Tumor infiltrating lymphocyte population as well as preparation method and application thereof

Abstract

The invention relates to a tumor infiltrating lymphocyte population as well as a preparation method and application thereof, and belongs to the technical field of biological medicine product manufacturing. The tumor infiltrating lymphocyte population provided by the invention has a stronger anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, specifically relating to a tumor-infiltrating lymphocyte population and its preparation method, a corresponding tumor-infiltrating lymphocyte preparation and its uses. Background Technology

[0002] Tumor-infiltrating lymphocytes (TILs) are used in the treatment of tumors due to their anti-tumor effects.

[0003] Disclosed clinical trial data confirms that the quantity of tumor-infiltrating lymphocyte cell preparations is directly proportional to patient prognosis. During the in vitro isolation and culture of tumor-infiltrating lymphocytes, due to the long culture period, cells undergo progressive differentiation and exhaustion, resulting in a high proportion of differentiated cells in the final tumor-infiltrating lymphocyte cell preparations, thus affecting the clinical treatment efficacy of tumors.

[0004] Therefore, there is an urgent need to develop new tumor-infiltrating lymphocyte cell preparations to overcome the aforementioned problems existing in current tumor-infiltrating lymphocyte cell preparations. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a tumor-infiltrating lymphocyte population, which, through a specific culture method, achieves the expansion of tumor-infiltrating lymphocytes while maintaining them in a low-differentiation state. The resulting effector tumor-infiltrating lymphocyte population can enhance the anti-tumor effect, improve the therapeutic effect and patient prognosis.

[0006] Specifically, in a first aspect, the present invention provides a method for preparing a population of tumor-infiltrating lymphocytes, comprising: (a) The tumor tissue was cleaned, digested, and centrifuged to extract tumor-infiltrating lymphocytes, thus obtaining the first cell population; (b) Adjust the cell density of the first cell population to 3 × 10⁻⁶. 6 ~4×10 6 Cells / ml, after culturing for 5-7 days, to obtain the second cell population; (c) The second cell population was initially sorted and enriched with T cells to achieve a cell density of 1 × 10⁻⁶. 5 ~1×10 6 Cells / ml, after culturing for 5-7 days, yielded the third cell population; (d) Secondary sorting and enrichment of tumor-infiltrating lymphocytes in the third cell population to achieve a cell density of 1×10⁻⁶. 5 ~1×10 6 Cells / ml, after culturing for 5-7 days, yielded the fourth cell population; (e) Whole-exome sequencing was performed on tumor tissue and normal tissue of the same origin to obtain whole-exome genome data. The whole-exome genome data of tumor tissue and normal tissue were compared with the human reference genome to screen for mutated genes. HLA typing was then performed on peripheral blood of the same origin to obtain HLA typing results. Based on the mutated genes and HLA typing results, the binding affinity of the peptides corresponding to the mutated genes to HLA molecules was predicted, and the affinity was calculated using IC50. 50 A polypeptide with a molecular weight below 550 nM was used as a tumor neoantigen polypeptide, and this tumor neoantigen polypeptide was synthesized. (f) Obtain PBMCs from the same source, separate and process them to obtain dendritic cells (DCs), and load the dendritic cells with tumor neoantigen peptides, wherein the proportion of dendritic cells loaded with tumor neoantigen peptides is 5~7×10 13 Dendritic cells: 1 mole of tumor neoantigen polypeptide, or 5~10×10 6 Each dendritic cell: 50~200 μg of tumor neoantigen polypeptide; (g) Dendritic cells loaded with tumor neoantigen peptides are co-cultured with the fourth cell population for 5-7 days to obtain a tumor-infiltrating lymphocyte population.

[0007] In this invention, "same source" refers to tissues originating from the same individual. For example, "normal tissues of the same source" means that normal tissues and tumor tissues originate from the same source, i.e., both are from the same subject; "PBMCs of the same source" means that PBMCs and tumor tissues originate from the same source, i.e., both are from the same subject. In some preferred embodiments, the subject is a human.

[0008] In the preparation method of the present invention, (a) to (g) are only used to conveniently distinguish the operation steps and are not intended to limit the order of operation. For example, during preparation, the operation can be performed sequentially in the order of (a) to (g), or in the order of (a)-(b)-(e)-(f)-(c)-(d)-(g), or in the order of (e)-(f)-(a)-(b)-(c)-(d)-(g), etc. Among them, (e)-(f) can be performed before or after any of (a), (b), (c), and (d), as long as (g) is the last step among (a) to (g).

[0009] In some implementations, (a) includes the steps of: washing the tumor tissue with a tumor washing solution; and cutting the tumor tissue into 1-3 mm pieces. 3After the tissue fragments were removed, digestion solution was added to digest the cells to obtain a cell suspension. Digestion was terminated by adding RPMI 1640 containing 10 wt% human AB serum. The filtrate was sieved and collected. The cell suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in RPMI 1640 medium containing 10 wt% human AB serum, 1 wt% penicillin and antibiotics, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HERPES, and 6000 IU / ml IL-2 to obtain a single-cell suspension, which is the first cell population.

[0010] In some implementation schemes, (a) the required time is 1-3 days.

[0011] In some implementations, (b) includes the step of adjusting the cell density of the first cell population to 4 × 10⁻⁶ cells using a culture medium. 6 Cells were cultured at 37°C and 5% CO2 for 7 days to obtain the second cell population.

[0012] In some embodiments, in (b), the culture medium used to adjust cell density and the culture medium are RPMI 1640 medium containing 1-10 wt% human AB serum, 0.5-1.5 wt% penicillin-dextrin antibiotics, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, and 3000-8000 IU / ml IL-2. Preferably, the culture medium is RPMI 1640 medium containing 10 wt% human AB serum, 1 wt% penicillin-dextrin antibiotics, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HERPES, and 6000 IU / ml IL-2. GlutaMAX is a solution containing L-glutamine and its derivatives, and 1:100 GlutaMAX refers to a volume ratio of GlutaMAX to the culture medium of 1:100.

[0013] In some implementations, (c) includes the following steps: initial sorting of the second cell population using MACS CD3 (TIL) MicroBead Kits to enrich T cells, and adjusting the cell density to 1 × 10⁻⁶ cells using culture medium. 6 Cells / ml, cultured for 7 days, yielding the third cell population. The initial sorting yielded a relatively broad T cell population, containing various T cell subsets with different functions and characteristics, of which only a portion were true tumor-infiltrating lymphocytes (TILs).

[0014] In some embodiments, in (c), the culture medium used to adjust cell density and the culture medium used for culturing are RPMI 1640 medium containing 0-10 wt% human AB serum, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, 3000-8000 IU / ml IL-2, 20-40 ng / mg IL-7, 10-20 ng / mg IL-21, and 3-8 ng / ml TGF-β. Preferably, the culture medium is RPMI 1640 medium containing 10 wt% human AB serum, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HERPES, 6000 IU / ml IL-2, 30 ng / mg IL-7, 15 ng / mg IL-21, and 5 ng / ml TGF-β.

[0015] In some implementations, (d) includes the following steps: secondary sorting of the third cell population using MACS CD4 (TIL) and CD8 (TIL) MicroBead Kits to enrich TIL cells, and adjusting the cell density to 1 × 10⁻⁶ cells using culture medium. 6 Cells were cultured at a density of [number] cells / ml for 7 days to obtain the fourth cell population. Secondary sorting further enriched tumor-infiltrating lymphocytes based on the third cell population. The TIL cells in the fourth cell population obtained through secondary sorting were obtained after two enrichments. Compared to the T cell population obtained from the first sorting, the TILs had higher purity and were more representative of the tumor-killing T cell subset, making them more suitable for subsequent tumor research and therapeutic applications.

[0016] In some embodiments, in (d), the culture medium used to adjust cell density and the culture medium used for culturing are RPMI 1640 medium containing 0-10 wt% human AB serum, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, 3000-8000 IU / ml IL-2, 20-40 ng / mg IL-7, 10-20 ng / mg IL-21, and 3-8 ng / ml TGF-β. Preferably, the culture medium is RPMI 1640 medium containing 10 wt% human AB serum, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HERPES, 6000 IU / ml IL-2, 30 ng / mg IL-7, 15 ng / mg IL-21, and 5 ng / ml TGF-β.

[0017] In some implementations, (e) includes the following steps: (1) Whole exome sequencing and data analysis; (2) Screening and functional annotation of mutant genes; (3) HLA typing and neoantigen prediction; (4) Synthesis and quality control of tumor neoantigen peptides.

[0018] In some preferred embodiments, in step (1), whole-exome sequencing is performed using NGS (Nest Gene Sequence) technology. After whole-exome sequencing is completed, the raw sequencing data is first quality controlled through a bioinformatics analysis process to remove low-quality reads, adapter sequences, and contaminating sequences. Then, the sequencing data of tumor tissue and normal tissue are compared with the human reference genome (the human reference genome known by the software BWA). Sequence alignment software known in the art, such as BWA (Burrows-Wheeler Aligner), can be used for alignment. Based on the alignment results, somatic mutations in tumor tissue are identified using mutation detection tools (such as MuTect2, VarScan2, etc.). These tools comprehensively consider factors such as base quality, coverage, and mutation frequency to accurately identify different types of mutations, such as single nucleotide variants (SNVs), insertions / deletions (Indels), and copy number variations (CNVs).

[0019] In some preferred embodiments, the normal tissue is adjacent to cancer tissue.

[0020] In some preferred embodiments, in step (2), mutant genes with potential functional impacts are screened from the large number of somatic mutations identified in step (1). For single nucleotide variants, the focus is on whether they lead to changes in the amino acid sequence (missense mutations), premature appearance of stop codons (speechless mutations), or affect splicing sites. Insertion-deletion mutations, if occurring in coding regions, may lead to frameshift mutations, severely affecting protein structure and function. Then, the screened mutant genes are functionally annotated using public databases (such as dbSNP, COSMIC, etc.) and functional prediction software (such as SIFT, PolyPhen-2, etc.). SIFT can predict the impact of amino acid substitutions on protein function, while PolyPhen-2 assesses the degree of harm of mutations to protein structure and function. Through these analyses, it is further determined which mutant genes may be closely related to the occurrence and development of tumors, providing more accurate candidate genes for subsequent neoantigen prediction.

[0021] In some preferred embodiments, in step (3), peripheral blood is used for HLA typing. The detection methods can include PCR-SSP (polymerase chain reaction-sequence-specific primers), PCR-SBT (polymerase chain reaction-sequencing typing), etc. These methods accurately determine the molecular types of the patient's major histocompatibility complex (MHC) class I alleles, such as HLA-A, HLA-B, and HLA-C. The screened mutant gene information and HLA typing results are input into neoantigen prediction software (such as Net-MHC4.0) to predict the binding affinity of the mutant gene-encoded peptide to HLA molecules (the calculation of binding affinity considers factors such as the amino acid sequence and length of the peptide, as well as the structural characteristics of the HLA molecule). Based on the binding affinity values ​​of each peptide to different HLA molecules output by the software (usually expressed as half-maximal inhibition concentration (IC50)...), the binding affinity is calculated... 50 (or percentile ranking), selecting those with high binding affinity (IC). 50 Peptides corresponding to mutated genes that are less than 500 nM and meet other immunogenicity criteria (such as appropriate peptide length and not being highly expressed in normal tissues) are used as tumor neoantigens.

[0022] In some preferred embodiments, in step (4), after identifying the tumor neoantigen, the polypeptide is synthesized. The synthesis method can be any method known in the art, such as solid-phase polypeptide synthesis. After synthesis, the polypeptide is subjected to quality control measures such as purification, purity testing, and endotoxin testing to obtain the tumor neoantigen.

[0023] In some specific embodiments, the steps for synthesizing tumor neoantigen peptides using solid-phase peptide synthesis include: using Fmoc (9-fluorenylmethoxycarbonyl) protected amino acids as starting materials, covalently linking the carboxyl terminus of the first amino acid to a solid support (such as Wang resin). Using activators such as DCC (N,N'-dicyclohexylcarbodiimide) or EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and with the synergistic effect of auxiliary reagents such as HOBt (1-hydroxybenzotriazole), subsequent amino acids are sequentially linked to the amino groups of the amino acids already linked to the solid support. After each amino acid linking, the Fmoc protecting group is removed using a 20% piperidine / DMF (N,N-dimethylformamide) solution to prepare for the linking of the next amino acid, ultimately yielding the tumor neoantigen peptide.

[0024] In some specific implementations, quality control of peptides, including purification, purity testing, and endotoxin detection, includes: lysing the peptide from a solid-phase support (e.g., using a lysis buffer containing trifluoroacetic acid (TFA)). The lysed peptide solution undergoes preliminary impurity removal via ether precipitation and centrifugation, followed by purification by high-performance liquid chromatography (HPLC). HPLC uses a reversed-phase column with acetonitrile and water as the mobile phase. By adjusting the ratio and flow rate of the mobile phase, peptides of different purities are separated on the column. The elution peak of the target peptide is collected and lyophilized to obtain a high-purity tumor neoantigen peptide. The synthesized tumor neoantigen peptide is then subjected to purity testing (HPLC purity should reach ≥95%), amino acid sequence determination (using mass spectrometry or other methods to verify the correctness of the amino acid sequence), and endotoxin detection (ensuring that the endotoxin content meets relevant standards, e.g., less than 5 EU per microgram of peptide).

[0025] In some implementations, (f) obtaining PBMCs from the same source includes the following steps: obtaining fresh anticoagulated whole blood from the same source, diluting the anticoagulated whole blood with physiological saline at a 1:1 volume ratio, slowly adding the diluted anticoagulated whole blood lymphocytes to a centrifuge tube containing cell separation medium at a 1:1 volume ratio (do not mix with the separation medium; the two should clearly separate into layers), and centrifuging; after centrifugation, the liquid surface in the centrifuge tube from top to bottom consists of a diluted plasma layer, a PBMC layer, a separation medium layer, and a red blood cell layer. The PBMC layer (i.e., the white membrane layer) cells are aspirated, and physiological saline is added at a 1:1 volume ratio for washing and centrifugation. The washing and centrifugation steps are repeated 1-2 times to obtain PBMCs.

[0026] In some embodiments, (f) the separation process includes the following steps: mixing the PBMC suspension by pipetting and adjusting the cell density to 1×10⁻⁶ cells / mL with AIM-V culture medium. 6 After incubating at 37°C and 5% CO2 for 90 minutes, the supernatant and non-adherent cells were discarded. Adherent cells were washed with RPMI 1640 medium and then cultured in AIM-V medium for 60 minutes. The supernatant and non-adherent cells were discarded, and DC medium (RPMI 1640 medium + 5% human serum + 1% penicillin antibiotics, 1:100 glutamine, 800 IU / ml GM-CSF, 200 U / ml IL-4) was added and cultured for 2-4 days.

[0027] In some embodiments, (f) loading the dendritic cells with the tumor neoantigen peptide includes the following steps: dissolving the tumor neoantigen peptide synthesized in (e) in PBS and mixing it with the dendritic cells, wherein the ratio of the dendritic cells loaded with the tumor neoantigen peptide is 5~7×10⁻⁶. 13 Dendritic cells: 1 mole of tumor neoantigen polypeptide, or 5~10×106 Dendritic cells: 50-200 μg tumor neoantigen polypeptide, preferably 7×102 13 Dendritic cells: 1 mole of tumor neoantigen polypeptide or 1 × 10 7 Dendritic cells: 100 μg tumor neoantigen peptide, cultured for 6-12 h to complete loading.

[0028] In some embodiments, in (g), the ratio of the fourth cell population to the dendritic cells loaded with the tumor neoantigen polypeptide is (15-30):(0.5-1.5), preferably 20:1; and the total density of the fourth cell population and the dendritic cells loaded with the tumor neoantigen polypeptide is 1×10⁻⁶. 5 ~1×10 6 Cells / ml, preferably 1×10 6 per ml.

[0029] In some embodiments, (g) the culture medium used for co-culture is a mixed culture medium of AIM-V CTS medium and RPMI1640 medium mixed at a ratio of (0.5-1.5):(0.5-1.5), preferably mixed at a ratio of 1:1. The RPMI 1640 medium contains 0-10 wt% human AB serum, 0.5-1.5 wt% penicillin-antibody, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, 3000-8000 IU / ml IL-2, 20-40 ng / ml OKT-3, 20-40 ng / mg IL-7, 10-20 ng / mg IL-21, and 3-8 ng / ml TGF-β; preferably, it contains 5 wt% human AB serum, 1 wt% penicillin-antibody, 10 mg / ml gentamicin, 1:10 GlutaMAX, 25 mM HERPES, 3000 IU / ml IL-2, 30 ng / ml OKT-3, 30 ng / mg IL-7, 15 ng / mg IL-21, and 5 ng / ml TGF-β.

[0030] In some preferred embodiments, the culture time required to obtain tumor-infiltrating lymphocyte populations using the preparation method of the present invention is 21-35 days.

[0031] In some embodiments, the tumor-infiltrating lymphocyte population obtained in (g) can be cryopreserved or expanded and cultured as needed. Therefore, the preparation method further includes (h): expanding and culturing the tumor-infiltrating lymphocyte population obtained in (g) in a bioreactor for 5-7 days to obtain expanded tumor-infiltrating lymphocyte population; and / or (i): aliquoting or cryopreserving the tumor-infiltrating lymphocyte population obtained in (g) or the expanded tumor-infiltrating lymphocyte population obtained in (h) to obtain a direct formulation of tumor-infiltrating lymphocytes or a cryopreserved formulation of tumor-infiltrating lymphocytes.

[0032] In some preferred embodiments, in (i), the tumor-infiltrating lymphocyte population obtained in (g) is cryopreserved, comprising the following steps: (1) Perform counting centrifugation, replace the supernatant with CS10 cryopreservation solution containing 0.5% HSA, and adjust the cell density in the cryopreservation solution to 1×10⁻⁶ cells / mL. 8 Quantities / ml, dispensed into cryovials; (2) Transfer the cryovials to a programmed cooling system where the chamber temperature has been reduced to 4°C for cryopreservation. The parameters of the programmed cooling system are as follows: 1. Wait at 4.0℃ 2. Ramp 1.0℃ / min until Sample = -10.0℃ 3. Ramp 15.0℃ / min until Chamber = -35.0℃ 4. Ramp 10.0℃ / min until Chamber = -22.0℃ 5. Ramp 1.0℃ / min until Sample= -40.0℃ 6. Ramp 10.0℃ / min until Chamber = -90.0℃ 7. End (3) After the program cooling device is finished, take out the cell cryopreservation tube and transfer it to the gas phase liquid nitrogen tank for long-term cryopreservation.

[0033] In some preferred embodiments, in (h), the tumor-infiltrating lymphocyte population obtained in (g) is expanded and cultured using the Xuri-W25 cell expansion system, including the following steps: (1) Xuri-Day 0~2: The tumor-infiltrating lymphocyte population was transferred to the Xuri-W25 cell expansion system for culture. ① If the total number of cells ≥ 2E9, the entire cell suspension was transferred to the Xuri-W25 cell expansion system for culture, with a final volume of 2 L, and the cell density was calculated at this time; ② If the total number of cells < 2E9, the entire cell suspension was transferred to the Xuri-W25 cell expansion system for culture, and according to the counting results, an appropriate amount of complete culture medium (AIM-V CTS medium containing 5 wt% human AB serum, 1 wt% penicillin antibody, 3000 IU / ml IL-2, 30 ng / ml OKT, 15 ng / mg IL-21, 5 ng / ml TGF-β, 100 ng / ml CD185) was added to adjust the cell density to 1×10 6 Cells / ml. Recorded as Xuri-Day 0; Xuri-W25 cell expansion system parameters were set as follows: CO2 concentration 5%, O2 concentration 21%, temperature 37℃, swing speed 0, and angle 0.

[0034] (2) Xuri-Day 2~4: When culturing TIL cells in the Xuri-W25 cell expansion system to Xuri-Day 2, ① if the total volume is less than 2 L, add fluid to 2 L; ② if the total volume is equal to 2 L, no fluid addition is required; start perfusion at a rate of 1 L / day. The parameters of the Xuri-W25 cell expansion system are set as follows: CO2 concentration of 5%, O2 concentration of 21%, temperature of 37℃, swing speed of 2, and angle of 2.

[0035] (3) Xuri-Day 4~7: Adjust the perfusion rate to 2 L / day. The parameters of the Xuri-W25 cell expansion system were set as follows: CO2 concentration of 5%, O2 concentration of 21%, temperature of 37℃, swing speed of 4, and angle of 4.

[0036] In some implementations, in (i), the expanded tumor-infiltrating lymphocyte population obtained in (h) is aliquoted or cryopreserved according to clinical needs to obtain a direct formulation or a cryopreserved formulation, including: (1) Direct formulation: Continuous flow centrifugation was performed using a Gentle Flex Pro high-throughput fully enclosed automated cell processing system. The supernatant was replaced with physiological saline containing 0.5% HSA, and the cell density was adjusted to 5-8 × 10⁻⁶ cells / year. 7 Cells / ml; Dispensed using the GentleP-Pac high-precision automated dispensing system into cell reinfusion bags, 5-8×10⁶ cells / ml. 9 / 100ml / bag, transfer to a 4℃ transport box for clinical use.

[0037] (2) Cryopreservation preparation: The cell density was adjusted to 5 × 10⁶ cells by continuous flow centrifugation using a Gentle Flex Pro high-throughput fully enclosed automated cell processing system, replacing the supernatant with CS10 cryopreservation solution containing 0.5% HSA. 7 Cells / ml. After cell aliquoting is complete, remove all cryopreservation bags and transfer them to a temperature-programmed freezer (TCF) with the chamber temperature already reduced to 4°C for cryopreservation. The TCF parameters are shown below: 1. Wait at 4.0℃ 2. Ramp 1.0℃ / min until Sample = -10.0℃ 3. Ramp 15.0℃ / min until Chamber = -35.0℃ 4. Ramp 10.0℃ / min until Chamber = -22.0℃ 5. Ramp 1.0℃ / min until Sample= -40.0℃ 6. Ramp 10.0℃ / min until Chamber = -90.0℃ 7. End After the cooling process is complete, remove the cell cryopreservation tubes and transfer them to a gas phase liquid nitrogen tank for long-term cryopreservation.

[0038] In a second aspect, the present invention provides a tumor-infiltrating lymphocyte population prepared by the preparation method described in the first aspect.

[0039] In some embodiments, the cells in the tumor-infiltrating lymphocyte population are in a poorly differentiated state and possess tumor-resident memory T cell characteristics (CD69). high CD49 high CD103 high CD28 low CD27 low CD62L low ).

[0040] In a third aspect, the present invention provides a tumor-infiltrating lymphocyte preparation, which is prepared by the preparation method described in the first aspect.

[0041] In some embodiments, the tumor-infiltrating lymphocyte preparation is an injectable solution.

[0042] In a fourth aspect, the present invention provides the use of the tumor-infiltrating lymphocyte population prepared by the preparation method of the first aspect, the tumor-infiltrating lymphocyte population of the second aspect, or the tumor-infiltrating lymphocyte preparation of the third aspect in the preparation of a medicament for treating tumors.

[0043] In some implementations, the drug for treating tumors is a TIL injection.

[0044] It is understood that the tumor-infiltrating lymphocyte populations or tumor-infiltrating lymphocyte preparations provided by this invention can be used to treat various tumors. In some specific embodiments, the tumor is a liquid tumor or a solid tumor.

[0045] The tumor-infiltrating lymphocyte population or tumor-infiltrating lymphocyte preparation provided by this invention has the following advantages: (1) In the process of culturing tumor-infiltrating lymphocyte populations, the present invention can maintain tumor-infiltrating lymphocytes in a low-differentiation state by adding a specific combination of cytokines, which is beneficial to improving the clinical treatment effect. (2) This invention predicts and synthesizes novel tumor antigen peptides, and co-cultures dendritic cells loaded with these novel tumor antigen peptides with tumor-infiltrating lymphocytes. This facilitates personalized and precise immune activation, effectively overcomes immune escape, and stimulates a stronger immune response, while reducing the occurrence of adverse reactions. Specifically, firstly, tumor neoantigens are generated by somatic mutations specific to the subject's tumor cells and have high individual specificity. Each subject's tumor produces different neoantigens due to its unique gene mutation spectrum. Known tumor antigens presented by dendritic cells (DCs) are usually common antigens that are prevalent in various tumor patients, such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). However, the presentation of tumor neoantigens by DCs can precisely activate the immune system based on the unique mutation characteristics of each subject's tumor cells, making the immune response more targeted. This personalized immune activation can more effectively identify and kill tumor cells in the subject's body, improve the treatment effect, and reduce the immune attack on normal tissues, thus reducing the risk of adverse reactions. Secondly, in order to evade the body's immune surveillance, tumor cells often downregulate or lose the expression of known tumor antigens. For example, in some melanoma patients, tumor cells may reduce the expression of known tumor antigens such as MART-1, thus significantly reducing the effectiveness of traditional immunotherapy based on known antigens. Tumor neoantigens, on the other hand, are newly generated by tumor cells, which have not yet had sufficient time to develop immune escape mechanisms against them. When dendritic cells (DCs) take up and present tumor neoantigens, they can activate immune responses against these unique neoantigens, breaking the tumor cells' immune escape strategies, reactivating the immune system's killing effect on tumor cells, and enhancing the effectiveness of immunotherapy. Third, because tumor neoantigens are absent in normal tissues, the immune system treats them as foreign substances, exhibiting high immunogenicity. When DCs take up and present them to T cells, they more easily trigger a strong immune response, especially activating cytotoxic T lymphocytes (CTLs). Once activated, CTLs can proliferate rapidly and specifically recognize and kill tumor cells expressing the corresponding neoantigens. In contrast, known tumor antigens, due to their long-term presence in the body, have developed a certain degree of tolerance to the immune system, resulting in relatively weaker immune activation. For example, in some patients with chronic cancer, the body's immune response to known antigens such as CEA gradually weakens, while dendritic cells (DCs) presenting neoantigens can revitalize the immune system and produce a stronger anti-tumor immune effect. Furthermore, DCs loaded with neoantigens can more effectively present antigen information to T cells, promoting T cells to develop into tumor-resident memory T cells (CD69). high CD49 high CD103 high CD28 low CD27 low CD62L lowFourth, the addition of IL-2, a cytokine that promotes the proliferation of tumor reactive T cells during the culture process, can greatly promote the growth and reproduction of TIL cells, which is beneficial to further enhance their anti-tumor effect. Attached Figure Description

[0046] Figure 1 A flowchart illustrating the preparation process of the tumor-infiltrating lymphocyte preparation in Example 1 is shown. Detailed Implementation

[0047] The materials, methods, and embodiments described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from this specification, the drawings, and the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0048] Unless otherwise specified, all cells, culture media, reagents, raw materials, drugs, instruments, etc. used in the following examples are commercially available.

[0049] Example 1 This embodiment provides a specific preparation process for a tumor-infiltrating lymphocyte preparation, the flowchart of which is shown below. Figure 1 As shown.

[0050] I. Extraction of TILs from Tumor Tissue 1. Cleaning: Under aseptic conditions, ex vivo tumor tissue was obtained from a non-small cell lung cancer patient, rinsed with tumor cleaning solution (50 μg / ml gentamicin Hanks balanced salt solution), and necrotic tissue and yellow adipose tissue were removed.

[0051] 2. Digestion: Cut the tumor tissue into 1-3 mm pieces. 3 Tissue fragments were added to 10 ml of a mixed digestion solution (10 ml of 2 wt% IV collagenase working solution + 10 ml of 0.25 wt% trypsin + 20 ml of physiological saline) at 37°C for 30 min. The digestion status was observed, and the fragments were mechanically minced and placed back into the 37°C water bath for 30 min. This process was repeated 3-4 times to obtain a cell suspension.

[0052] 3. Termination of digestion: Add 3 ml of 10% human AB serum in RPMI 1640 to each tube to terminate digestion.

[0053] 4. Collect the filtrate: Sieve the filtrate through a 70 μm cell sieve and collect the filtrate.

[0054] 5. Centrifugation: Centrifuge at 2100 rpm with the brake off for 10 min. After centrifugation, discard the supernatant and resuspend the cells in complete culture medium (RPMI 1640 containing 10 wt% human AB serum, 1 wt% penicillin and antibiotics, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HEPES, and 6000 IU / ml IL-2) to obtain a single-cell suspension. The cells obtained at this time are called the first cell population.

[0055] 6. Cell counting and plating: Mix the single-cell suspension by pipetting, count the cells, and adjust the cell density to 4 × 10⁶ cells / mL with complete culture medium. 6 Cells per ml were seeded into T25 bottles, 5 ml per bottle, and incubated at 37°C with 5% CO2 for 7 days. The resulting cells were termed the second cell population.

[0056] II. Sorting of T cells 1. Initial sorting: The second cell population was sorted using MACS CD3(TIL) MicroBead Kits to enrich T cells. Cell counts were performed, and the cell density was adjusted to 1×10⁻⁶ cells using complete culture medium (RPMI 1640 containing 10 wt% human AB serum, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HEPES, 6000 IU / ml IL-2, 30 ng / mg IL-7, 15 ng / mg IL-21, and 5 ng / ml TGF-β). 6 Fill the bottle with the solution of 100 cells / ml; replenish the solution every other day to maintain a density of 1×10⁻⁶. 6 Cells per ml, cultured for 7 days, are called the third cell population.

[0057] 2. Secondary sorting: The third cell population was sorted using MACS CD4 (TIL) and CD8 (TIL) MicroBead Kits to enrich TIL cells. Cell counts were performed, and the cell density was adjusted to 1×10⁻⁶ cells using complete culture medium (RPMI 1640 containing 10 wt% human AB serum, 10 mg / ml gentamicin, 1:100 GlutaMAX, 25 mM HEPES, 6000 IU / ml IL-2, 30 ng / mg IL-7, 15 ng / mg IL-21, and 5 ng / ml TGF-β). 6 Fill the bottle with the solution of 100 cells / ml; replenish the solution every other day to maintain a density of 1×10⁻⁶. 6 Cells / ml; after culturing for 7 days, the obtained cells are called the fourth cell population.

[0058] III. Prediction and Synthesis of Neoantigens in Cancer Patients 1. Sample processing and sequencing analysis Samples were collected from ex vivo normal lung tissue (taken from a location distant from the tumor and confirmed pathologically free of lesions) from the same patient as the tumor tissue. Whole-exome sequencing was performed on both tumor and normal lung tissues at a depth of 300X to ensure high-quality data acquisition. The processed tumor and normal lung tissue sequencing data were aligned to a human reference genome (e.g., GRCh38) using BWA software. BWA, through the construction of an FM index, can quickly and accurately locate sequencing reads to the reference genome and generate alignment files. Bioinformatics analysis detected 2000 somatic mutations. Among these mutations, a single nucleotide variant (c.2573T>G) was found on the EGFR gene. This variant results in the substitution of threonine (T) at position 858 of the EGFR protein for arginine (R) (p.T858R).

[0059] 2. HLA typing and neoantigen prediction HLA typing of peripheral blood samples from patients was performed using PCR-SBT, identifying HLA-A02:01 as the predominant MHC class I allele. Neoantigen prediction of the p.T858R mutant peptide of the EGFR gene was performed using Net-MHC 4.0 software. The results showed that this mutant peptide had a high binding affinity to the HLA-A02:01 molecule, with an IC50 value of [missing value]. 50 The value is 200 nM, which meets the screening criteria for neoantigens.

[0060] 3. Synthesis of neoantigen polypeptides The neoantigen peptide was synthesized using a solid-phase peptide synthesis method. Fmoc-protected arginine (Fmoc - Arg(Pbf)-OH) was used as the starting amino acid and ligated to Wang resin. EDC and HOBt were used as activators and auxiliary reagents to promote the coupling reaction between amino acids. After each coupling reaction, the Fmoc protecting group was removed using a 20% piperidine / DMF solution. After multiple rounds of reactions, a neoantigen peptide containing the EGFR p.T858R mutation site was successfully synthesized (sequence: Ala-Val-Leu-Arg-Ala-Val-Leu (SEQ ID NO: 1), where Arg is the mutated amino acid at position 4, corresponding to the EGFR p.T858R mutation site). The synthesized peptide was purified by HPLC to a purity of 98%, yielding a high-quality EGFR p.T858R neoantigen peptide for subsequent DC cell loading and immunotherapy studies. In subsequent in vitro experiments, this neoantigen peptide effectively activated the patient's T cells, enabling them to significantly kill tumor cells expressing the EGFR p.T858R mutation, demonstrating good immunotherapy potential.

[0061] IV. Extraction of PBMC 1. Draw fresh anticoagulated whole blood from the patient. Dilute the anticoagulated whole blood with physiological saline at a ratio of 1:1 and mix well. Calculate the amount of lymphocyte separation solution to be used based on a 1:1 ratio of peripheral blood to lymphocyte separation solution. Take the corresponding volume of lymphocyte separation solution and place it into a 50 ml centrifuge tube. Slowly add the diluted anticoagulated blood to the 50 ml centrifuge tube (do not mix with the separation solution; the two should separate into distinct layers). Centrifuge at 700 g for 20 min at 20℃, with an acceleration of 6 and a deceleration of 0.

[0062] 2. After centrifugation, the liquid levels in the tube, from top to bottom, are: diluted plasma layer, PBMC layer, separation fluid layer, and red blood cell layer. Discard the plasma layer, carefully aspirate the PBMC layer (i.e., the white membrane layer), and transfer it to a 15 mL centrifuge tube. Add physiological saline to the PBMC cells at a 1:1 volume ratio, mix well, and centrifuge at 2100 rpm for 10 min in a benchtop refrigerated centrifuge at 20°C. Repeat this washing step 1-2 times to obtain PBMCs.

[0063] V. Preparation of DC cells and loading of new antigens 1. Mix the PBMC suspension by pipetting, count the cells, and adjust the cell density to 1×10⁶ cells / mL using AIM-V culture medium. 6Cells were cultured at a density of 10 ml per 10 cm cell culture dish. The dish was incubated at 37°C with 5% CO2 for 90 minutes. The supernatant and non-adherent cells were then discarded. Adherent cells were washed with RPMI 1640 medium and then cultured in AIM-V medium for 60 minutes. The supernatant and non-adherent cells were then discarded. DC medium (RPMI 1640 medium + 5 wt% human serum + 1 wt% penicillin antibody, 1:100 glutamine, 800 IU / ml GM-CSF, 200 U / ml IL-4) was added. After four days of culture, neoantigen loading was performed.

[0064] 2. Change the medium for DC cells, dissolve the synthesized neoantigen peptide in PBS, add it to the DC cell culture dish, the final concentration of the neoantigen peptide is 10 μg / ml, and after culturing overnight, obtain DCs loaded with neoantigen.

[0065] VI. Presentation of DC-loaded neoantigen peptides and rapid amplification of TILs 1. Co-culturing neoantigen-loaded dendritic cells (DCs) with the fourth cell population to activate triglycerides (TILs): DCs loaded with neoantigens were counted and co-cultured with the fourth cell population at a TIL:DC ratio of 20:1 in a 1:1 mixed medium (AIM-V CTS medium with 5 wt% human AB serum, 1 wt% penicillin-dextrin, 10 mg / ml gentamicin, 1:10 GlutaMAX, 25 mM HEPES, 3000 IU / ml IL-2, 30 ng / ml OKT-3, 30 ng / ml IL-7, 15 ng / mg IL-21, and 5 ng / ml TGF-β), with a total density of 1×10⁻⁶ cells. 6 Cells / ml; on the third day, take half of the total cell culture suspension, centrifuge at 1800 rpm / min for 5 minutes, discard the supernatant, add an equal volume of mixed culture medium and continue culturing; replenish the medium every other day to maintain the density at 1×10⁶ cells / ml. 6 Cells / ml; when the total cell volume exceeds 100 ml, transfer to a cell culture bag for 7 days of culture. The resulting cells are termed tumor-infiltrating lymphocytes. They can be cryopreserved or further expanded using Xuri-W25 culture, depending on requirements.

[0066] 2. Cryopreservation: (1) Perform counting centrifugation, replace the supernatant with CS10 cryopreservation solution containing 0.5% HSA, and adjust the cell density in the cryopreservation solution to 1×10⁻⁶ cells / mL. 8 Dispense the vials / ml into cryovials and transfer them to a 4°C temporary storage refrigerator.

[0067] (2) After the cells are dispensed, take all cryopreservation tubes out of the 4°C temporary storage refrigerator, insert them into the cryopreservation rack, and transfer them to the programmed cooling instrument where the chamber temperature has been reduced to 4°C for cryopreservation.

[0068] (3) The parameters of the programmable cooling device are as follows: Profile Steps: 1. Wait at 4.0℃ 2. Ramp 1.0℃ / min until Sample = -10.0℃ 3. Ramp 15.0℃ / min until Chamber = -35.0℃ 4. Ramp 10.0℃ / min until Chamber = -22.0℃ 5. Ramp 1.0℃ / min until Sample= -40.0℃ 6. Ramp 10.0℃ / min until Chamber = -90.0℃ 7. End After the cooling process is complete, open the hatch, remove the cell cryopreservation tubes, and transfer them to a gas phase liquid nitrogen tank for long-term cryopreservation.

[0069] VII. Bioreactor Expansion of TIL Cells Xuri-W25 amplification culture: 1. Xuri-Day 0-2: Transfer the tumor-infiltrating lymphocyte population to the Xuri-W25 cell expansion system for culture. ① If the total cell count is ≥2E9, transfer all the cell suspension in the bag to the Xuri-W25 cell expansion system for culture, with a final volume of 2 L, and calculate the cell density at this point; ② If the total cell count is <2E9, transfer all the cell suspension in the bag to the Xuri-W25 cell expansion system for culture, and add an appropriate amount of complete culture medium according to the counting results to adjust the cell density to 1×10⁹ / L. 6 Cells / ml. Labeled Xuri-Day0, the medium contained 5% human AB serum, 1% penicillin antibody, 3000 IU / ml IL-2 in AIM-V CTS medium, 30 ng / ml OKT, 15 ng / mg IL-21, 5 ng / ml TGF-β, and 100 ng / ml CD185; the Xuri-W25 cell expansion system parameters were set as follows: CO2 concentration 5%, O2 concentration 21%, temperature 37℃, swing speed 0, and angle 0.

[0070] 2. Xuri-Day 2–4: When culturing TIL cells in the Xuri-W25 cell expansion system to Xuri-Day 2, ① if the total volume is less than 2 L, add fluid to 2 L; ② if the total volume is equal to 2 L, no fluid addition is needed; start perfusion at a rate of 1 L / day. The Xuri-W25 cell expansion system parameters are set as follows: CO2 concentration 5%, O2 concentration 21%, temperature 37℃, swing speed 2, and angle 2.

[0071] 3. Xuri-Day 4-7: Adjust the perfusion rate to 2 L / day. The Xuri-W25 cell expansion system parameters were set as follows: CO2 concentration 5%, O2 concentration 21%, temperature 37℃, swing speed 4, and angle 4. VIII. Formulation and Packaging of TIL Injection It can be formulated directly for clinical use or frozen for later use, depending on clinical needs.

[0072] 1. Direct formulation (1) Continuous flow centrifugation was performed using a Gentle Flex Pro high-throughput fully enclosed automated cell processing system. The supernatant was replaced with physiological saline containing 0.5% HSA, and the cell density was adjusted to 5-8 × 10⁻⁶ cells / mL. 7 per ml.

[0073] (2) Dispensing: Dispensing is performed using the Gentle P-Pac high-precision automatic dispensing system, dispensing the cells into cell reinfusion bags with a specification of 5-8×10. 9 100ml / bag, transfer to 4℃ transport box for clinical use.

[0074] 2. Frozen preparations (1) The cells were centrifuged continuously using a Gentle Flex Pro high-throughput fully enclosed automated cell processing system. The supernatant was replaced with CS10 cryopreservation solution containing 0.5% HSA, and the cell density was adjusted to 5 × 10⁶ cells / year. 7 per ml.

[0075] (2) After the cells are dispensed, take out all the cryopreservation bags from the 4°C temporary storage refrigerator, put them into the cryopreservation clip, insert them into the cryopreservation rack, and transfer them to the programmed cooling instrument where the chamber temperature has been reduced to 4°C for cryopreservation.

[0076] (3) The parameters of the programmable cooling device are as follows: Profile Steps: 1. Wait at 4.0℃ 2. Ramp 1.0℃ / min until Sample = -10.0℃ 3. Ramp 15.0℃ / min until Chamber = -35.0℃ 4. Ramp 10.0℃ / min until Chamber = -22.0℃ 5. Ramp 1.0℃ / min until Sample= -40.0℃ 6. Ramp 10.0℃ / min until Chamber = -90.0℃ 7. End After the cooling process is complete, open the hatch, remove the cell cryopreservation bag, and transfer it to a gas phase liquid nitrogen tank for long-term cryopreservation.

[0077] Example 2 This embodiment analyzes the biomarkers of the tumor-infiltrating lymphocyte preparation obtained in Example 1.

[0078] 1. Preparation of experimental materials: Obtain the tumor-infiltrating lymphocyte preparation obtained in Example 1, and use ordinary TIL cell samples as controls. Purchase fluorescently labeled antibodies against markers such as CD69, CD49, CD103, CD28, CD27, and CD62L, as well as flow cytometry buffer and 4% paraformaldehyde fixative.

[0079] 2. Experimental Procedure: Wash the cell samples twice with flow cytometry buffer, centrifuging at 1500 rpm for 5 minutes each time, and discard the supernatant. Count the washed cells using a hemocytometer and adjust the cell concentration to 1×10⁻⁶. 6 per ml.

[0080] Take 100 μl of cell suspension (approximately 1 × 10⁻⁶ cells) 5 Add cells to flow cytometry tubes and add anti-human CD69-FITC, anti-human CD49-PE, anti-human CD103-APC, anti-human CD28-PerCP-Cy5.5, anti-human CD27-BV421, and anti-human CD62L-BV510 antibodies, respectively. Add antibodies according to the working concentration recommended in the antibody's instructions; generally, add 5-10 μl of each antibody. Gently mix and incubate at 4°C in the dark for 30 minutes.

[0081] After incubation, add 1-2 ml of flow cytometry buffer to the flow cytometry tube, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and repeat the washing twice. After washing, add 200 μl of 4% paraformaldehyde to fix the cells and fix them at room temperature for 15 minutes. After fixation, wash once more with buffer, discard the supernatant, and prepare for flow cytometry analysis.

[0082] The processed cell samples were loaded onto the flow cytometer for analysis. Appropriate detection channels were set up to detect fluorescence signals for FITC, PE, APC, PerCP-Cy5.5, BV421, and BV510, respectively. At least 1 × 10⁻⁶ cells were collected. 4 Data for each cell.

[0083] 3. The experimental results are shown in Table 1.

[0084] Table 1. Raw data and statistical analysis of flow cytometry

[0085] As can be seen from Table 1, compared with the control group, the tumor-infiltrating lymphocyte preparation of the present invention can effectively induce tumor-infiltrating lymphocytes to exhibit the characteristics of tumor-resident memory T cells by loading tumor neoantigens onto DCs.

[0086] Example 3 1. Preparation of experimental materials Cell samples: A second cell population was obtained by extraction and culture following the steps in Example 1. The difference from Example 1 is that the second cell population was divided into three groups, each cultured to obtain a fourth cell population. In Group 1 (experimental group), the same cytokines were added during the culture of the second cell population to obtain the fourth cell population as in Example 1. In Group 2 (control group), a conventional combination of cytokines (IL-2: 10 ng / mL, IL-7: 5 ng / mL, IL-15: 5 ng / mL) was added during the culture of the second cell population to obtain the fourth cell population. In Group 3 (blank group), basal medium was used for the culture of the second cell population to obtain the fourth cell population. All other culture conditions were the same.

[0087] 2. Cytokines and reagents: Prepare flow cytometry-specific buffer (PBS containing 0.5% BSA and 2 mM EDTA), cell fixation solution (4% paraformaldehyde), and fluorescently labeled antibodies against markers of poorly differentiated T cells (such as CD45RA and CCR7) and markers of well-differentiated T cells (such as CD45RO and CD27): anti-human CD45RA-FITC, anti-human CCR7-PE, anti-human CD45RO-APC, anti-human CD27-PerCP-Cy5.5, etc.

[0088] 3. Experimental Procedure Cell phenotype analysis (flow cytometry): Cells were collected from three groups on days 7, 14, and 21 of culture. After washing the cells with PBS buffer, flow cytometry antibodies targeting markers of poorly differentiated T cells (such as CD45RA and CCR7) and those targeting markers of well-differentiated T cells (such as CD45RO and CD27) were added. Following the antibody manufacturer's instructions, 5-10 μl of each antibody was added, gently mixed, and incubated at 4°C in the dark for 30 minutes. The cells were then washed again with PBS, and the expression of cell surface markers was detected by flow cytometry. Appropriate detection channels were set up to detect fluorescence signals such as FITC, PE, APC, and PerCP-Cy5.5, and at least 1 × 10⁻⁶ cells were collected. 4 Data for each cell. 4. Experimental Results Results of the experimental group: On day 14 of culture, flow cytometry data analysis revealed that the proportion of cells expressing markers of poorly differentiated T cells (such as CD45RA and CCR7) was significantly higher than that of the control group and the blank group. For example, CD45RA... + The cell ratio reached 65%, CCR7 + The proportion of cells expressing highly differentiated T cell markers (such as CD45RO and CD27) was 60%; however, the proportion of cells expressing these markers was significantly lower than that of the control group and the blank control group. + The cell proportion was 25%, CD27 + The cell ratio of 30% indicates that this application can effectively maintain TIL in a low-differentiation state by adding a specific combination of cytokines during the culture process.

[0089] Control group results: Detected on day 14 of culture, CD45RA + The cell proportion was 40%, CCR7 + The cell proportion was 35%; CD45RO + The cell proportion was 45%, CD27 + The cell ratio was 40%. It can be seen that although some cytokines can maintain TILs in a certain low-differentiation state, their effect is not as good as the cytokine combination of this application.

[0090] Results in the control group: Under basal culture medium alone, TIL cells grew slowly and showed lower expression rates of low-differentiated T cell markers, including CD45RA. + The cell proportion was only 20%, CCR7 + The proportion of cells was 15%; the expression rate of markers of highly differentiated T cells was high, CD45RO + The cell proportion was 60%, CD27 +The cell ratio was 55%, indicating that it is difficult to maintain the low differentiation state of TILs without a specific combination of cytokines.

[0091] This embodiment, through comparison with the control group and the blank group, further demonstrates that the specific combination of cytokines used in this application has significant advantages in inducing tumor-infiltrating lymphocytes to maintain a low-differentiation state.

Claims

1. A method for preparing a population of tumor-infiltrating lymphocytes, comprising: (a) The tumor tissue was cleaned, digested, and centrifuged to extract tumor-infiltrating lymphocytes, thus obtaining the first cell population; (b) Adjust the cell density of the first cell population to 3 × 10⁻⁶. 6 ~4×10 6 Cells / ml, after culturing for 5-7 days, to obtain the second cell population; (c) The second cell population was initially sorted and enriched with T cells to achieve a cell density of 1 × 10⁻⁶. 5 ~1×10 6 Cells / ml, after culturing for 5-7 days, yielded the third cell population; (d) Secondary sorting and enrichment of tumor-infiltrating lymphocytes in the third cell population to achieve a cell density of 1×10⁻⁶. 5 ~1×10 6 Cells / ml, after culturing for 5-7 days, yielded the fourth cell population; (e) Whole-exome sequencing was performed on tumor tissue and normal tissue of the same origin to obtain whole-exome genome data. The whole-exome genome data of tumor tissue and normal tissue were compared with the human reference genome to screen for mutated genes. HLA typing was then performed on peripheral blood of the same origin to obtain HLA typing results. Based on the mutated genes and HLA typing results, the binding affinity of the peptides corresponding to the mutated genes to HLA molecules was predicted, and the affinity was calculated using IC50. 50 A polypeptide with a molecular weight below 550 nM was used as a tumor neoantigen polypeptide, and this tumor neoantigen polypeptide was synthesized. (f) Obtain PBMCs from the same source, separate and process them to obtain dendritic cells, and load the dendritic cells with tumor neoantigen peptides, wherein the proportion of dendritic cells loaded with tumor neoantigen peptides is 5~7×10 13 Dendritic cells: 1 mole of tumor neoantigen polypeptide, or 5~10×10 6 Each dendritic cell: 50~200 μg of tumor neoantigen polypeptide; (g) Dendritic cells loaded with tumor neoantigen peptides are co-cultured with the fourth cell population for 5-7 days to obtain a tumor-infiltrating lymphocyte population.

2. The preparation method according to claim 1, wherein, (b) The culture medium used for the culture is RPMI 1640 medium containing 1-10 wt% human AB serum, 0.5-1.5 wt% penicillin and antibiotics, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, and 3000-8000 IU / ml IL-2.

3. The preparation method according to claim 1, wherein, (c) The culture medium used for the culture is RPMI 1640 medium containing 0-10 wt% human AB serum, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, 3000-8000 IU / ml IL-2, 20-40 ng / mg IL-7, 10-20 ng / mg IL-21, and 3-8 ng / ml TGF-β.

4. The preparation method according to claim 1, wherein, (d) The culture medium used for the culture is RPMI 1640 medium containing 0-10 wt% human AB serum, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, 3000-8000 IU / ml IL-2, 20-40 ng / mg IL-7, 10-20 ng / mg IL-21, and 3-8 ng / ml TGF-β.

5. The preparation method according to claim 1, wherein, In (g), the ratio of the fourth cell population to the dendritic cells loaded with the tumor neoantigen polypeptide is (15-30):(0.5-1.5); and the total density of the fourth cell population and the dendritic cells loaded with the tumor neoantigen polypeptide is 1×10⁻⁶. 5 ~1×10 6 per ml.

6. The preparation method according to claim 1, wherein, In (g), the culture medium used for co-culture is a mixed culture medium of AIM-VCTS medium and RPMI 1640 medium mixed at a ratio of (0.5-1.5):(0.5-1.5), wherein the RPMI 1640 medium contains 0-10 wt% human AB serum, 0.5-1.5 wt% penicillin and antibiotics, 5-15 mg / ml gentamicin, 1:(50-150) GlutaMAX, 15-35 mM HERPES, 3000-8000 IU / ml IL-2, 20-40 ng / ml OKT-3, 20-40 ng / mg IL-7, 10-20 ng / mg IL-21, and 3-8 ng / ml TGF-β.

7. The preparation method according to any one of claims 1-6, wherein, The preparation method further includes: (h): The tumor-infiltrating lymphocyte population obtained from (g) is expanded and cultured in a bioreactor for 5-7 days to obtain the expanded tumor-infiltrating lymphocyte population; and / or (i): The tumor-infiltrating lymphocyte population obtained in (g) or the expanded tumor-infiltrating lymphocyte population obtained in (h) is aliquoted or cryopreserved to obtain a direct preparation of tumor-infiltrating lymphocytes or a cryopreserved preparation of tumor-infiltrating lymphocytes.

8. A tumor-infiltrating lymphocyte population, prepared by the preparation method according to any one of claims 1-6.

9. A tumor-infiltrating lymphocyte preparation, which is prepared by the preparation method according to any one of claims 1-6.

10. Use of the tumor-infiltrating lymphocyte population prepared by the preparation method according to any one of claims 1-6, the tumor-infiltrating lymphocyte population according to claim 8, or the tumor-infiltrating lymphocyte preparation according to claim 9 in the preparation of a medicament for treating tumors.

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