[0150] Using T2 cells loaded with tumor antigen peptides as a stimulus, they were co-cultured with cells obtained in Examples 2 and 3, respectively, to detect the number of cells capable of secreting IFN-γ in the samples. Following the instructions of the IFN-γ ELISPOT kit, the test cells and peptide-loaded T2 cells (test group) were co-cultured in ELISPOT wells for 20 h, and then the IFN-γ secreting cells were stained and detected according to the kit instructions. The negative control group consisted of test cells co-cultured with unloaded T2 cells, while the positive control group consisted of test cells with PMA. The detection results are shown in Table 4. Figure 1 .

[0151] Table 4. Results of IFN-γ ELISPOT detection in TILs cells in Examples 2 and 3

[0152] Example 2 156 15 824 Example 3 297 4 945

[0153] Example 6: Tetramer detection of tumor antigen peptide-specific T cell content in Examples 2 and 3

[0154] 1. Take 6 × 10 5 The cells obtained in Example 2 or Example 3 were resuspended in 300 μL of DPBS containing 2% FBS, centrifuged at 400g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 200 μL of DPBS containing 2% FBS.

[0155] 2. Take 50 μL of cell suspension and place it into two 1.5 mL centrifuge tubes, one as the control group and the other as the experimental group. The control group was left untreated, while the experimental group was treated with 2 μL of tetramer prepared from tumor antigen peptide (APC-labeled).

[0156] 3. Add 2 μL of PE anti-human CD8 (Biolegend) staining solution to each of the two tubes, mix well, and incubate at room temperature in the dark for 20 min.

[0157] 4. After staining, add DPBS to the centrifuge tube, mix well, centrifuge at 400g for 5 min, and discard the supernatant. Disperse the cell pellet and resuspend the cells in 200μL of DPBS.

[0158] 5. Add 5 μL of 7-ADD (Biolegend) staining solution to each tube, incubate at room temperature in the dark for 5 min, and then perform the analysis using a flow cytometer (Beckman).

[0159] 6. First, select the 7-ADD negative cell population. Then, select APC+ / PE+ double positive cells from the 7-ADD negative cell population. The ratio of these cells is the specific T cell ratio.

[0160] The test results are shown in Table 5.

[0161] Table 5. Results of tumor antigen peptide-specific T-cell peptide-tetramer detection in Examples 2 and 3.

[0162] Proportion of peptide-tetramer positive cell population 3.4％ 48.9％

[0163] Example 7: Detection of the cytotoxic ability of cells from Example 2 and Example 3 against T2 cells loaded with antigen peptides using CFSE and 7-AAD dual labeling techniques.

[0164] T2 cells in logarithmic growth phase were divided into a tumor antigen peptide-loaded group and a wild-type peptide group, and cultured overnight at 37°C in a 5% CO2 incubator. The overnight cultured T2 cells were washed, and 0.5 μM CFSE solution was added, followed by treatment at 37°C for 20 min. Immediately after staining, the cells were removed, staining was stopped, and the cells were washed 2-3 times with DPBS. Cells were counted and resuspended to a concentration of 2 × 10⁻⁶ cells / mL. 5Cells / mL. Cells obtained in Example 2 or Example 3 were co-cultured with T2 cells loaded with tumor antigen peptides or wild-type peptides at a specific effective-to-target ratio (see Table 6). The mixture was incubated at 37°C in a 5% CO2 incubator for 20-22 hours. The cell mixture was then resuspended, and all cells were removed. 7-ADD was added, and the cells were stained in the dark for 5 minutes to label dead cells. The killing rate was then detected by flow cytometry. CSFE(+)7-AAD(+) double-positive cells were considered killed target cells, and CFSE(+)7ADD(-) cells were considered unkilled target cells. The killing activity of target cells against T2 cells was calculated based on these ratios. The killing activity of wild-type peptide-specific CD8(+) T cells was detected using the same protocol. Kill rate (%) = (Percentage of dead cells in experimental target cells - Percentage of dead cells in negative control target cells) / (1 - Percentage of dead cells in negative control target cells). Results are shown in Table 6 and... Figure 2 .

[0165] Table 6. Target cells that specifically recognize and kill experimentally presented peptides.

[0166]

[0167] Example 8: Pharmacodynamic study of tumor-bearing mouse model with tumor antigen peptide stabilized by intravenous injection of cells from Examples 2 and 3 (Examples 2 and 3)

[0168] In vivo efficacy evaluation of TILs cells prepared in Examples 2 and 3 was conducted in mice. A subcutaneous tumor model was constructed using 64 7-9 week old immunodeficient NOG mice and tumor cell lines stably transfected with tumor-specific antigen peptides. Each mouse was subcutaneously inoculated with 2 × 10⁶ cells. 6 100 cervical cancer siHa tumor cells were inoculated into mice. After inoculation, tumor growth and size were monitored regularly, with tumors reaching 50-100 mm in size. 3 The mice were then randomly grouped according to tumor size and body weight, and administered the drug via tail vein.

[0169] The cells were divided into eight groups: 1) PBS adjuvant group, 2) low-dose group (Example 2), 3) medium-dose group (Example 2), 4) high-dose group (Example 2), 5) low-dose group (Example 3), 6) medium-dose group (Example 3), 7) high-dose group (Example 3), and 8) high-dose Mock-T cell group, with eight cells in each group. The high-dose group contained 2 × 103 cells. 7 / Dosage, medium dose group was 7×10 6 / Dosage, low-dose group: 2×10 6 The dosage is administered twice, with the second dose given 7 days after the first dose. It is also combined with IL-2 (50,000 IU / dose, 3 doses daily).

[0170] See results Figure 3After tumor formation was confirmed (5-8 days post-inoculation), the length and width of the tumor were measured every 2 days, and the relative tumor inhibition rate (TGI) was calculated to evaluate the efficacy. Results showed that, after administration 5 days post-tumor formation, by day 32, the TGIs of the low-, medium-, and high-dose groups in Example 3 were 88%, 93%, and 99%, respectively, significantly inhibiting tumor growth in NOG mice and exhibiting a dose-response relationship. In Example 2, the TGIs of the low-, medium-, and high-dose groups were 47%, 63%, and 79%, respectively, while the TGI of the Mock-T group was 29%.

[0171] This invention uses dendritic cells loaded with tumor antigen peptides to stimulate initially cultured tumor-inhibiting lymphocytes (TILs). Subsequently, the antigen-specific activation marker CD137 is used as a marker to sort tumor antigen-specific TILs. The culture time is no more than 5 weeks. A combination of cytokines is used to expand young TILs. This method significantly increases the number and proportion of tumor antigen-specific T cells in the final TIL product. Therapeutic young TIL products can be obtained within 5 weeks, which are more suitable for clinical use. They have strong in vivo proliferation and specific anti-tumor effects. The culture process is simple, widely applicable, and easy to carry out large-scale production.

[0172] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A tumor antigen peptide, characterized in that, The amino acid sequence of the tumor antigen peptide is shown in SEQ ID NO:

1.

2. A method for preparing tumor antigen-specific T cells, characterized in that, The method includes the following steps: A) Co-cultured cells were obtained by co-culturing DC cells loaded with tumor antigen peptides with primary TILs cells; B) Subsequently, tumor antigen-specific T cells were sorted using antigen-specific activation markers to obtain marker-positive cells; and, C) Amplify the marker-positive cells; The preparation process of the DC cells includes the following steps: (1) After filtering and centrifuging the suspension of peripheral blood mononuclear cells, CD14 magnetic beads were added and incubated. After sorting, CD14 positive cells were obtained. (2) The CD14 positive cells were resuspended in DC cell culture medium, and then differentiated by adding directed differentiation factor and maturation factor. (3) Add the tumor antigen peptide as described in claim 1 for co-incubation to obtain DC cells loaded with tumor antigen peptide.

3. The method as described in claim 2, characterized in that, The marker is CD137, CD107a, or CD134.

4. The method as described in claim 3, characterized in that, The marker is CD137.

5. The method as described in claim 2, characterized in that, In step (1), the peripheral blood mononuclear cells are derived from blood cells of a tumor patient; and / or, The filter size is 35-45µm; and / or, The centrifugation conditions are 300-500g, 5-10min; and / or, The incubation temperature is 2-4℃; In step (2), the DC cell culture medium is a DC cell culture medium containing autologous serum or HSA; and / or, The targeted differentiation factor is one or more of rhGM-CSF and rhIL-4; and / or, The maturation factors are one or more of rhGM-CSF, rhIL-4, rhIL-1β, rhIL-6, rhTNF-α, PGE-2, and Poly I:C; and / or, The differentiation process lasts 3-5 days; and / or, The culture period is 1-6 days; In step (3), the co-incubation period is 6 days.

6. The method as described in claim 2, characterized in that, The preparation process of the primary TILs cells includes: (a) The tumor tissue was digested to obtain a single-cell suspension; (b) Mix the above single-cell suspension with HIPP medium A containing 1%-5% autologous plasma or serum substitute and 500-6000 IU / mL IL-2; (c) Change the medium using the HIPP medium A, and then culture to obtain primary TILs cells.

7. The method as described in claim 6, characterized in that, In step (c), the HIPP medium A is a HIPP medium containing 500 IU / mL IL-2 and 2% autologous plasma or serum substitute; and / or, The fluid replacement is a half-volume replacement; and / or, The fluid change frequency is once every 2-3 days; and / or, The total duration of the re-culturing is 1-3 weeks.

8. The method as described in claim 7, characterized in that, The total duration of the re-culturing is 2 weeks.

9. The method as described in claim 2, characterized in that, The method includes the following steps: In step A), the DC cells and the primary TILs cells are mixed at a ratio of 1:4 to 1:20, resuspended in HIPP medium B containing 1%-5% HSA, and the tumor antigen peptide and rhIL-21 as described in claim 1 are added for co-culture, and the co-cultured cells are collected. In step B), tumor antigen-specific T cells are separated using a CD137-positive cell sorting reagent to obtain marker-positive cells.

10. The method as described in claim 9, characterized in that, In step A), the ratio is 1:10; and / or, The HIPP medium B is a HIPP medium containing 2.5% HSA; and / or, The final concentration of the tumor antigen peptide is 1-20 µg / mL; and / or, The final concentration of rhIL-21 is 30 ng / mL; and / or, The co-culture duration is 12-48 hours.

11. The method as described in claim 10, characterized in that, In step A), the final concentration of the tumor antigen peptide is 10 μg / mL; and / or, The co-culture duration was 18 hours.

12. The method as described in claim 2, characterized in that, In step C), the amplification includes one or more of the following conditions: (i) Irradiated allogeneic PBMC cells and TILs cells were mixed at a ratio of 1:20 to 1:100 to obtain mixed cells; (ii) The mixed cells were resuspended in HIPP medium 1 containing 1%-5% autologous plasma or serum substitutes and cultured with one or more of anti-CD3 antibody, anti-CD28 antibody, rhIL-2, rhIL-7 and rhIL-15. (iii) Change the medium using HIPP medium 2 containing 1%-5% autologous plasma or serum substitute; (iv) Continue culturing at a rate of 0.5-2 × 10⁻⁶. 6 Cells were resuspended at a density of 1 / mL in serum-free HIPP-T009 lymphocyte culture medium containing 1%-5% autologous plasma or serum substitutes, and cytokines were added.

13. The method as described in claim 12, characterized in that, The fluid replacement is a half-volume replacement.

14. The method as described in claim 12, characterized in that, The amplification also includes the following steps: (v) Supplement the cytokines every 2-3 days, repeating 2-5 times.

15. The method as described in claim 12, characterized in that, Step C) includes one or more of the following conditions: In step (i), the radiation is X-ray radiation, and / or, The ratio is 1:100; In step (ii), the HIPP medium 1 is a HIPP medium containing 2.5% autologous plasma or serum substitute. The concentration of the anti-CD3 antibody is 30-600 ng / mL. The concentration of the anti-CD28 antibody is 30-600 ng / mL. The concentration of rhIL-2 is 100-1000 IU / mL. The concentration of rhIL-7 was 10-100 ng / mL. The concentration of rhIL-15 is 10-100 ng / mL, and / or, The culture period is 4-6 days; In step (iii), the HIPP culture medium 2 contains 100-1000 IU / mL rhIL-2, 10-100 ng / mL rhIL-7, and 10-100 ng / mL rhIL-15, and contains 2% autologous plasma or serum substitutes, and / or, In step (iv), the duration of the continued culture is 2-5 days; The HIPP-T009 lymphocyte serum-free culture medium is a HIPP-T009 lymphocyte serum-free culture medium containing 2% autologous plasma, and / or, The cytokines are rhIL-2 at 100-1000 IU / mL, rhIL-7 at 10-100 ng / mL, and rhIL-15 at 10-100 ng / mL.

16. The method as described in claim 15, characterized in that, In step (i), the radiation dose is 200 Gy.

17. The method as described in claim 15, characterized in that, In step (iv), the duration of the continued culture is 2 days.

18. The method as described in claim 15, characterized in that, In step (ii), the concentration of the anti-CD3 antibody is 600 ng / mL. The concentration of the anti-CD28 antibody was 600 ng / mL. The concentration of rhIL-2 was 200 IU / mL. The concentration of rhIL-7 was 10 ng / mL. The concentration of rhIL-15 was 10 ng / mL. In step (iii), the HIPP medium 2 contains 200 IU / mL rhIL-2, 10 ng / mL rhIL-7, and 10 ng / mL rhIL-15, and / or, In step (iv), the cytokines are 200 IU / mL rhIL-2, 10 ng / mL rhIL-7 and 10 ng / mL rhIL-15.

19. A tumor antigen peptide as described in claim 1 in the preparation of HLA-B Application in a kit for detecting tumor antigen-specific T cells in cervical cancer patients with the 40:01 genotype.

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