Use of modified TIL cells in combination with CAR-NK cells for the treatment of solid tumors

Abstract

The application develops a treatment method of a modified TIL cell combined with a CAR-NK cell on a solid tumor, the modified TIL cell comprises a nucleic acid encoding a perforin anchoring structure; the perforin anchoring structure comprises: a perforin gene, a GPI anchoring gene and a Neotag gene; the Neotag gene has at least 90% sequence identity with a nucleotide sequence selected from SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; the modification on the tumor infiltrating lymphocyte can improve its survival time, proliferation and killing capacity, and promote the infiltration of NK cells to the solid tumor, and synergistically mobilize the anti-tumor response of the immune cells of the patient; the CAR-NK cell targeting the NeoTag enhances the survival time, proliferation and killing capacity of the CAR-NK, and a suicide gene of the CAR-NK cell is designed, which can effectively target and eliminate tumor cells and reduce side effects.

Description

Technical Field

[0001] This invention relates to the field of medical immunology, and more specifically, to the application of modified TIL cells in combination with CAR-NK cells in the treatment of solid tumors. Background Technology

[0002] Tumor-infiltrating lymphocytes (TILs) are immune cells capable of entering the tumor microenvironment and directly contacting tumor cells. They are primarily CD4+ and CD8+ T cells, but also include a small number of B cells and NK cells. These cells can recognize tumor cell antigens and exert immune surveillance through interaction with tumor cells. Although TILs play a crucial role in tumor immune surveillance, tumor cells can also inhibit their function through various mechanisms, such as suppressing T cell activity via the PD-1 / PD-L1 pathway. Therefore, while TILs can infiltrate tumors, their function may be suppressed.

[0003] Chimeric antigen receptor NK cells (CAR-NK cells) refer to NK cells artificially implanted with a CAR structure. The CAR structure includes an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular domain. It can specifically recognize target cell antigens, directly activate NK cells, and kill target cells. Because CAR-NK cells are not inhibited by immunosuppressive factors in the tumor microenvironment and are less susceptible to inhibition by "immune checkpoints" such as PD-1, they may exhibit stronger anti-tumor activity in the tumor microenvironment compared to traditional tumor cell-derived immunosorbent assays (TILs).

[0004] Although CAR-NK cells have shown remarkable efficacy in treating hematologic malignancies, their application in solid tumors faces challenges such as low CAR-NK cell infiltration and loss of target antigens, resulting in less than ideal therapeutic effects of traditional CAR-NK cells in clinical trials for solid tumors. Summary of the Invention

[0005] On the one hand, the present invention provides the following technical solution:

[0006] The modified TIL cells include nucleic acids encoding a perforin anchoring structure; the perforin anchoring structure includes a perforin gene, a GPI anchoring gene, and a Neotag gene; the Neotag gene has at least 90% sequence identity with a nucleotide sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Wherein, at least 90% sequence identity includes at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 100% sequence identity.

[0007] On the other hand, the present invention provides the following technical solution:

[0008] The application of modified TIL cells combined with CAR-NK cells in the treatment of solid tumors includes the modified TIL cells and CAR-NK cells mentioned above.

[0009] The CAR structure of the CAR-NK cell includes an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular domain.

[0010] The antigen-binding domain of the CAR structure includes scFv; the hinge domain includes CD8 or CD28; the transmembrane domain includes CD8, CD28, NKG2D or proMP; the intracellular domain includes a co-stimulatory domain and a signal transduction domain, wherein the co-stimulatory domain includes CD28, 4-1BB or 2B4, and the signal transduction domain includes CD3 zeta.

[0011] In summary, the present invention has the following beneficial effects:

[0012] 1. This invention provides a method for modifying tumor-infiltrating lymphocytes (TILs), which can improve their survival time, proliferation and killing ability, and promote NK cell infiltration into solid tumors, thereby synergistically mobilizing the anti-tumor response of the patient's own immune cells;

[0013] 2. This invention provides a method for designing and preparing NeoTag-targeted CAR-NK cells, enhancing CAR-NK cell survival time, proliferation and killing ability, and designing a suicide gene for CAR-NK cells, which can effectively target and eliminate tumor cells and reduce side effects;

[0014] 3. This invention provides modified tumor-infiltrating lymphocytes (TILs) combined with CAR-NK cells for the treatment of various solid tumors, which can effectively kill tumor cells, generate a stronger and more durable immune response, and has high safety. Attached Figure Description

[0015] Figure 1 Image of perforin anchoring structure plasmid vector;

[0016] Figure 2 Designed for CAR structure;

[0017] Figure 3 Image of CAR structural plasmid vector;

[0018] Figure 4 For the statistical analysis of anti-tumor results. Detailed Implementation

[0019] The invention will be further described with reference to the accompanying drawings.

[0020] This invention provides modified TIL cells, including the introduction of nucleic acids encoding perforin anchoring structures and nucleic acids regulating TIL cell gene expression; the regulation of TIL cell gene expression includes upregulating endogenous gene expression and downregulating endogenous gene expression.

[0021] Preferably, the perforin anchoring structure includes: the perforin gene, the GPI (glycosylphosphatidyl alcohol) anchoring sequence, and the artificially designed Neotag sequence.

[0022] Preferably, the GPI anchoring sequence consists of acetylglucosamine (GlcNAc), phosphatidylinositol, mannose (Man), and ethanolamine (Etn). Ethanolamine is linked to the C-terminus of perforin via a phosphodiester bond, and the hydrophobic tail of phosphatidylinositol is embedded in the cell membrane, ensuring that perforin attaches to the target cell surface via the GPI anchoring sequence.

[0023] Preferably, NeoTag is a highly specific, artificially designed small molecule sequence that does not affect perforin activity, is sufficiently recognized by specific antibodies, and does not significantly affect normal cellular function; it uses a combination of non-natural amino acids to reduce the likelihood of being recognized by the normal immune system, ensuring that it is not expressed in normal tissues.

[0024] Preferably, the nucleic acids introduced to regulate gene expression include those that upregulate endogenous gene expression and those that downregulate endogenous gene expression.

[0025] Preferably, the step of upregulating endogenous gene expression includes inserting a recombinant nucleic acid sequence encoding one or more of the first target genes, namely CXCL9, CXCL10, and CCL5, into a perforin anchoring target structure before introducing it into TIL cells.

[0026] Preferably, the method of introduction includes lentiviral or retroviral infection.

[0027] Preferred methods for downregulating endogenous gene expression include knocking out a second target gene sequence using CRISPR / Cas9 technology, including PD-1, CTLA-4, LAG-3, TIM-3, TGFBR2, PTEN, Fas, SOCS1, and Cbl-b.

[0028] On the other hand, this invention provides a CAR structure design for CAR-NK cells. The antigen-binding domain of the CAR is composed of a specific scFv designed for the NeoTag sequence, which can specifically recognize and bind to target cells with NeoTag labels on their surface. The hinge domain is composed of one of CD8 and CD28. The transmembrane domain is composed of one of CD8, CD28, NKG2D, and programmed membrane protein (proMP). The intracellular domain includes a co-stimulatory domain composed of one of CD28, tumor necrosis factor receptor superfamily member 9 recombinant protein (4-1BB), and 2B4, and a signal transduction domain composed of CD3 zeta. CD28 provides short-term, potent killing ability, while 4-1BB provides sustained killing ability.

[0029] Preparation methods of CAR-NK cells: NK cells are derived from peripheral blood, umbilical cord blood, NK-92, NK92MI and other cell lines or iPSCs.

[0030] Preferably, the introduction of CAR-encoding nucleic acids includes: transfecting NK cells with lentivirus or retrovirus to obtain CAR-NK cells. Alternatively, transfecting CAR-NK cells with lentivirus or retrovirus to introduce nucleic acids encoding IL15, IL7, CCR2, Bcl-2, MCL1, CD16, HSV-TK, iCasp9, and rituximab-binding epitopes to achieve upregulation and insertion of the corresponding genes.

[0031] Preferably, the steps of upregulating endogenous gene expression and adding suicide gene include: inserting a third target gene sequence and a suicide gene sequence after the CAR nucleic acid sequence to obtain the target gene sequence, and then transfecting CAR-NK cells with a lentivirus or retrovirus containing the target gene sequence.

[0032] Preferably, downregulating endogenous gene expression includes knocking out a fourth target gene sequence using CRISPR / Cas9 technology.

[0033] Preferably, the third target genes include: interleukin 15 (IL15), interleukin 7 (IL7), CC chemokine receptor 2 (CCR2), B-cell lymphoma factor 2 (Bcl-2), myeloid leukemia protein 1 (MCL1), and CD16.

[0034] Preferred suicide genes include: herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (iCasp-9), and rituximab-binding epitopes.

[0035] Preferably, the fourth target gene includes: estrogen receptor-binding fragment-associated antigen 9 (EBAG9) and RAS GTPase activator protein 2 (RASA2).

[0036] The specific embodiments of the present invention mainly include methods for preparing modified TIL cells and CAR-NK cells, as well as methods for combined treatment of solid tumor-related diseases.

[0037] I. This example uses melanoma tumor tissue as an example to isolate, proliferate, and prepare modified tumor-infiltrating lymphocytes (TILs).

[0038] (1) Isolation of tumor-infiltrating lymphocytes

[0039] Melanoma B16-F10 cells were subcutaneously injected into C57BL / 6 mice (purchased from Vital River Pharmaceuticals, Beijing) to induce tumor formation. Fresh melanoma tumor tissue was obtained on day 14, washed twice in PBS solution containing antibiotics, and then cut into 1-2 mm pieces using sterile scissors or a scalpel. 3 Small pieces were added to RPMI 1640 culture medium containing 1 mg / mL collagenase IV and 20 U / mL DNase I and digested at 37°C for 1 h. After complete digestion, the obtained melanoma tumor samples were lysed with ACK lysis buffer. The mixture was filtered through a 70 μm cell filter to remove undigested tissue. The filtered cell suspension was washed with RPMI 1640, centrifuged, and resuspended in PBS. Then, TIL cells were enriched by density gradient centrifugation.

[0040] (2) Proliferation of tumor-infiltrating lymphocytes

[0041] The isolated TILs were cultured in a complete medium containing RPMI 1640, 10% FBS, 1% Penicillin-Streptomycin, 2mM L-Glutamine, and 25mM HEPES. The cells were then supplemented with mouse IL-2 (10ng / mL) and IL-7 (10ng / mL) and cultured for another 2 days. The medium and cytokines were changed every two days to obtain the target TIL cells.

[0042] (3) Obtaining nucleic acids encoding perforin anchoring structures

[0043] The perforin anchoring structure consists of the mouse perforin gene, a GPI (glycosylphosphatidyl alcohol) anchoring sequence, and a designed Neotag sequence. At the leading edge of the anchoring structure is a CD8 signal peptide, which can secrete the pre-synthesized perforin anchoring structure from TIL cells extracellularly. The nucleic acid encoding the perforin anchoring structure is obtained using PCR and artificial gene synthesis methods based on the designed nucleic acid sequence.

[0044] (4) Upregulation of endogenous gene expression

[0045] After obtaining the primary target gene nucleic acid using PCR technology, it was inserted into the perforin anchoring structure nucleic acid sequence using enzyme digestion and ligation technology. Then, the perforin anchoring structure and the primary target gene were translated and expressed separately using the T2A sequence to obtain novel coding nucleic acids. The primary target genes include: CXCL9, CXCL10, and CCL5.

[0046] (5) Introducing nucleic acids encoding novel perforin anchoring structures into TIL cells

[0047] A novel nucleic acid encoding a perforin anchoring structure was obtained and introduced into a lentiviral or retroviral transfer plasmid (containing the genomic sequence of the lentiviral or retroviral virus) via enzyme digestion and ligation. Virus packaging followed. The transfer plasmid, packaging plasmid (containing lentiviral or retroviral capsid proteins, structural proteins, etc.), and membrane protein particles (containing the outer membrane of the lentiviral or retroviral virus, etc.) were transfected into 293FT cells together, finally yielding packaged lentivirus or retrovirus containing the novel perforin anchoring structure. TIL cells were then processed at a rate of 2 × 10⁻⁶. 6 Transfer the cells to a 24-well plate, add 8 μg / mL of polybrene to the culture medium, add the above-mentioned virus for transfection, and continue to culture TIL cells until they reach a resting state after transfection.

[0048] (6) Downregulation of the expression of endogenous genes in modified tumor-infiltrating lymphocytes (TILs) using CRISPR / Cas9 technology

[0049] After designing the gRNA sequence of the second target gene, the corresponding DNA fragment was obtained using artificial gene synthesis methods, and the Cas9 nucleic acid was obtained using PCR technology. The DNA fragment of one of the second target gene's gRNAs was ligated to the Cas9 nucleic acid using enzyme digestion and ligation technology, and then introduced into TIL cells via lentivirus or retrovirus, thereby downregulating the expression of endogenous genes in TIL cells. The second target genes include: PD-1, CTLA-4, LAG-3, TIM-3, TGFBR2, PTEN, Fas, SOCS1, and Cbl-b.

[0050] II. Preparation of CAR-NK cells

[0051] (1) Acquisition of NK cells

[0052] Lymphocytes are extracted from peripheral blood or umbilical cord blood of humans or animals. NK cells are sorted from the lymphocytes using CD56 magnetic beads. Alternatively, cell lines such as NK-92 or NK-92MI can be used directly. Cells are amplified in MEM medium containing 0.2 mM inositol, 0.1 mM β-mercaptoethanol, 200 U / mL interleukin-2 (IL2), 12.5% ​​horse serum, 12.5% ​​fetal bovine serum, and 1% penicillin-streptomycin (37°C, 95% air, 5% CO2).

[0053] (2) Obtaining the nucleic acid encoding CAR

[0054] The antigen-binding domain of the CAR is composed of the scFv corresponding to the anti-NeoTag. The scFv consists of one heavy chain and one light chain from the variable region of the corresponding antibody, linked by a linker peptide; the linker peptide is a polymer of glycine and serine. The hinge domain is composed of one of CD8 or CD28. The co-stimulatory domain of the CAR is composed of one of CD28, 4-1BB, or 2B4. The signal transduction domain of the CAR is composed of CD3 zeta.

[0055] In addition, the leading edge of the CAR contains a CD8 signal peptide, which transports the CAR synthesized by NK cells to the desired location. After selecting and designing the CAR nucleic acid sequence as needed, the nucleic acid encoding the CAR is obtained through artificial gene synthesis.

[0056] (3) Upregulation of endogenous gene expression and introduction of suicide genes

[0057] After obtaining the nucleic acids of the third target gene and the suicide gene using PCR technology, they were ligated into the CAR nucleic acid sequence using enzyme digestion and ligation technology. The CAR, the third target gene, and the suicide gene were then translated and expressed separately using T2A and P2A sequences to obtain novel nucleic acids encoding the CAR. The third target genes include: IL15, IL7, CCR2, Bcl-2, MCL1, and CD16. The suicide genes include: HSV-TK, iCasp-9, and rituximab-binding epitopes.

[0058] (4) Introducing novel CAR-encoding nucleic acids into NK cells

[0059] After obtaining the novel CAR-encoding nucleic acid, it was introduced into a lentiviral or retroviral transfer plasmid (containing the lentiviral or retroviral genome sequence, etc.) via enzyme digestion and ligation, followed by viral packaging. The transfer plasmid, packaging plasmid (containing lentiviral or retroviral capsid proteins, structural proteins, etc.), and membrane protein particles (containing the lentiviral or retroviral outer membrane, etc.) were transfected together into 293FT cells, finally yielding packaged lentiviruses or retroviruses containing the novel CAR-encoding nucleic acid.

[0060] NK cells were seeded into 24-well plates. Then, a virus containing novel CAR nucleic acid and polybrene (8 μg / ml) were added to transfect the NK cells. After centrifugation at 1000g for 1 hour at room temperature, the cells were placed in an incubator. After incubation overnight, the medium was replaced to expand the NK cells, ultimately obtaining CAR-NK cells.

[0061] (5) Downregulate the expression of endogenous genes in CAR-NK cells

[0062] The expression of endogenous genes in CAR-NK cells was downregulated using CRISPR / Cas9 technology. After designing the gRNA sequence of the fourth target gene, the corresponding DNA fragment was obtained using artificial gene synthesis methods, and the Cas9 nucleic acid was obtained using PCR. The DNA fragment of one of the fourth target gene's gRNAs was ligated to the Cas9 nucleic acid using enzyme digestion and ligation technology, and then introduced into CAR-NK cells via lentivirus or retrovirus, thereby downregulating the expression of endogenous genes in CAR-NK cells. The fourth target genes included EBAG9 and RASA2.

[0063] III. Example 1: Modified tumor-infiltrating lymphocytes (TILs) combined with CAR-NK cells for the treatment of solid tumor-related diseases:

[0064] 1×10 melanoma B16-F10 cells 5 Up to 1×10 6 Subcutaneous injection of C57BL / 6 mice (purchased from Vital River Pharmaceuticals, Beijing) resulted in observable tumor formation 10-14 days later. Modified TIL cells were isolated, expanded, and prepared from the tumor-forming mice. Tumors were then analyzed in the tumor-forming mice (tumor volume reaching 100 mm²). 3 Treatment was performed on the left and right sides. Modified tumor-infiltrating lymphocytes were injected via the tail vein at a dose of 1×10⁻⁶. 6 Up to 5×10 6 10 cells, injected twice (3 days apart); 3 days after the second injection of modified TIL cells, CAR-NK cells were injected via tail vein at a dose of 1×10⁻⁶. 5 Up to 1×10 7 1 cell, 2 injections (3 days apart).

[0065] Anti-tumor result detection

[0066] Subcutaneous tumors were measured using vernier calipers, and the longest diameter (a) and shortest diameter (b) of the tumor were recorded. The tumor volume was calculated as V = a × b × 0.5 × b (mm3). The changes in tumor volume in each group were measured and recorded at 2-3 day intervals, and the survival status of mice was recorded and statistically analyzed. Mice were sacrificed on day 28, and the proportion of CAR-NK and TIL infiltration and retention in the tumor was detected.

[0067] Experimental results: There was no significant difference in mouse body weight between the PBS control group and the treatment group; the mice in the treatment group achieved significant tumor growth inhibition, and a few mice achieved complete remission. At 28 days, the tumor CAR-NK infiltration was 15.8% ± 3.2, and the TIL cell infiltration was 18.4% ± 2.5. The mice in the treatment group achieved the highest survival rate.

[0068] Comparative Example 1:

[0069] Using the same method, 1×10 melanoma B16-F10 cells were... 5 Up to 1×10 6 C57BL / 6 mice were subcutaneously injected, and tumors formed (at a volume of 50-100 mm). 3 TIL cells were excised and isolated. Nucleic acid encoding a novel perforin anchoring structure and a DNA fragment encoding a second target gene were introduced into the TIL cells along with Cas9 nucleic acid via lentivirus or retrovirus, thus obtaining modified tumor-infiltrating lymphocytes (TILs). Tumors were then tested in tumor-forming mice (tumor volume reaching 100 mm²). 3 Treatment involved injecting modified tumor-infiltrating lymphocytes via the tail vein (approximately 1 × 10⁻⁶) into the left and right sides of the body. 6 Up to 5×10 6 Two injections of 100 cells were administered (3 days apart). After 4 weeks, some mice achieved tumor suppression, and the percentage of TIL cell infiltration and retention was 13.7% ± 0.8%.

[0070] Comparative Example 2:

[0071] Using the NK-92MI cell line, CARs were constructed using the same method, but the antigen-binding domain was changed from the scFv corresponding to the anti-NeoTag to the scFv targeting GD2. The CAR-encoding nucleic acid was then transfected into NK cells via lentivirus or retrovirus to obtain GD2-CAR-NK cells. After tumor formation in mice, GD2-CAR-NK cells were injected via tail vein at a dose of 1 × 10⁻⁶. 5 Up to 1×10 7Two injections of 100 cells were administered (3 days apart). Mice were continuously monitored for survival, and subcutaneous tumor size was measured. Mice were sacrificed on day 28, and tumor tissue was excised to examine GD2-CAR-NK infiltration. Overall tumor growth was inhibited after 28 days, with an average tumor volume of 500-800 mm². 3 The CAR-NK infiltration rate was 5.7% ± 1.1%.

[0072] The experimental results of Example 1 and Comparative Examples 1-2 are shown in Table 1:

[0073] Table 1

[0074]

[0075] In summary, this invention demonstrates that the combined application of modified tumor-infiltrating lymphocytes and CAR-NK cells can significantly enhance the tumor-killing ability in vivo.

[0076] The results showed that the combined application of modified tumor-infiltrating lymphocytes (TILs) and CAR-NK cells enhanced the infiltration and killing ability of TIL cells, and significantly improved the survival time, proliferation, and killing ability of CAR-NK cells, effectively killing tumor cells. Furthermore, the use of small molecule inducing drugs such as AP1903 or NeoTag-labeled saponins (Tag-saporin) induced apoptosis in CAR-NK cells or selectively killed saponin-labeled CAR-NK cells, enhancing the controllability of CAR-NK cells. The combined application of modified tumor-infiltrating lymphocytes (TILs) and CAR-NK cells could better kill and inhibit the development and progression of various solid tumors.

[0077] In addition, the GPI anchor sequence, NeoTag and other sequences are as follows:

[0078] GPI-anchored genes:

[0079] (CD59A)SDGSLGKTPLLGTSVLVAILNLCFLSHL, denoted as SEQ ID NO: 1;

[0080] (CD55)GGDRYIYGHTCLITLTVLHVMLSLIGYLT, denoted as SEQ ID NO: 2;

[0081] (Ly6A)GSTWTMAGVLLFSLSSVLLQTLL, recorded as SEQ ID NO: 3;

[0082] (CD48)SSGVCWTATWLVVTTLIIHRILLT, denoted as SEQ ID NO: 4;

[0083] (CD90)CGGISLLVQNTSWMLLLLLSLSLLQALDFISL, denoted as SEQ ID NO: 5;

[0084] NeoTag reference sequence:

[0085] NQVSLTCL, denoted as SEQ ID NO: 6;

[0086] GGSGYALD, denoted as SEQ ID NO: 7;

[0087] LSPGTVHF, denoted as SEQ ID NO: 8;

[0088] CD8 signal peptide:

[0089] 5'-ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCT CCACGCCGCTCGGCCC-3', recorded as SEQ ID NO: 9.

[0090] T2A:

[0091] 5'-AGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAAC CCTGGCCCC-3', recorded as SEQ ID NO: 10.

[0092] Perforin: NM_011073.3.

[0093] CXCL9:NM_008599.4.

[0094] CXCL10:NM_021274.2.

[0095] CCL5:NM_013653.3.

[0096] IL15: NM_001254747.2.

[0097] IL7: NM_001313888.1.

[0098] CCR2: NM_009915.2.

[0099] Bcl-2: NM_009741.5.

[0100] MCL1: NM_008562.3.

[0101] CD16: NM_001356511.2.

[0102] HSV-TK: GenBank:ACC91769.1.

[0103] PD-1gRNA: 5'-CCGCCATACTTTGTGGGCAT-3', denoted as SEQ ID NO: 11;

[0104] CTLA-4gRNA: 5'-CAGCTTGTCCAACTGGTCGG-3', recorded as SEQ ID NO: 12;.

[0105] LAG-3gRNA: 5'-TCGAGGCCTGGCCGACGCGC-3', recorded as SEQ ID NO: 13;.

[0106] TIM-3gRNA: 5'-TGTTGAGATCGCCCTTTAGC-3', denoted as SEQ ID NO: 14;

[0107] TGFBR2 gRNA: 5'-GATCACGGCGTTCCACGCGA-3', denoted as SEQ ID NO: 15;

[0108] PTEN gRNA: 5'-AACTTGTCCTCCCGCCGCGT-3', denoted as SEQ ID NO: 16;

[0109] Fas gRNA: 5'-CAGGTCCCAGAAATCGCCTA-3', recorded as SEQ ID NO: 17;.

[0110] SOCS1 gRNA: 5'-CCGATTACCGGCGCATCACG-3', denoted as SEQ ID NO: 18;

[0111] Cbl-b gRNA: 5'-GCACTTCGTGCCTTACCGCG-3', denoted as SEQ ID NO: 19;

[0112] EBAG9 gRNA:5'-CCCACAAGTGTAAAGATCGA-3', denoted as SEQ ID NO: 20;

[0113] RASA2 gRNA: 5'-ATGCAGCAACAGTCGTACAA-3', denoted as SEQ ID NO: 21;

Claims

1. Modified TIL cells, characterized by: Including nucleic acids encoding perforin anchoring structures; The perforin anchoring structure includes: the perforin gene, the GPI anchoring gene, and the Neotag gene; The Neotag gene has at least 90% sequence identity with the nucleotide sequence selected from SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

2. The modified TIL cells according to claim 1, characterized in that: The GPI-anchored genes include acetylglucosamine, phosphatidylinositol, mannose, and ethanolamine.

3. The modified TIL cells according to claim 1 or 2, characterized in that: The GPI anchoring gene is selected from the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

4. The modified TIL cells according to claim 3, characterized in that: The ethanolamine is attached to the C-terminus of the perforin.

5. The modified TIL cells according to claim 4, characterized in that: The modified TIL cells also include nucleic acids that regulate TIL cell gene expression, including those that upregulate endogenous gene expression and those that downregulate endogenous gene expression.

6. The modified TIL cells according to claim 5, characterized in that: The step of upregulating endogenous gene expression includes inserting a recombinant encoding a first target gene into a perforin anchoring target structure and then introducing it into TIL cells; the first target gene includes one or more of CXCL9, CXCL10, and CCL5.

7. The modified TIL cells according to claim 5 or 6, characterized in that: The downregulation of endogenous gene expression includes knocking out a second target gene using CRISPR / Cas9 technology, the second target gene including PD-1, CTLA-4, LAG-3, TIM-3, TGFBR2, PTEN, Fas, SOCS1, or Cbl-b.

8. The application of modified TIL cells combined with CAR-NK cells in the treatment of solid tumors is characterized by: Including the modified TIL cells as described in any one of claims 1 to 7, and CAR-NK cells; The CAR-NK cells are obtained by introducing nucleic acids encoding the CAR structure into NK cells, and the gene expression of the CAR-NK cells is regulated. The CAR structure includes an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular domain. The antigen-binding domain of the CAR structure includes scFv; the hinge domain includes CD8 or CD28; the transmembrane domain includes CD8, CD28, NKG2D or proMP; the intracellular domain includes a co-stimulatory domain and a signal transduction domain, wherein the co-stimulatory domain includes CD28, 4-1BB or 2B4, and the signal transduction domain includes CD3 zeta.

9. The application of the modified TIL cells of claim 8 combined with CAR-NK cells in the treatment of solid tumors, characterized in that: The CAR structure also includes a third target gene and a suicide gene; the third target gene includes IL15, IL7, CCR2, Bcl-2, MCL1 or CD16; the suicide gene includes HSV-TK, iCasp-9 or rituximab binding epitopes.

10. The application of the modified TIL cells of claim 9 combined with CAR-NK cells in the treatment of solid tumors, characterized in that: The fourth target gene sequence, including EBAG9 or RASA2, was knocked out using CRISPR / Cas9 technology.

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