Tumor infiltrating lymphocytes expressing membrane binding cytokines

Abstract

The invention provides a tumor infiltrating lymphocyte expressing a membrane binding cytokine, and particularly provides a modified TIL, and an expression membrane of the modified TIL anchors IL-7. The TIL can be effectively activated and proliferated, can be efficiently detected and sorted, and can effectively mediate a molecular braking effect.

Description

Tumor-infiltrating lymphocytes expressing membrane-bound cytokines Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a tumor-infiltrating lymphocyte expressing a membrane-bound cytokine and a preparation method thereof. Background Art

[0002] Tumor infiltrating lymphocyte (TIL) therapy is an emerging immune cell therapy that has initially demonstrated excellent efficacy against solid tumors. The TILs present in the tumor are a highly heterogeneous group that is subject to immunosuppression by the tumor microenvironment, with limited tumor-killing effects and a limited duration in the body. Current TIL therapies involve culturing reactivated and expanded TIL cell populations from the patient's solid tumor tissue in vitro, which are then re-infused into the patient's body to achieve enhanced targeted solid tumor-killing effects. However, the immunosuppressive microenvironment inside the tumor still has an inhibitory effect on the reinfused TILs, and the activity and duration of the reinfused TILs in the body still need to be improved.

[0003] Modifying the unmodified natural TILs to be reinfused with exogenous genes is one of the means to solve the above problems. However, there are currently few types of gene-modified TILs in this field. There is an urgent need for more modification technologies that can enhance TIL proliferation ability, tumor killing efficacy and prolong its in vivo persistence.

[0004] Summary of the Invention

[0005] In a first aspect, the present invention first provides a modified TIL that expresses a membrane-anchored cytokine, wherein the cytokine is membrane-anchored IL-7.

[0006] In one or more embodiments, the membrane-anchored IL-7 comprises IL-7 or a functional fragment thereof and a transmembrane region. Preferably, the IL-7 or a functional fragment thereof is located at the N-terminal end of the transmembrane region.

[0007] In one or more embodiments, the amino acid sequence of IL-7 is shown in SEQ ID NO: 7; its exemplary coding sequence is shown in SEQ ID NO: 8.

[0008] In one or more embodiments, the transmembrane region is a protein transmembrane domain. Preferably, the protein transmembrane domain comprises any one selected from the group consisting of the CD8 transmembrane region, the CD28 transmembrane region, and the IL-7Rα transmembrane region. Preferably, the CD8 transmembrane region is shown in SEQ ID NO: 3, and an exemplary coding sequence thereof is shown in SEQ ID NO: 4.

[0009] In one or more embodiments, the transmembrane region is a GPI anchor region. Preferably, the GPI anchor region comprises a membrane anchor region of one or more GPI membrane anchor proteins selected from the group consisting of CD44, CD56, CD73, CD55, Thy1, AchE, IAP, ALPP, CD59, CD14, CD16, CD24, CD28, CD48, CD52, CD58, CD66a, CD66c, CD66d, CD66e, CD67, CD87, CD108, CD157, uPAR, JMH protein, GDNFR, CNTFR, TAG-1, PrP, phosphatidylinositol protein, semaphorin 7, CEA, GFR, Ly6G, transferrin receptor, contactin (F3), and T-cadherin. More preferably, the GPI anchor region is selected from the membrane anchor region of one or more GPI membrane anchor proteins: CD52, CD48, CD55, ALPP, and CD90.

[0010] In one or more embodiments, the membrane-anchored IL-7 further comprises a membrane surface tag. Preferably, the membrane surface tag is located between the IL-7 and the transmembrane region. Preferably, the membrane-anchored IL-7 comprises, from the N-terminus to the C-terminus, IL-7, the membrane surface tag, and the transmembrane region.

[0011] In one or more embodiments, the membrane surface tag comprises the extracellular domain of a B cell surface antigen or a variant thereof that retains antibody binding function. Preferably, the B cell surface antigen is BCMA, and the membrane surface tag is the extracellular region of BCMA or a variant thereof that retains anti-BCMA antibody binding function. Preferably, the variant is a truncated variant, an insertion variant, a deletion variant, a substitution variant, or a combination thereof.

[0012] In one or more embodiments, the BCMA extracellular region is represented by SEQ ID NO:21.

[0013] In one or more embodiments, the BCMA extracellular region variant is a truncated variant, such as that shown in SEQ ID NO: 23.

[0014] In one or more embodiments, the BCMA extracellular region variant is a truncated substitution variant, such as that shown in SEQ ID NO: 25.

[0015] In one or more embodiments, the membrane-anchored IL-7 further comprises a linker positioned between the IL-7 and the membrane surface tag. In one or more embodiments, the linker is a rigid linker or a flexible linker, preferably a rigid linker. Preferably, the sequence of the rigid linker is selected from any one of SEQ ID NOs: 9, 11, 15, and 17.

[0016] In one or more embodiments, the membrane-anchored IL-7 further comprises a hinge region or linker located at the N-terminus of the transmembrane region (e.g., between the membrane surface tag and the transmembrane region). Hinge regions include, but are not limited to, CD4 extracellular hinge regions, CD8 extracellular hinge regions, CD28 extracellular hinge regions, IgG1 Fc hinge regions, and IgG4 Fc hinge regions. Preferably, the hinge region is the CD8 extracellular hinge region. Preferably, the sequence of the CD8 extracellular hinge region is as shown in SEQ ID NO: 19. The linker is a rigid linker or a flexible linker, preferably a rigid linker. Preferably, the sequence of the rigid linker is selected from any one of SEQ ID NOs: 9, 11, 15, and 17.

[0017] Preferably, the linker is located between IL-7 and the membrane surface tag and / or between the membrane surface tag and the transmembrane region.

[0018] In one or more embodiments, the membrane-anchored IL-7 further comprises a signal peptide. The signal peptide is located at the N-terminus of the membrane-anchored IL-7. Preferably, the signal peptide is a CD52 signal peptide or an IL-7 signal peptide. Preferably, the CD52 signal peptide is set forth in SEQ ID NO: 1; an exemplary coding sequence thereof is set forth in SEQ ID NO: 2. Preferably, the IL-7 signal peptide is set forth in SEQ ID NO: 5; an exemplary coding sequence thereof is set forth in SEQ ID NO: 6.

[0019] In one or more embodiments, the membrane-anchored IL-7 comprises, linked in sequence: an optional signal peptide, IL-7, an optional linker, a BCMA extracellular region or a variant thereof, an optional hinge region or linker, and a transmembrane region, wherein the signal peptide is a CD52 signal peptide or an IL-7 signal peptide, and the transmembrane region is a CD8 transmembrane region or CD52.

[0020] In one or more embodiments, the membrane-anchored IL-7 comprises sequentially linked: a signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, and a transmembrane region.

[0021] In one or more embodiments, the membrane-anchored IL-7 comprises, linked in sequence:

[0022] CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region,

[0023] CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD52,

[0024] CD52 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, hinge region or linker, and CD52,

[0025] CD52 signal peptide, IL-7, BCMA extracellular domain or its variant, hinge region or linker, and CD52,

[0026] IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region,

[0027] IL-7 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, and CD52.

[0028] In one or more embodiments, the amino acid sequence of the membrane-anchored IL-7 is any one or more selected from SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, and 47.

[0029] In one or more embodiments, the nucleic acid sequence encoding the membrane-anchored IL-7 is any one or more of the nucleic acid sequences encoding SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, and 47. Preferably, the nucleic acid sequence encoding the membrane-anchored IL-7 is any one or more selected from SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, 46, and 48.

[0030] In a first group of embodiments of the first aspect, the TIL is prepared by the following method: incubating a sample containing TIL with a TIL seed cell culture medium to obtain a TIL cell population.

[0031] In one or more embodiments, the sample is selected from the group consisting of ascites, surgically resected primary lesion samples, synchronous and metachronous surgically resected metastatic lesion samples, puncture samples, and body fluids.

[0032] In one or more embodiments, the sample is a tissue containing tumor cells. Preferably, the tissue containing tumor cells is pre-treated, including fragmentation and / or dissociation of tumor tissue. The tumor cells are derived from a tumor selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer and osteosarcoma. Preferably, the tissue containing tumor cells is a tumor tissue or body fluid of a cancer subject.

[0033] In one or more embodiments, the body fluid comprises blood, pleural effusion, interstitial fluid, lymphatic fluid, and / or ascites.

[0034] In one or more embodiments, the seed cell culture medium includes the following components: cell culture components, cytokines and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include IL-2, the immune checkpoints include: PD-1, LAG-3, TIGIT and / or CTLA-4, and the cell culture components are serum culture medium or serum-free culture medium.

[0035] In one or more embodiments, the TIL seed cell culture medium includes any one of the following combinations of components: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α and basal culture medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody and basal culture medium; 4) IL-1β, IL-2, IL-7, G-CSF, GM-CSF, IFN-γ, LAG3 antibody, PD-1 antibody 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody and basal culture medium; 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α and basal culture medium; 7) IL-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1 antibody and basal culture medium; 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, CD40 antibody, OX-40 antibody, TIGIT antibody , PD-1 antibody and basal culture medium; 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α and basal culture medium; 10) IL-2, IL-7, IL-12, G-CSF, GM-CSF, IFN-α, IFN-γ, CD28 antibody, CD40 antibody, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101 and basal culture medium; 12) IL-2, IL-7, IL-15, GM-CSF, M-CS F, PD-1 antibody, CNI-1493 and basal culture medium; 13) IL-2, IL-7, IL-15, CD137 antibody, CD28 antibody, LAG3 antibody, PD-1 antibody, dasatinib and basal culture medium; 14) IL-2, IL-6, IL-12, G-CSF, M-CSF, IFN-β, IFN-γ, CTLA-4 antibody, PD-1 antibody, dasatinib, LYC-55716, GENE-1858 and basal culture medium; 15) IL-1α, IL-2, IL-9, IL-15, GM-CSF, CD137 antibody, CD28 antibody, LAG3 antibody, TIGIT antibody, CNI-1493 and basal culture medium;16) IL-2, IL-7, IL-12, IL-15, IL-21, G-CSF, M-CSF, IFN-γ, CD28 antibody, CD40 antibody, LAG3 antibody, PD-1 antibody, CNI-1493, dasatinib, GNE-1858, and basal culture medium. Preferably, the combination of the above components also includes serum and / or antibiotics.

[0036] In a second group of embodiments of the first aspect, the TIL is prepared by the following method: (1) incubating a sample containing TIL with a TIL seed cell culture medium to obtain a first TIL cell population, and (2) incubating the first TIL cell population with a TIL cell expansion culture medium to obtain a second TIL cell population.

[0037] In one or more embodiments, the sample is selected from the group consisting of ascites, surgically resected primary lesion samples, synchronous and metachronous surgically resected metastatic lesion samples, puncture samples, and body fluids.

[0038] In one or more embodiments, the sample is a tissue containing tumor cells. Preferably, the tissue containing tumor cells is pre-treated, including fragmentation and / or dissociation of tumor tissue. The tumor cells are derived from a tumor selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer and osteosarcoma. Preferably, the tissue containing tumor cells is a tumor tissue or body fluid of a cancer subject.

[0039] In one or more embodiments, the body fluid comprises blood, pleural effusion, interstitial fluid, lymphatic fluid, and / or ascites.

[0040] In one or more embodiments, the TIL seed cell culture medium in step (1) includes any one of the following combinations of components: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α and basal culture medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody and basal culture medium; 4) IL-1β, IL-2, IL-7, G-CSF, GM-CSF, IFN-γ, LAG3 antibody, PD-1 antibody, TNF-α and basal culture medium; 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody and basal culture medium; 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α and basal culture medium; 7) IL-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1 antibody and basal culture medium; 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, CD40 antibody, OX-40 antibody, TIGIT antibody, PD-1 antibody and basal culture medium; 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 Antibody, PD-1 antibody, TNF-α and basal culture medium; 10) IL-2, IL-7, IL-12, G-CSF, GM-CSF, IFN-α, IFN-γ, CD28 antibody, CD40 antibody, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101 and basal culture medium; 12) IL-2, IL-7, IL-15, G M-CSF, M-CSF, PD-1 antibody, CNI-1493, and basal culture medium; 13) IL-2, IL-7, IL-15, CD137 antibody, CD28 antibody, LAG3 antibody, PD-1 antibody, dasatinib, and basal culture medium; 14) IL-2, IL-6, IL-12, G-CSF, M-CSF, IFN-β, IFN-γ, CTLA-4 antibody, PD-1 antibody, dasatinib, LYC-55716, GENE-1858, and basal culture medium;15) IL-1α, IL-2, IL-9, IL-15, GM-CSF, CD137 antibody, CD28 antibody, LAG3 antibody, TIGIT antibody, CNI-1493, and basal culture medium; 16) IL-2, IL-7, IL-12, IL-15, IL-21, G-CSF, M-CSF, IFN-γ, CD28 antibody, CD40 antibody, LAG3 antibody, PD-1 antibody, CNI-1493, dasatinib, GNE-1858, and basal culture medium. Preferably, the combination of the above components also includes serum and / or antibiotics.

[0041] In one or more embodiments, the TIL cell expansion medium in step (2) comprises IL-2, IL-7, IL-15 and an immune checkpoint antibody or an antigen-binding fragment thereof, preferably, the immune checkpoint antibody comprises a PD-1 antibody.

[0042] In one or more embodiments, the TIL cell expansion medium further comprises platelets or platelets. The platelets are allogeneic platelets or the subject's own platelets.

[0043] In one or more embodiments, the TIL cell expansion medium in step (2) comprises any one of the following combinations of components: 17) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody and GM-CSF; 18) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody, CD137 antibody and GITR antibody; 19) IL-2, IL-7, IL-15, PD-1 antibody, CD3 antibody and CD28 antibody, preferably, the CD3 antibody and CD28 antibody Antibodies coupled to a matrix; 20) IL-2, IL-7, IL-15, PD-1 antibodies, IL-21, IL-12, CD3 antibodies, CD28 antibodies, and GM-CSF, preferably, the CD3 antibodies and CD28 antibodies are coupled to a matrix; 21) IL-2, IL-7, IL-15, PD-1 antibodies, CD3 antibodies, CD28 antibodies, and CD137 antibodies, preferably, the CD3 antibodies, CD137 antibodies, and CD28 antibodies are coupled to a matrix; 22) IL-2, IL-7, IL-15, PD-1 antibodies, GITR antibodies, CD3 antibodies, CD28 antibodies, TWS119, and C D137 antibody, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix; 23) IL-2, IL-7, IL-15, PD-1 antibody, TIGIT antibody, CD3 antibody, CD28 antibody, CD137 antibody and GM-CSF, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix; 24) IL-2, IL-7, IL-15, LAG3 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody, CD137 antibody and GITR antibody, preferably, the CD3 antibody, CD137 antibody and CD28 Antibodies are coupled to a matrix; 25) IL-2, IL-7, IL-15, PD-1 antibodies, TIGIT antibodies, CD3 antibodies, CD28 antibodies, CD137 antibodies, CD40 antibodies, OX-40 antibodies, and autologous platelets, preferably, the CD3 antibodies, CD137 antibodies, and CD28 antibodies are coupled to a matrix; 26) IL-2, IL-7, IL-15, PD-1 antibodies, CTLA-4 antibodies, CD3 antibodies, CD28 antibodies, CD137 antibodies, GITR antibodies, GM-CSF, TWS119, and allogeneic platelets, preferably, the CD3 antibodies and CD28 antibodies are coupled to a matrix.

[0044] In one or more embodiments, the TIL cell expansion medium further comprises serum and / or platelets, and a basal medium.

[0045] In one or more embodiments, the basal culture medium in the seed cell culture medium and the expansion culture medium is selected from any one or more of AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizer™, and FUJIFILM Irvin MHM-C.

[0046] In one or more embodiments, the serum is selected from human AB serum, autologous serum of a subject, or serum of animal origin.

[0047] The present invention also provides a method for preparing the modified TIL described in any embodiment herein, comprising introducing the nucleic acid sequence encoding the membrane-anchored IL-7 into the TIL.

[0048] In one or more embodiments, prior to introduction, the TIL does not express the membrane-anchored IL-7.

[0049] In one or more embodiments, the TIL that does not express the membrane-anchored IL-7 is an unmodified TIL.

[0050] In one or more embodiments, the unmodified TIL is prepared by the method for preparing TIL described in any one of the first or second group of embodiments of the first aspect of the present invention.

[0051] In one or more embodiments, the method comprises: introducing into TILs a nucleic acid construct, such as an expression vector or mRNA, that expresses the membrane-anchored IL-7. Preferably, the expression vector is a viral vector or a non-viral vector. Preferably, the expression vector is an integrative vector or a non-integrative vector. Preferably, the viral vector is a lentiviral vector.

[0052] In one or more embodiments, the non-viral vector is a vector based on a transposon system. Preferably, the non-viral vector is a vector based on the PiggyBac transposition system or the Sleeping Beauty transposition system. Preferably, the non-viral vector is a pKB20 or pNB328 vector.

[0053] In one or more embodiments, the expression vector comprises the nucleic acid sequence encoding the membrane-anchored IL-7 described in any embodiment of the first aspect of the present invention. Preferably, the expression vector is a pKB20 expression vector comprising the nucleic acid sequence encoding the membrane-anchored IL-7 described in any embodiment of the first aspect of the present invention.

[0054] In one or more embodiments, the method comprises introducing the nucleic acid construct into unmodified TILs by electroporation or liposome nanoparticles (LNPs).

[0055] In one or more embodiments, the method comprises: 1) incubating a sample containing TIL with the TIL seed cell culture medium described in any one of the second group of embodiments of the first aspect of the present invention to obtain a first TIL cell population; 2) introducing the nucleic acid construct expressing membrane-anchored IL-7 into the first TIL cell population by electroporation or liposome nanoparticles to obtain a second TIL cell population; and 3) incubating the second TIL cell population with the TIL cell expansion medium described in any one of the second group of embodiments of the first aspect of the present invention to obtain a third TIL cell population. Other features of the method are as described in any one of the second group of embodiments of the first aspect of the present invention.

[0056] The present invention also provides a modified TIL prepared by the method for preparing a modified TIL described in any embodiment herein.

[0057] The present invention also provides use of the modified TIL described in any embodiment herein in the preparation of a medicament for preventing or treating tumors.

[0058] The present invention also provides a method for preventing or treating tumors, comprising administering the modified TIL described in any embodiment herein to a subject in need thereof.

[0059] In one or more embodiments, the tumor is selected from one or more of the following: melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer and osteosarcoma.

[0060] The present invention also provides a polypeptide comprising: an optional signal peptide, IL-7, an optional linker, a BCMA extracellular region or a variant thereof, an optional hinge region or linker, and a transmembrane region connected in sequence, wherein the signal peptide is a CD52 signal peptide or an IL-7 signal peptide, and the transmembrane region is a CD8 transmembrane region or CD52.

[0061] In one or more embodiments, the polypeptide comprises, linked in sequence:

[0062] CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region,

[0063] CD52 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, hinge region or linker, and CD52,

[0064] CD52 signal peptide, IL-7, BCMA extracellular domain or its variant, hinge region or linker, and CD52,

[0065] IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region,

[0066] IL-7 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, and CD52.

[0067] Advantages of the present invention:

[0068] The present invention anchors the cytokine IL-7 on the surface of TILs through GPI-anchored proteins, which can effectively activate and proliferate TILs, significantly enhancing their tumor-killing effector function. Compared with conventional amino acid transmembrane domain fixation, the expression intensity of IL-7 on the membrane surface through GPI anchoring is significantly improved. At the same time, the present invention introduces wild-type or mutant sequences of BCMA, a small molecular weight protein not present on the surface of T cells, into the structure of membrane-anchored IL-7 as a detection and sorting tag. In addition to effectively detecting and sorting TILs that express membrane-anchored IL-7, the present invention can also effectively mediate ADCC and CDC effects in vivo in the presence of antibodies, promptly killing TILs expressing membrane-anchored IL-7 containing the BCMA extracellular region, acting as a molecular brake, and greatly improving the clinical safety of TILs expressing membrane-anchored IL-7. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1. RTCA killing curves of T002-TIL and T002-2 cells against homologous melanoma cells;

[0070] Figure 2. Tumor volume change curve of T014-9 cells killing SKOV-3320CDX mouse model in vivo;

[0071] Figure 3. In vivo fluorescence imaging results of T014-9 cells killing the SKOV-3320CDX mouse model;

[0072] Figure 4. Tumor volume change curves of T011-TIL and T011-9 cells killing syngeneic PDX mouse models in vivo;

[0073] Figure 5. Imaging evaluation results of tumor lesions in patient T011 before and after TIL infusion therapy;

[0074] Figure 6. Imaging evaluation results of tumor lesions in patient T012 before and after TIL infusion therapy;

[0075] Figure 7. Imaging evaluation results of tumor lesions in patient T013 before and after TIL infusion therapy;

[0076] Figure 8. Imaging evaluation results of tumor lesions in patient T014 before and after TIL reinfusion therapy. DETAILED DESCRIPTION

[0077] The inventors found that by partially anchoring the cytokines IL-7 and BCMA on the surface of TIL, they can effectively activate and proliferate TIL, detect and sort TIL that express positive membrane-anchored IL-7, and effectively mediate the molecular brake effect.

[0078] Definition of terms

[0079] In this application, the term "interferon" generally refers to a family of proteins with high species specificity that inhibit viral replication and cell proliferation and regulate immune responses. Typical suitable interferons include, but are not limited to, recombinant interferon alpha-2b, recombinant interferon alpha-2a, recombinant interferon alpha-2c, interferon alpha-n1, consensus alpha interferon, or interferon alpha-n3.

[0080] In this application, the term "colony stimulating factor" generally refers to a class of secreted glycoproteins that bind to receptor proteins on the surface of hematopoietic stem cells, thereby activating intracellular signal transduction pathways, which can lead to cell proliferation and differentiation into specific types of blood cells (usually white blood cells, for red blood cell formation, see erythropoietin). They can be artificially synthesized and administered exogenously.

[0081] In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is referred to as an "epitope" as described above. As described above, an antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily comprise both. An Fd fragment, for example, has two VH regions and generally retains some antigen-binding functions of a complete antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) a Fab fragment, a monovalent fragment having a VL, VH, constant light chain (CL), and CH1 domains; (2) a F(ab')2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is incorporated herein by reference in its entirety), which has a VH domain; (6) isolated complementarity determining regions (CDRs), and (7) single-chain Fv (scFv), e.g., derived from an scFV-library. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined using recombinant methods by synthetic linkers, which allow them to be prepared as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions are paired to form a monovalent molecule (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same manner as for intact antibodies. In this application, the term "antigen-binding fragment" also includes ligands that specifically bind to antigens, particularly cell surface receptor molecules. A ligand is generally a polypeptide or compound that can bind to a receptor protein (e.g., the antigen) with high affinity and specificity to elicit a functional response.For example, the ligand may include CD40L, a ligand of CD40, CD137L, a ligand of CD137, CD80, a ligand of CD28, and / or OX-40L, a ligand of OX-40.

[0082] In this application, the term "immune checkpoint" generally refers to a group of molecules on the cell surface of CD4+ and CD8+ T cells. These molecules can effectively act as "brakes" to downregulate or inhibit anti-tumor immune responses. These inhibitory molecules can be inhibited by suppressing DNA, RNA or protein levels.

[0083] In this application, the term "activating antibody" generally refers to antibodies targeting surface receptors of immune cells such as T cells, such as TCR and / or co-stimulatory receptors. These antibodies can activate the activation signaling pathways downstream of these receptors by binding to TCR and / or co-stimulatory receptors, thereby significantly increasing the activation and proliferation levels of the immune cells.

[0084] In this application, the term "co-stimulatory receptor" generally refers to a class of receptors expressed on the surface of immune cells, such as T cells, that activate immune responses. In the presence of antigen-presenting cells and the provision of a first signal, co-stimulatory receptors activate co-stimulatory signals by binding to their corresponding activating antibodies, ligands, or antigen-binding fragments, thereby fully activating immune effector cells. The activation of co-stimulatory receptor-mediated signaling pathways generally plays a key role in the effective occurrence of immune responses. Common co-stimulatory receptors include, but are not limited to, CD28, CD40, CD137, and OX-40.

[0085] In this application, the term "tumor necrosis factor TNF" generally refers to the TNF protein and is not the superfamily of TNF ligands. The Genbank accession number for human TNFα is NP_000585.2. The term "TNFα" encompasses precursor forms of TNFα, pro-TNFα (pro-TNFα), full-length TNFα, and any form of TNFα produced by intracellular processes. The term also encompasses naturally occurring and non-naturally occurring variants of TNFα, such as splice variants, allelic variants, and isoforms. TNFα can bind to two receptors, TNFR1 (TNF receptor type 1; CD120a; p55 / 60) and TNFR2 (TNF receptor type 2; CD120b; p75 / 80). TNFα acts as a proinflammatory cytokine, for example, in neuroinflammation.

[0086] In this application, the term "tumor infiltrating lymphocytes" generally refers to infiltrating lymphocytes isolated from tumor tissue. Immunohistochemical staining shows that tumor infiltrating lymphocytes can include CD3+T cells, CD20+, CD79α+B cells, plasma cells and CD56+NK cells; they can also include T cells, a small number of B cells, NK cells, macrophages and dendritic cells. The tumor infiltrating lymphocytes can produce highly efficient and specific anti-tumor killing effects through direct cytotoxic T lymphocytes and secretion of cytokines. In this application, the tumor infiltrating lymphocytes can include mononuclear leukocytes, which leave the bloodstream and migrate to a tumor. The tumor infiltrating lymphocytes can be selected from the group consisting of T cells, B cells, natural killer cells, and natural killer T cells.

[0087] In this application, the term "seed cells" generally refers to tumor-infiltrating lymphocytes cultured from tumor tissue using any suitable culture medium, including the culture medium described herein. These cells can serve as a starting cell population for subsequent expansion and culture, or as a cell population for an end-use, such as a cell population for transfusion therapy into an individual subject. The seed cells can be obtained from tumor tissue after any length of culture, which can be determined based on the subsequent use of the seed cells and is not limited to a specific culture duration.

[0088] In this application, the term "IL-15" generally refers to a proinflammatory cytokine that acts as a potent growth, survival, and activation factor for T cells, particularly intestinal intraepithelial lymphocytes (IELs) and natural killer (NK) cells, and may also be referred to as "IL-15 antigen" and "interleukin 15." Increased expression of IL-15 has been demonstrated in a variety of inflammatory diseases, including CD, rheumatoid arthritis (RA), and psoriasis (Malamut et al., 2010). IL-15 is considered a central regulator of CD immunopathology and a non-redundant driver of lymphomagenesis in RCD.

[0089] In this application, the term "IL-2" generally refers to a protein derived from a lymphokine that is produced by normal peripheral blood lymphocytes and present in low concentrations in the body. IL-2 was originally described by Morgan et al. (1976) Science 193:1007-1008 and was initially referred to as a T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes. The term also includes proteins that have been modified after expression of IL-2, such as by glycosylation, acetylation, phosphorylation, etc.

[0090] In this application, the term "IL-7" generally refers to a protein encoded by the IL-7 gene. IL-7 is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. It can be produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells. IL-7 may be involved in effects on T cells and B cells.

[0091] Throughout this application, the term "PD-1" generally refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells and binds to two ligands, PD-1 and PD-2. The complete hPD-1 sequence can be found in GenBank Accession No. U64863.

[0092] In this application, the term "LYC-55716" generally refers to a RORγ activator with a CAS number of 2055536-64-4.

[0093] In this application, the term "GNE-1858" generally refers to an HPK1 inhibitor with CAS number T11438.

[0094] In this application, the term "TWS119" generally refers to a GSK-3 inhibitor with CAS number 601514-19-6.

[0095] In this application, the term "RRX-001" generally refers to an epigenetic modulator with radiosensitizing activity. RRX-001 can downregulate the CD47 / SIRPα axis and repolarize TAMs and other immunosuppressive cells in the tumor microenvironment to an immunostimulatory phenotype. Its CAS number is 925206-65-1.

[0096] In this application, the term "CNI-1493" generally refers to a compound that can inhibit the production of inflammatory cytokines (including TNF), and its CAS number is 164301-51-3.

[0097] In this application, the term "anchoring" refers to fixing the moiety of interest to a substance through intermolecular interactions (chemical bonds, van der Waals forces, and hydrogen bonds). Membrane-anchored cytokines refer to cytokines that are anchored to the cell membrane.

[0098] In this article, the term "induced TIL" refers to the process of isolating TIL cells from animal tumors. After the TIL cells are expanded to a certain number in vitro, they can be infused back into the animal to control tumor growth and metastasis.

[0099] The present invention is suitable for isolating TILs from any tumor tissue, including tumor tissue obtained by open or minimally invasive surgery. Exemplary tumor tissues include gastric cancer, thyroid tumors, gallbladder cancer, bile duct cancer, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, osteosarcoma, and other cancers.

[0100] Detailed Description of the Invention

[0101] The present invention first provides a modified TIL expressing membrane-anchored IL-7. Membrane-anchored IL-7 is a polypeptide comprising one or more IL-7 or functional fragments thereof and a transmembrane region, optionally including a membrane surface tag. The sequence of IL-7 is shown in SEQ ID NO: 7. Functional fragments of IL-7 have common meanings in the art, and their sequences are known to those skilled in the art.

[0102] Although the inventors have found that GPI-anchored membrane-surface IL-7 expression intensity is significantly improved, the use of conventional protein transmembrane domains can also achieve certain effects. The transmembrane region can be the transmembrane region of the membrane surface tag or the transmembrane region of other transmembrane proteins, as long as it can anchor the membrane-anchored IL-7 to the cell membrane surface and does not negatively affect its activity and function. Therefore, those skilled in the art can select an appropriate transmembrane region as needed (e.g., based on the structure or composition of the membrane). The membrane spanning region includes but is not limited to CD28, CD8, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R β, IL-2R γ, IL-4R α, IL-7R α, IL-10R, IL-12R, IL-15R, IL-21R, CD27 and CD40. Any membrane spanning region. Exemplary membrane spanning region includes CD28 membrane spaning region, CD8 membrane spaning region, IL-7R membrane spaning region. The membrane spaning region is connected to its adjacent structure (such as membrane surface tag) by hinge region or joint. Therefore, the polypeptide can be a membrane surface protein or a membrane spaning protein.

[0103] Of course, the transmembrane region is preferably a GPI anchor region, which may include one or more membrane anchor regions of GPI membrane anchor proteins selected from the group consisting of CD44, CD56, CD73, CD55, Thy1, AchE, IAP, ALPP, CD59, CD14, CD16, CD24, CD28, CD48, CD52, CD58, CD66a, CD66c, CD66d, CD66e, CD67, CD87, CD108, CD157, uPAR, JMH protein, GDNFR, CNTFR, TAG-1, PrP, phosphatidylinositol protein, semaphorin 7, CEA, GFR, Ly6G, transferrin receptor, contactin (F3), and T-cadherin. Preferably, the GPI anchor region is selected from one or more membrane anchor regions of GPI membrane anchor proteins selected from the group consisting of CD52, CD48, CD55, ALPP, and CD90. The amino acid sequence of the GPI anchor region of CD52 is shown in SEQ ID NO: 29, and an exemplary coding sequence thereof is shown in SEQ ID NO: 30.

[0104] The membrane surface tag is located at the N-terminus or C-terminus of the IL-7. Therefore, the membrane surface cytokine may have a structure of: membrane surface tag-IL-7-transmembrane region, or IL-7-membrane surface tag-transmembrane region. In embodiments of multiple IL-7s, the membrane surface tag may be located at the N-terminus and C-terminus of any IL-7. For example, in the case of two IL-7s, the membrane surface cytokine may have a structure of: membrane surface tag-first IL-7-second IL-7-transmembrane region, or first IL-7-membrane surface tag-second IL-7-transmembrane region, or first IL-7-second IL-7-membrane surface tag-transmembrane region. And so on. Preferably, the IL-7 is located at the N-terminus of the membrane surface tag.

[0105] In the embodiment of multiple membrane surface tags, the multiple membrane surface tags can be connected in series or dispersed at any extracellular position of the membrane surface cytokine (for example, the N-terminus and C-terminus of any IL-7), as long as they are all located at the N-terminus of the transmembrane region and do not affect the function of the membrane surface tag and the membrane surface cytokine.

[0106] The N-terminus of the transmembrane region (e.g., between the membrane surface tag and the transmembrane region) may have a hinge region or a linker, or may have any sequence that does not affect the function of the membrane surface cytokine, such as a signal peptide or other polypeptide that needs to be expressed on the membrane surface. Hinge regions include, but are not limited to, the CD4 extracellular hinge region, the CD8 extracellular hinge region, the CD28 extracellular hinge region, the IgG1 Fc hinge region, and the IgG4 Fc hinge region. Preferably, the hinge region is the CD8 extracellular hinge region. Preferably, the sequence of the CD8 extracellular hinge region is shown in SEQ ID NO: 19; its exemplary coding sequence is shown in SEQ ID NO: 20. The linker is a rigid linker or a flexible linker, and its sequence is shown in SEQ ID NO: 9, 11, 13, 15, 17; its exemplary coding sequence is shown in SEQ ID NO: 10, 12, 14, 16, or 18, respectively. Preferably, the sequence of the rigid linker is any one selected from SEQ ID NO: 9, 11, 15, 17.

[0107] In embodiments containing a membrane surface tag, a linker may be provided between the IL-7 and the membrane surface tag. In one or more embodiments, the linker is a rigid linker or a flexible linker, preferably a rigid linker. Preferably, the sequence of the rigid linker is selected from any one of SEQ ID NOs: 9, 11, 15, and 17.

[0108] In certain embodiments, there is a linker between the IL-7 and the membrane surface tag and between the membrane surface tag and the transmembrane region.

[0109] Herein, the membrane surface tag (or membrane surface tag) includes the BCMA extracellular domain or a variant thereof that retains the function of binding to an anti-BCMA antibody. The BCMA extracellular domain is shown in SEQ ID NO: 21; its exemplary coding sequence is shown in SEQ ID NO: 22. The variant can be a truncated variant, an insertion variant, a deletion variant, a substitution variant, or a combination thereof that retains the function of binding to an anti-BCMA antibody. The sequence of the anti-BCMA antibody is not limited and can be any anti-BCMA antibody known to those skilled in the art. Exemplarily: a truncated variant of the BCMA extracellular domain is shown in SEQ ID NO: 23, and its exemplary coding sequence is shown in SEQ ID NO: 24; a substitution variant of the BCMA extracellular domain is shown in SEQ ID NO: 25, and its exemplary coding sequence is shown in SEQ ID NO: 26; a truncated substitution variant of the BCMA extracellular domain is shown in SEQ ID NO: 27, and its exemplary coding sequence is shown in SEQ ID NO: 28.

[0110] The membrane-anchored IL-7 may further include a signal peptide. Those skilled in the art can select an appropriate signal peptide based on the desired expression format. The signal peptide may be cleaved during secretion of the polypeptide. Exemplarily, the signal peptide is located at the N-terminus of the membrane-anchored IL-7. Preferably, the signal peptide is a CD52 signal peptide or an IL-7 signal peptide. Preferably, the CD52 signal peptide is set forth in SEQ ID NO: 1; an exemplary coding sequence thereof is set forth in SEQ ID NO: 2. Preferably, the IL-7 signal peptide is set forth in SEQ ID NO: 5; an exemplary coding sequence thereof is set forth in SEQ ID NO: 6.

[0111] In some embodiments, the present invention provides a polypeptide or a TIL expressing the polypeptide, wherein the polypeptide is a membrane-anchored IL-7, comprising sequentially linked: an optional signal peptide, IL-7, an optional linker, a BCMA extracellular region or a variant thereof, an optional hinge region or linker, and a transmembrane region, wherein the signal peptide is a CD52 signal peptide or an IL-7 signal peptide, and the transmembrane region is a CD8 transmembrane region or CD52. In some preferred embodiments, the membrane-anchored IL-7 comprises sequentially linked: a signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, and a transmembrane region. In some preferred embodiments, the membrane-anchored IL-7 comprises sequentially linked: (1) CD52 signal peptide, IL-7, a linker, the extracellular region of BCMA or a variant thereof, and a CD8 transmembrane region, (2) CD52 signal peptide, IL-7, a linker, the extracellular region of BCMA or a variant thereof, and CD52, (3) CD52 signal peptide, IL-7, a linker, the extracellular region of BCMA or a variant thereof, a hinge region or a linker, and CD52, (4) CD52 signal peptide, IL-7, the extracellular region of BCMA or a variant thereof, a hinge region or a linker, and CD52, (5) IL-7 signal peptide, IL-7, a linker, the extracellular region of BCMA or a variant thereof, and a CD8 transmembrane region, or (6) IL-7 signal peptide, IL-7, a linker, the extracellular region of BCMA or a variant thereof, and CD52. In an exemplary embodiment, the amino acid sequence of the membrane-anchored IL-7 is any one or more selected from SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, and 45. An exemplary encoding nucleic acid sequence thereof is any one or more selected from SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, and 46.

[0112] The present invention also includes fragments, derivatives, and analogs of the above-mentioned polypeptides. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the polypeptide. A fragment, derivative, or analog of a polypeptide or domain can be (i) a polypeptide or domain in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide or domain having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide or domain with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide or domain sequence (such as a leader sequence or secretory sequence, a sequence used to purify the polypeptide, a proprotein sequence, or a fusion protein formed with an antigenic IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0113] The term "variant" or "mutant" refers to a peptide or polypeptide that has an amino acid sequence that has been altered by insertion, deletion, or substitution of one or more amino acids compared to a reference sequence, but retains at least one biological activity. The mutants described in any embodiment herein include amino acid sequences that have at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and preferably at least 97% sequence identity to a reference sequence (e.g., an amino acid sequence encoded by SEQ ID NO: 2, 4, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 12, or 14 as described herein) and retain the biological activity of the reference sequence (e.g., as a chimeric antigen receptor, a surface cytokine expression protein, or a signal transduction receptor). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. Mutants also include amino acid sequences that have one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of a reference sequence while still retaining the biological activity of the reference sequence. The multiple mutations generally refer to within 1-10, such as 1-8, 1-5, or 1-3. Substitutions are preferably conservative substitutions. For example, in the art, conservative substitutions with amino acids with similar or similar properties generally do not alter the function of the protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in a polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0114] Truncated variants of the polypeptide or domain can also be used in the present invention, as long as the variant retains substantially the same biological function or activity as the polypeptide or domain, for example, a truncated variant of the BCMA extracellular domain that retains the antigen-antibody reactivity of the BCMA extracellular domain with an antibody.

[0115] The polypeptides described herein may be modified polypeptides. Modifications (generally without altering the primary structure) include chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation during polypeptide synthesis and processing or during further processing steps. Such modifications can be accomplished by exposing the polypeptide to a glycosylation enzyme (e.g., a mammalian glycosylase or deglycosylase). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or optimize their solubility.

[0116] Nucleic acid molecules encoding the polypeptides or domains are also within the scope of this invention. The nucleic acid molecules can be in the form of DNA or RNA. The nucleic acid molecules described herein include sequences that have been altered by codon optimization, as long as the amino acid sequence encoded by the nucleic acid molecule remains unchanged. Codon-optimized sequences can exhibit more suitable expressivity for a specific species. Methods for codon optimization of nucleic acid molecule sequences are well known in the art. The coding region sequence encoding the mature polypeptide can be a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence that encodes the amino acid sequence or a domain fragment thereof encoded by the nucleic acid sequence shown in the sequence listing, but differs from the corresponding nucleic acid sequence.

[0117] Nucleic acid molecules encoding mature polypeptides include: coding sequences encoding only the mature polypeptide; coding sequences encoding the mature polypeptide and various additional coding sequences; coding sequences encoding the mature polypeptide (and optional additional coding sequences) and non-coding sequences. The term "nucleic acid molecule encoding a polypeptide" may include a nucleic acid molecule encoding the polypeptide or a nucleic acid molecule further including additional coding and / or non-coding sequences.

[0118] The nucleic acid molecules of the present invention can be the coding sequence of a polypeptide or a domain, or the expression cassette of a polypeptide or a domain.Herein, the coding sequence refers to the part of the amino acid sequence that directly determines its protein product (such as CAR, single-chain antibody, hinge region, transmembrane region, intracellular signaling region or cytokine protein) in the nucleic acid sequence. The boundary of the coding sequence is usually determined by the ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of mRNA 5' end and the transcription termination sequence immediately downstream of the open reading frame of mRNA 3' end. The coding sequence may include, but is not limited to DNA, cDNA and recombinant nucleic acid sequences.Herein, expression cassette refers to the complete elements required for expressing a gene of interest, including promoter, gene coding sequence and Poly A tailing signal sequence. In certain embodiments, the coding sequence or expression cassette is integrated into the genome of the cell. Therefore, in these embodiments, the genome of the cell described herein is stably integrated with an expression cassette comprising an encoding polypeptide described herein.

[0119] The present invention also relates to variants of the aforementioned nucleic acid molecules, which encode polypeptides or fragments, analogs, and derivatives thereof having the same amino acid sequence as the present invention. These polynucleotide variants may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded polypeptide.

[0120] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80%, 85%, 90%, or 95% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0121] The present invention also relates to complementary sequences of the above sequences or nucleic acid fragments that hybridize with the above sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding desired polypeptides or domains. "Hybridization" as described herein mainly refers to the pairing of nucleic acid sequences under stringent conditions. Exemplary stringent conditions are hybridization and membrane washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0122] In certain embodiments, the nucleic acid molecule is a nucleic acid construct comprising a coding sequence for a polypeptide as described herein and one or more regulatory sequences operatively linked to these sequences. The polynucleotides of the present invention can be manipulated in a variety of ways to ensure expression of the polypeptide. The nucleic acid construct can be manipulated prior to insertion into a vector depending on the differences or requirements of the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0123] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used herein. The regulatory sequence can also be a suitable leader sequence, an untranslated region of an mRNA that is important for host cell translation. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention.

[0124] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. Specifically, the coding sequence of the polypeptide herein can be cloned into many types of vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequence of the polypeptide of the present invention. Expression vectors can be provided to cells in the form of vectors. Expression of the polynucleotide of the present invention is generally achieved by operably linking the coding sequence of the present invention to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence. The vector can be a virus, and viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome.

[0125] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.

[0126] Suitable promoters include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter sequence, elongated growth factor-1α (EF-1α), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.

[0127] In order to assess the expression of polypeptide, the expression vector of importing cell also can comprise selectable marker gene, so that from the cell group that seeks to be transfected or infected by viral vector, identify and select expressing cell.Selectable marker can be carried on single section DNA and used for co-transfection procedure.Selectable marker both flanks can have suitable regulatory sequence, so that can be expressed in host cell.Useful selectable marker comprises for example antibiotic resistance gene, such as neo etc.

[0128] The polynucleotides described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates to obtain the relevant sequences. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification into the correct order. Alternatively, the nucleic acid molecules described herein can be directly synthesized.

[0129] Methods for introducing nucleic acid molecules into cells and expressing genes into cells are known in the art. Nucleic acid molecules such as vectors or mRNA can be easily introduced into host cells, for example, mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means. Physical methods include calcium phosphate precipitation, lipid nanoparticle transfection, particle bombardment, microinjection, electroporation (electroporation) and the like. Biological methods include the use of DNA and RNA vectors. Chemical means include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes.

[0130] As described herein, host cells express polypeptides as described herein or contain nucleic acid molecules as described herein. Host cells include both final target cells (e.g., TILs) and various cells used in the production of target cells, such as E. coli cells, for example, to provide the coding sequence of a protein of the present invention or to provide a vector as described herein. In certain embodiments, a TIL stably expressing a polypeptide as described herein (membrane-anchored IL-7) is provided herein. Typically, prior to the introduction of membrane-anchored IL-7, the TIL does not express membrane-anchored IL-7, or the TIL is a TIL without any modification.

[0131] TILs suitable for use in the present invention can be various types of TILs from various sources, for example, derived from tissue containing tumor cells. Typically, the tissue containing tumor cells is pretreated, including fragmentation and / or dissociation of tumor tissue. The diameter of the fragmented and / or dissociated tumor tissue is about 1 mm to about 10 mm, for example, about 2 mm to about 10 mm, about 3 mm to about 10 mm, about 4 mm to about 10 mm, about 5 mm to about 10 mm, about 6 mm to about 10 mm, about 7 mm to about 10 mm, or about 8 mm to about 10 mm. The tumor cells are derived from a tumor selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the tissue containing tumor cells is tumor tissue or body fluid of a cancer subject. The body fluid includes blood, pleural effusion, tissue fluid, lymph and / or ascites. For example, the body fluid may include peritoneal effusion (e.g., ascites from a patient with gastric cancer or ascites from a patient with breast cancer) and / or pleural effusion (e.g., ascites from a patient with endometrial stromal sarcoma).

[0132] The method for preparing TIL is not limited. In a preferred embodiment, TIL is obtained using the method described in PCT / CN2021 / 133059, which is incorporated herein by reference in its entirety. In an exemplary embodiment, the TIL is prepared by the following method: a sample containing TIL is incubated with a TIL seed cell culture medium to obtain a TIL cell population.

[0133] The TIL seed cell culture medium herein includes any TIL seed culture medium known in the art, such as the TIL seed cell culture medium described in PCT / CN2021 / 096585 or PCT / CN2021 / 133059, which are incorporated herein by reference in their entirety. In a specific embodiment, the TIL seed cell culture medium includes the following components: cell culture components, cytokines, and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include IL-2, and the immune checkpoints include: PD-1, LAG-3, TIGIT, and / or CTLA-4, and the cell culture components are serum culture medium or serum-free culture medium. In one or more embodiments, the concentration of IL-2 is about 3000 IU / mL or less. The cytokines may also include one or more of IL-7, IL-15, TNFα, GM-CSF, G-CSF, M-CSF, IFN-γ, IFN-α, IFN-β, IL-4, IL-1α, IL-1β, IL-6, IL-9, IL-18, IL-12, IL-21, and IL-10. Exemplarily, the concentration of IL-7 is about 200 to about 1000 U / mL; the concentration of IL-15 is about 200 to about 500 U / mL; the concentration of TNFα is about 10 to about 100 pg / mL; and the concentration of GM-CSF is about 200 to about 5000 U / mL. The concentration of G-CSF is about 300 U / mL to about 1000 U / mL; the concentration of M-CSF is about 300 U / mL to about 800 U / mL; the concentration of IFN-γ is about 10 to about 1000 ng / mL; the concentration of IFN-γ is about 300 to about 1000 U / mL; the concentration of IFN-α is about 500 U / mL to about 1000 U / mL; the concentration of IFN-β is about 200 U / mL to about 500 U / mL; the concentration of IL-4 is about 200 to about 500 U / mL; The concentration of IL-1α is about 200-about 500 U / mL; the concentration of IL-1β is about 200-about 500 U / mL; the concentration of IL-6 is about 200-about 500 U / mL; the concentration of IL-9 is about 200-about 500 U / mL; the concentration of IL-18 is about 200-about 500 U / mL; the concentration of IL-12 is about 200-about 1000 U / mL; the concentration of IL-21 is about 200-about 500 U / mL; and the concentration of IL-10 is about 200-about 500 U / mL.

[0134] The immune checkpoint antibody or antigen-binding fragment thereof in the TIL seed cell culture medium can be a human PD-1 antibody or antigen-binding fragment thereof at a concentration of about 1 to about 100 μg / mL, or a LAG-3 antibody or antigen-binding fragment thereof at a concentration of about 3 to about 10 μg / mL, or a TIGIT antibody or antigen-binding fragment thereof at a concentration of about 1 to about 25 μg / mL, or a CTLA-4 antibody or antigen-binding fragment thereof at a concentration of about 1 to about 100 μg / mL.

[0135] The TIL seed cell culture medium may further include a co-stimulatory receptor antibody or an antigen-binding fragment thereof, such as a CD40 antibody or an antigen-binding fragment thereof, an OX-40 antibody or an antigen-binding fragment thereof, a CD137 antibody or an antigen-binding fragment thereof, and / or a CD28 antibody or an antigen-binding fragment thereof. The concentration of the CD137 antibody or an antigen-binding fragment thereof is about 1 to about 100 μg / mL; the concentration of the CD28 antibody or an antigen-binding fragment thereof is about 1 to about 10 μg / mL; the concentration of the CD40 antibody or an antigen-binding fragment thereof is about 5 to about 10 μg / mL; and the concentration of the OX-40 antibody or an antigen-binding fragment thereof is about 3 to about 10 μg / mL.

[0136] In addition, the seed cell culture medium may further include an M2 macrophage inhibitor, such as RRx001 and / or CNI-1493. The concentration of the M2 macrophage inhibitor is about 0.1 to about 100 μM. The seed cell culture medium may also include a regulatory T cell inhibitor, such as CAL-101, dasatinib, imatinib, and / or Panobinostat. The concentration of the regulatory T cell inhibitor is about 0.1 to about 100 μM. The seed cell culture medium may also include a bone marrow-derived suppressor cell (MDSC) inhibitor, such as AG490, decitabine, sunitinib, and / or BBI608. The concentration of the bone marrow-derived suppressor cell inhibitor is about 0.1 to about 100 μg / mL. The seed cell culture medium also includes a T cell activator, such as LYC-55716, GNE-1858, and / or methylene blue. The concentration of the T cell activator is about 1 to about 10 μM. The seed cell culture medium may further include a T cell differentiation inhibitor, such as TWS119. The concentration of the T cell differentiation inhibitor is about 1 to about 10 μM.

[0137] In a specific embodiment, the TIL seed cell culture medium includes any one of the following combinations of components: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α and basal culture medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody and basal culture medium; 4) IL-1β, IL-2, IL-7, G-CSF, GM-CSF, IFN-γ, LAG3 antibody, PD-1 antibody, TN F-α and basal culture medium; 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody and basal culture medium; 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α and basal culture medium; 7) IL-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1 antibody and basal culture medium; 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, CD40 antibody, OX-40 antibody, TIGIT antibody, P D-1 antibody and basal culture medium; 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α and basal culture medium; 10) IL-2, IL-7, IL-12, G-CSF, GM-CSF, IFN-α, IFN-γ, CD28 antibody, CD40 antibody, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101 and basal culture medium; 12) IL-2, IL-7, IL-15, GM-CSF, M-CSF , PD-1 antibody, CNI-1493 and basal culture medium; 13) IL-2, IL-7, IL-15, CD137 antibody, CD28 antibody, LAG3 antibody, PD-1 antibody, dasatinib and basal culture medium; 14) IL-2, IL-6, IL-12, G-CSF, M-CSF, IFN-β, IFN-γ, CTLA-4 antibody, PD-1 antibody, dasatinib, LYC-55716, GENE-1858 and basal culture medium; 15) IL-1α, IL-2, IL-9, IL-15, GM-CSF, CD137 antibody, CD28 antibody, LAG3 antibody, TIGIT antibody, CNI-1493 and basal culture medium;16) IL-2, IL-7, IL-12, IL-15, IL-21, G-CSF, M-CSF, IFN-γ, CD28 antibody, CD40 antibody, LAG3 antibody, PD-1 antibody, CNI-1493, dasatinib, GNE-1858, and basal culture medium. Preferably, the combination of the above components also includes serum and / or antibiotics. The serum concentration is about 1 to about 10% (v / v). Antibiotics include penicillin and / or streptomycin (e.g., PS dual-antibody). The antibiotic concentration is about 1 to about 200 U / mL.

[0138] In other embodiments, the TILs are prepared by the following method: (1) incubating a sample containing TILs with a TIL seed cell culture medium to obtain a first TIL cell population, and (2) incubating the first TIL cell population with a TIL cell expansion medium to obtain a second TIL cell population. The TIL seed cell culture medium is as described above.

[0139] TIL cell expansion medium can be any culture medium in the art that can proliferate TIL cells, such as a culture composition for amplifying TIL cells described in PCT / CN2021 / 133059 and a TIL cell expansion medium containing the culture composition, which is incorporated herein by reference in its entirety. In a specific embodiment, the TIL cell expansion medium comprises IL-2, IL-7, IL-15, an immune checkpoint antibody or its antigen-binding fragment and a basal medium. In addition, the expansion medium also includes any one, two, three or four of IL-21, IL-12, GM-CSF, and a small molecule that increases the ratio of memory T cells. Preferably, the expansion medium also includes IL-21 and IL-12, or IL-21, IL-12 and GM-CSF, or GM-CSF and a small molecule that increases the ratio of memory T cells. Preferably, the small molecule that increases the ratio of memory T cells is TWS119. The immune checkpoint antibodies include any one or more selected from PD-1 antibodies, CTLA-4 antibodies, LAG3 antibodies, TIM3 antibodies, TIGIT antibodies and BTLA antibodies.

[0140] The TIL cell expansion culture medium may also include autologous platelets or allogeneic platelets. The TIL cell expansion culture medium may also include any one or more selected from CD3 antibodies, CD28 antibodies, CD137 antibodies, OX40 antibodies, GITR antibodies, ICOS antibodies, CD206 antibodies and CD40 antibodies. Preferably, the culture composition also includes any one, two, three or four selected from CD3 antibodies, CD28 antibodies, CD137 antibodies, GITR antibodies. For example, the expansion culture medium also includes (1) CD3 antibodies and CD28 antibodies, or (2) CD3 antibodies, CD28 antibodies, CD137 antibodies and GITR antibodies.

[0141] In one or more embodiments, the TIL cell expansion medium comprises any one of the following combinations of components: 17) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody and GM-CSF; 18) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody, CD137 antibody and GITR antibody; 19) IL-2, IL-7, IL-15, PD-1 antibody, CD3 antibody and CD28 antibody, preferably, the CD3 antibody and CD28 antibody are combined with Matrix coupling; 20) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody and GM-CSF, preferably, the CD3 antibody and CD28 antibody are coupled to the matrix; 21) IL-2, IL-7, IL-15, PD-1 antibody, CD3 antibody, CD28 antibody and CD137 antibody, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to the matrix; 22) IL-2, IL-7, IL-15, PD-1 antibody, GITR antibody, CD3 antibody, CD28 antibody, TWS119 and CD1 37 antibody, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix; 23) IL-2, IL-7, IL-15, PD-1 antibody, TIGIT antibody, CD3 antibody, CD28 antibody, CD137 antibody and GM-CSF, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix; 24) IL-2, IL-7, IL-15, LAG3 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody, CD137 antibody and GITR antibody, preferably, the CD3 antibody, CD137 antibody and CD28 antibody 25) IL-2, IL-7, IL-15, PD-1 antibody, TIGIT antibody, CD3 antibody, CD28 antibody, CD137 antibody, CD40 antibody, OX-40 antibody and autologous platelets, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to the matrix; 26) IL-2, IL-7, IL-15, PD-1 antibody, CTLA-4 antibody, CD3 antibody, CD28 antibody, CD137 antibody, GITR antibody, GM-CSF, TWS119 and allogeneic platelets, preferably, the CD3 antibody and CD28 antibody are coupled to the matrix.

[0142] In one or more embodiments, the TIL cell expansion medium comprises a component selected from any one of the following and a basal medium, optionally further comprising serum and / or platelets: (1) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, and 3-100 μg / mL of PD-1 antibody, or an equivalent concentrate of these components; (2) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, and 1-100 μg / mL of PD-1 antibody, or an equivalent concentrate of these components; (3) 200-60 00 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 3-100 μg / mL of PD-1 antibody, 5-100 ng / mL of IL-21, 5-100 ng / mL of IL-12, 1-10 μg / mL of CD3 antibody, 1-10 μg / mL of CD28 antibody and 200-5000 U / mL of GM-CSF, or an equivalent concentrate of said components; (4) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 3-100 μg / mL of PD-1 antibody, 5-1 00 ng / mL of IL-21, 5-100 ng / mL of IL-12, 1-10 μg / mL of CD3 antibody, 0.5-10 μg / mL of CD28 antibody and 200-5000 U / mL of GM-CSF, or equipotent concentrates of said components; (5) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 3-100 μg / mL of PD-1 antibody, 5-100 ng / mL of IL-21, 5-100 ng / mL of IL-12, 1-10 μg / mL of CD3 antibody, 1-10 μg / mL of CD28 antibody, 1 -100 μg / mL CD137 antibody and 1-100 μg / mL GITR antibody, or equipotent concentrates of these components; (6) 200-6000 IU / mL IL-2, 5-100 ng / mL IL-7, 5-100 ng / mL IL-15, 1-100 μg / mL PD-1 antibody, 5-100 ng / mL IL-21, 5-100 ng / mL IL-12, 0.5-10 μg / mL CD3 antibody, 1-10 μg / mL CD28 antibody, 1-100 μg / mL CD137 antibody and 1-100 μg / mL GITR antibody, or equipotent concentrates of these components;(7) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 3-100 μg / mL of PD-1 antibody, 1-10 μg / mL of CD3 antibody, 1-10 μg / mL of CD28 antibody, 1-100 μg / mL of CD137 antibody, 1-100 μg / mL of GITR antibody, 200-5000 U / mL of GM-CSF and 1-500 μM of TWS119, or equivalent concentrates of these components; (8) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL -15, 1-100 μg / mL of PD-1 antibody, CD3 antibody conjugated to matrix, CD28 antibody conjugated to matrix, 1-100 μg / mL of CD137 antibody and 1-100 μg / mL of GITR antibody, or equivalent concentrates of these components; (9) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 1-100 μg / mL of PD-1 antibody, 5-100 ng / mL of IL-21, 5-100 ng / mL of IL-12, CD3 antibody conjugated to matrix, CD28 antibody conjugated to matrix, 1-100 μg / mL of CD 137 antibody and 1-100 μg / mL GITR antibody, or an equivalent concentrate of these components; (10) 200-6000 IU / mL IL-2, 5-100 ng / mL IL-7, 5-100 ng / mL IL-15, 1-100 μg / mL PD-1 antibody, CD3 antibody coupled to a matrix, CD28 antibody coupled to a matrix and CD137 antibody coupled to a matrix, or an equivalent concentrate of these components; (11) 200-6000 IU / mL IL-2, 5-100 ng / mL IL-7, 5-100 ng / mL IL-15, 1-100 μg / mL PD-1 antibody, CD3 antibody, CD28 antibody conjugated to matrix, CD137 antibody conjugated to matrix, 1-100 μg / mL GITR antibody and 1-500 μM TWS119, or equivalent concentrates of these components; (12) 200-6000 IU / mL IL-2, 5-100 ng / mL IL-7, 5-100 ng / mL IL-15, 1-100 μg / mL PD-1 antibody, CD3 antibody conjugated to matrix, CD28 antibody conjugated to matrix, CD137 antibody conjugated to matrix, 1-100 μg / mL TIGIT antibody and 200-5000 U / mL GM-CSF, or equivalent concentrates of these components;(13) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 1-100 μg / mL of LAG-3 antibody, 5-100 ng / mL of IL-21, 5-100 ng / mL of IL-12, 0.5-10 μg / mL of CD3 antibody, 0.5-10 μg / mL of CD28 antibody, 1-100 μg / mL of CD137 antibody, and 1-100 μg / mL of GITR antibody, or Equivalent concentrates of these components; (14) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 1-100 μg / mL of PD-1 antibody, matrix-conjugated CD3 antibody, matrix-conjugated CD28 antibody, matrix-conjugated CD137 antibody, 1-100 μg / mL of TIGIT antibody, 1-100 μg / mL of CD40 antibody, 1-100 μg / mL of OX-40 antibody, and 1×10; 8 -5×10 8 / mL of autologous platelets, or concentrates of these components in equal proportions; (15) 200-6000 IU / mL of IL-2, 5-100 ng / mL of IL-7, 5-100 ng / mL of IL-15, 1-100 μg / mL of PD-1 antibody, 1-100 μg / mL of CTLA-4 antibody, matrix-coupled CD3 antibody, matrix-coupled CD28 antibody, 1-100 μg / mL of CD137 antibody, 1-100 μg / mL of GITR antibody, 200-5000 U / mL of GM-CSF, and 1×10 8 -5×10 8 / mL of allogeneic platelets, or concentrates of these components in equal proportions.

[0143] The antibodies in the culture medium herein can be antibodies or recombinant antibodies from any species, preferably monoclonal antibodies. These antibodies are well known in the art, for example, PD-1 antibody BeiGene, PD-1 antibody Sindili, PD-1 antibody Tepli, PD-1 antibody Erica, CD3 antibody ab86883, CD28 antibody MAB342, GITR antibody as shown in SEQ ID NO: 104 and SEQ ID NO: 105 in CN103951753B, CD137 antibody as shown in SEQ ID NO: 10 and SEQ ID NO: 11 in CN111182919A. These patent documents or disclosures are incorporated herein by reference in their entirety. One or more antibodies in the TIL cell seed culture medium and / or expansion culture medium described herein can be coupled to a matrix, such as a microsphere, a well plate, a petri dish, a cell culture flask, or a cell culture bag.

[0144] The serum described herein can be any serum known in the art to be useful for animal cell (e.g., TIL cell) culture, such as human AB serum, subject autologous serum, or animal-derived serum. The concentration of the serum is 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values.

[0145] The platelets described herein may be platelets derived from the subject's blood or platelets derived from allogeneic blood. The density of platelets is 0.5×10 8 / mL-3×10 8 / mL, for example 0.5×10 8 / mL, 1×10 8 / mL, 1.5×10 8 / mL, 2×10 8 / mL, 2.5×10 8 / mL, 3×10 8 / mL, or the range between any two of the above values.

[0146] The "basal medium" described herein is any medium known in the art that can be used for TIL cell culture, including but not limited to any one or more of AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizer™, and FUJIFILM Irvin MHM-C. The components and amounts of these media are well known in the art, and their sources are described in the Examples.

[0147] The concentration of TIL (non-electroporated or electroporated) inoculated into TIL cell expansion medium is not limited and can be selected by those skilled in the art. In a preferred embodiment, 2.0×10 5 -5.0×10 5 TILs are inoculated into TIL cell expansion medium at a concentration of about 2.0×10 5 , about 2.5×10 5 , about 3.0×10 5 , about 4.5×10 5 / mL or the range between any two values.

[0148] The time of " cultivating " or " incubation " described herein can be about 3-20 days. For example, the cultivation or incubation time can be about 3-15 days, for example, can be about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days or about 20 days or the range between any two values. During the use of TIL cell culture (for example, during the incubation of TIL cells using TIL cell expansion medium), the corresponding culture medium can be updated every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0149] For example, the temperature of the culture or incubation can be about 30-42° C. For example, it can be at least about 30° C., at least about 31° C., at least about 32° C., at least about 33° C., at least about 34° C., at least about 35° C., at least about 36° C., at least about 37° C., at least about 38° C., at least about 39° C., at least about 40° C., at least about 40° C., or at least about 42° C.

[0150] For example, the CO2 concentration of the culture or incubation is about 1%-10%, for example, it can be at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%.

[0151] The present invention includes a method for preparing the modified TIL and the modified TIL obtained therefrom, the method comprising the step of introducing the membrane-anchored IL-7 or its encoding nucleic acid molecule described herein into the TIL obtained by culture as above. In some embodiments, the introduction is achieved by electroporation. The cells after the introduction (e.g., electroporation) of the coding sequence may be activated (or activated) before subsequent operations (e.g., culture). Typically, activation is performed by incubating the electroporated cells in a TIL cell expansion medium for at least 1 day. The antibodies in the TIL cell expansion medium may be coated on the surface of the container or suspended in the culture medium. The incubation is preferably 1-10 days, for example, 1-5 days. The incubation conditions can be determined by those skilled in the art, for example, 37°C 5% CO2. In some embodiments, during the activation incubation, the resuspension concentration of the electroporated cells in the TIL cell expansion medium is 1.0×10 5 -1.0×10 6 / mL, for example 1.0×10 5 , 2.0×10 5 , 2.5×10 5 , 5.0×10 5 , 5.2×10 5 , 1.0×10 6 / mL or the range between any two values.

[0152] Therefore, in one or more embodiments, the present invention provides a modified TIL that expresses the membrane-anchored IL-7 described herein, and the modified TIL is prepared by the following method: 1) incubating a sample containing TIL with the TIL seed cell culture medium described herein to obtain a first TIL cell population, 2) introducing (e.g., by electroporation) a nucleic acid molecule encoding the membrane-anchored IL-7 (e.g., an expression vector) into the first TIL cell population to obtain a second TIL cell population that stably expresses membrane-anchored IL-7, and optionally 3) activating the electroporated cells.

[0153] In one or more embodiments, the present invention provides a modified TIL that expresses the membrane-anchored IL-7 described herein, and the modified TIL is prepared by the following method: 1) incubating a sample containing TIL with the TIL seed cell culture medium described herein to obtain a first TIL cell population, 2) incubating the first TIL cell population with the TIL cell expansion medium described herein to obtain a second TIL cell population, 3) introducing (e.g., by electroporation) a nucleic acid molecule encoding the membrane-anchored IL-7 (e.g., an expression vector) into the second TIL cell population to obtain a third TIL cell population that stably expresses membrane-anchored IL-7, and optionally 4) activating the electroporated cells.

[0154] In one or more embodiments, the present invention provides a modified TIL that expresses the membrane-anchored IL-7 described herein, and the modified TIL is prepared by the following method: 1) incubating a sample containing TIL with the TIL seed cell culture medium described herein to obtain a first TIL cell population, 2) introducing (e.g., by electroporation) a nucleic acid molecule encoding the membrane-anchored IL-7 (e.g., an expression vector) into the first TIL cell population to obtain a second TIL cell population, optionally 3) activating the electroporated cells, and 4) incubating the second TIL cell population with the TIL cell expansion medium described herein to obtain a third TIL cell population that stably expresses membrane-anchored IL-7.

[0155] In some embodiments of preparing the modified TILs herein, the TIL seed cell culture medium is selected from any one or more of the following (a), (b) and (c): TIL seed cell culture medium (a): IL-2 (3000 U / mL), IL-7 (50 ng / mL), IL-15 (50 ng / mL), IFN-γ (1000 U / mL), anti-CD137 activating monoclonal antibody (5 μg / mL), anti-CD40 activating monoclonal antibody (5 μg / mL), anti-OX-40 activating monoclonal antibody (5 μg / mL), anti-TIGIT blocking monoclonal antibody (5 μg / mL), anti-PD-1 blocking monoclonal antibody (15 μg / mL), PS double antibody (100 U / mL) and X-VIVO supplemented to the final volume. 15 basal medium; TIL seed cell medium (b): IL-2 (3000 U / mL), IL-7 (20 ng / mL), IL-15 (20 ng / mL), GM-CSF (500 U / mL), IFN-γ (1000 U / mL), anti-CD137 activating monoclonal antibody (3 μg / mL), anti-CD28 activating monoclonal antibody (3 μg / mL), anti-PD-1 blocking monoclonal antibody (3 μg / mL), TNF-α (10 pg / mL) and X-VIVO to the final volume 15 basal medium; TIL seed cell medium (c): IL-2 (3000U / mL), IL-7 (20ng / mL), IL-15 (30ng / mL), anti-CD137 activating antibody (12μg / mL), anti-CD28 activating monoclonal antibody (5μg / mL), anti-LAG-3 blocking monoclonal antibody (5μg / mL), anti-PD-1 blocking monoclonal antibody (3μg / mL), dasatinib (3μM), 5v / v% human AB serum and X-VIVO 15 basal medium to the final volume. Alternatively or in addition, in some embodiments of preparing the modified TILs herein, the TIL cell expansion medium is selected from any one or more of the following (d) and (e): TIL cell expansion medium (d): IL-2 (500 U / mL), IL-7 (5 ng / mL), IL-15 (10 ng / mL), anti-PD-1 blocking monoclonal antibody (3 μg / mL), anti-CD3 activating monoclonal antibody, anti-CD28 activating monoclonal antibody, 5 v / v% human AB serum and X-VIVO 15 basal medium supplemented to the final volume.The anti-CD3 activating monoclonal antibody and the anti-CD28 activating monoclonal antibody are coated on the surface of the culture vessel; TIL cell expansion medium (e): IL-2 (500U / mL), IL-7 (7ng / mL), IL-15 (30ng / mL), anti-PD-1 blocking monoclonal antibody (1μg / mL), anti-CD3 activating monoclonal antibody, anti-CD28 activating monoclonal antibody, anti-CD137 activating monoclonal antibody, 5v / v% human AB serum and X-VIVO 15 basic medium supplemented to the final volume, the anti-CD3 activating monoclonal antibody, anti-CD137 activating monoclonal antibody and anti-CD28 activating monoclonal antibody are coated on the surface of the culture vessel. In a specific embodiment of preparing the modified TIL herein, a combination of the following TIL seed cell culture medium and TIL cell expansion medium is used: (a) and (d), (a) and (e), (b) and (d), (b) and (e), (c) and (d), or (c) and (e).

[0156] In a specific embodiment of the modified TIL or its preparation method, the sample containing TIL is derived from gastric cancer tissue, melanoma, ovarian cancer tissue, cervical cancer tissue, intestinal cancer tissue, lung cancer tissue, osteosarcoma, or recurrent bladder metastasis of cervical cancer; the membrane-anchored IL-7 comprises: (i) CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region, (ii) CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD52, (iii) CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, hinge region or linker, and CD52, (iv) CD52 signal peptide, IL-7, BCMA extracellular region or its variant, hinge region or linker, and CD52, (v)(vi) IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region, or (vii) IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD52; the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a) or (b) or (c); the culture time in the seed cell culture medium is about 11-15 days; the TIL cell expansion medium is the aforementioned TIL cell expansion medium (d) or (e); the culture time in the expansion medium is about 11-15 days; the cell seeding density of the expansion medium is 2.0×10 5 -4.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in any one or more of SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43 and 45.

[0157] In the first set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from gastric cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 11 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 2.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 31.

[0158] In a second set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from melanoma, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (b), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (d), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 2.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 33.

[0159] In a third set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from ovarian cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 2.0×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 33.

[0160] In a fourth set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from cervical cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 2.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 31.

[0161] In a fifth set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from ovarian cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 11 days, and the cell seeding density of the expansion medium is 2.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 35.

[0162] In the sixth set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from colorectal cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a BCMA extracellular domain or a variant thereof, a linker, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 13 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 11 days, and the cell seeding density of the expansion medium is 2.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 39.

[0163] In the seventh set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from lung cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 3.0×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 41.

[0164] In an eighth set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from osteosarcoma tissue, the membrane-anchored IL-7 comprises an IL-7 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 15 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 15 days, and the cell seeding density of the expansion medium is 2.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 45.

[0165] In the ninth set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from recurrent bladder metastasis tissue of cervical cancer, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 2.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 35.

[0166] In the tenth set of embodiments of the modified TIL or its preparation method, the sample containing TIL is derived from ovarian cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a BCMA extracellular region or a variant thereof, a CD8 extracellular hinge region, and CD52 connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 4.5×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 37.

[0167] In an eleventh embodiment of the modified TIL or its preparation method, the sample containing TIL is derived from ovarian cancer tissue, the membrane-anchored IL-7 comprises a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and a CD8 transmembrane domain connected in sequence, the seed cell culture medium for preparing TIL is the aforementioned TIL seed cell culture medium (a), the culture time in the seed cell culture medium is about 12 days, the TIL cell expansion medium is the aforementioned TIL cell expansion medium (e), the culture time in the expansion medium is about 12 days, and the cell seeding density of the expansion medium is 2.0×10 5 Preferably, the sequence of membrane-anchored IL-7 is shown in SEQ ID NO: 43.

[0168] The TIL cell population prepared by the method of the present invention can be used to prepare a drug for treating cancer. Therefore, the present invention also provides a pharmaceutical composition containing the modified TIL described herein and a pharmaceutically acceptable excipient. Herein, a pharmaceutically acceptable excipient refers to a carrier, diluent and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers and preservatives. More specifically, suitable pharmaceutically acceptable excipients can be excipients commonly used in the art for TIL cell transfusion (e.g., intravenous infusion).

[0169] Typically, a pharmaceutical composition contains a therapeutically effective amount of modified TILs. A therapeutically effective amount refers to a dose that can treat, prevent, alleviate, and / or alleviate a disease or condition in a subject. The therapeutically effective amount can be determined based on factors such as the patient's age, gender, underlying condition and its severity, and other physical conditions. As used herein, a subject or patient generally refers to a mammal, and more particularly, a human.

[0170] The present invention also includes a cell therapy comprising administering to a subject the modified TILs, wherein the TILs are genetically modified to express the membrane-anchored IL-7 described herein. The administered cells are capable of killing tumor cells in the recipient. Additionally, the TIL cell-mediated immune response can be part of an adoptive immunotherapy procedure.

[0171] Herein, diseases suitable for treatment using the pharmaceutical compositions described herein are related to modified TILs. The diseases described herein include solid tumors and hematological tumors, such as adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, pancreatic cancer, and prostate cancer, as well as leukemias and lymphomas, such as B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0172] The modified TIL of the present invention can be administered alone or as a pharmaceutical composition in a manner suitable for treating (or preventing) a disease. The quantity and frequency of administration will be determined by various factors, such as the patient's condition and the type and severity of the patient's disease. The administration of the composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The composition described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the polypeptide or cell or pharmaceutical composition of the present invention is administered to the patient by intradermal or subcutaneous injection (e.g., intravenous injection). Alternatively, the polypeptide or cell or pharmaceutical composition can be directly injected into a tumor, lymph node or infection site.

[0173] In some embodiments of the present invention, the modified TILs of the present invention may be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressive agents. For example, treatment may be combined with radiotherapy or chemotherapy agents known in the art for treating tumor antigen-mediated diseases.

[0174] In certain embodiments, a kit is also provided herein, comprising a nucleic acid construct, such as a vector, as described herein. The kit may also contain various reagents suitable for transfecting the nucleic acid construct into cells, and optionally instructions for guiding those skilled in the art to transfect the nucleic acid construct into cells.

[0175] In certain embodiments, the present invention also provides a cell cryopreservation preparation comprising the modified TIL of the present invention and a cryopreservation solution. The cryopreservation solution herein can be a tissue cryopreservation solution or a cell cryopreservation solution, which can maintain the viability of the TIL cells of the present invention under low-temperature conditions, such as dry ice freezing or liquid nitrogen freezing. Preferably, the cryopreservation solution can be a serum-free cryopreservation solution, and after the cell cryopreservation preparation is revived from low temperature, it can be directly re-infused into the individual without removing the cryopreservation solution without causing significant toxic side effects to the individual.

[0176] The present invention will be described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, conventional materials and methods in the art.

[0177] Example

[0178] Example 1 Construction of membrane-anchored IL-7 expression vector

[0179] Synthesis of the coding sequences of membrane-anchored IL-7 in Table 1

[0180] Table 1

[0181] The pKB20 vector was constructed according to the method described in Example 1 on page 21 of PCT application WO2022078310A1. According to the method described in this example, pKB20 vectors containing expression cassettes for mbIL-7-1, mbIL-7-2, mbIL-7-3, mbIL-7-4, mbIL-7-5, mbIL-7-6, mbIL-7-7, mbIL-7-8, and mbIL-7-9 were constructed and designated pKB20-mbIL-7-1, pKB20-mbIL-7-2, pKB20-mbIL-7-3, pKB20-mbIL-7-4, pKB20-mbIL-7-5, pKB20-mbIL-7-6, pKB20-mbIL-7-7, pKB20-mbIL-7-8, and pKB20-mbIL-7-9, respectively. The obtained recombinant plasmids were transformed into E. coli (DH5c). After correct sequencing, the plasmids were extracted and purified using a plasmid purification kit from Qiagen to obtain high-quality plasmids of each recombinant expression vector.

[0182] Example 2 Preparation of TIL expressing membrane-anchored IL-7

[0183] The components and numbers of the TIL seed cell culture medium and TIL cell expansion culture medium used are as follows:

[0184] TIL seed cell culture medium:

[0185] (a) IL-2 (3000 U / mL), IL-7 (50 ng / mL), IL-15 (50 ng / mL), IFN-γ (1000 U / mL), anti-CD137 activating monoclonal antibody (5 μg / mL), anti-CD40 activating monoclonal antibody (5 μg / mL), anti-OX-40 activating monoclonal antibody (5 μg / mL), anti-TIGIT blocking monoclonal antibody (5 μg / mL), anti-PD-1 blocking monoclonal antibody (15 μg / mL), PS double antibody (100 U / mL), and X-VIVO 15 basal medium supplemented to the final volume;

[0186] (b) IL-2 (3000 U / mL), IL-7 (20 ng / mL), IL-15 (20 ng / mL), GM-CSF (500 U / mL), IFN-γ (1000 U / mL), anti-CD137 activating monoclonal antibody (3 μg / mL), anti-CD28 activating monoclonal antibody (3 μg / mL), anti-PD-1 blocking monoclonal antibody (3 μg / mL), TNF-α (10 pg / mL), and X-VIVO 15 basal medium supplemented to the final volume;

[0187] (c) IL-2 (3000 U / mL), IL-7 (20 ng / mL), IL-15 (30 ng / mL), anti-CD137 activating antibody (12 μg / mL), anti-CD28 activating monoclonal antibody (5 μg / mL), anti-LAG-3 blocking monoclonal antibody (5 μg / mL), anti-PD-1 blocking monoclonal antibody (3 μg / mL), dasatinib (3 μM), 5 v / v% human AB serum and X-VIVO 15 basal medium to the final volume.

[0188] TIL cell expansion medium

[0189] (d) IL-2 (500 U / mL), IL-7 (5 ng / mL), IL-15 (10 ng / mL), anti-PD-1 blocking monoclonal antibody (3 μg / mL), anti-CD3 activating monoclonal antibody, anti-CD28 activating monoclonal antibody, 5 v / v% human AB serum, and X-VIVO 15 basal medium supplemented to the final volume. The anti-CD3 activating monoclonal antibody and anti-CD28 activating monoclonal antibody are coated on the culture vessel surface;

[0190] (e) IL-2 (500 U / mL), IL-7 (7 ng / mL), IL-15 (30 ng / mL), anti-PD-1 blocking monoclonal antibody (1 μg / mL), anti-CD3 activating monoclonal antibody, anti-CD28 activating monoclonal antibody, anti-CD137 activating monoclonal antibody, 5 v / v% human AB serum and X-VIVO 15 basal medium supplemented to the final volume, wherein the anti-CD3 activating monoclonal antibody, anti-CD137 activating monoclonal antibody and anti-CD28 activating monoclonal antibody are coated on the surface of the culture vessel.

[0191] Solid tumor sample processing and preparation of membrane-anchored IL-7 expressing TILs:

[0192] 1) Prepare normal saline solution containing a final concentration of 100 U / mL penicillin, 100 μg / mL streptomycin, and 50 μg / mL gentamicin.

[0193] 2) Under a sterile environment in a Class II biosafety cabinet, wash the freshly isolated tumor tissue sample in a 10 cm dish containing 30 mL of the saline prepared in step 1). Then, wash the sample in a fresh 10 cm dish containing 30 mL of the saline prepared in step 1). Repeat this three times.

[0194] 3) Use a sterile surgical blade to remove fat tissue and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm. 3 Take two G-REX100 culture jars (purchased from Wilsonwolf), place 42 randomly selected tumor tissue blocks in each G-REX100 culture jar, and add one of the above-prepared TIL seed cell culture media a, b, or c as the seed cell culture medium to the culture jar; the excess tumor tissue blocks are frozen in liquid nitrogen using CryoStor10 (purchased from BioLifeSolutions) freezing solution;

[0195] 4) Add 1 L of TIL seed cell culture medium to the G-REX100 culture jar containing the tumor tissue fragments from 3) and culture the tumor tissue fragments at 37°C in 5% CO2. Remove half the volume of the old seed cell culture medium every 4 days and replace with half the volume of fresh seed cell culture medium. Harvest the TIL seed cells by centrifugation on days 11-15, and count the total cell count and viability.

[0196] 5) Take the TIL seed cells harvested in 4) and collect 1×10 8 -2×10 8 The TIL seed cell population of living cells was transfected with the expression vector expressing membrane-anchored IL-7 prepared in Example 1 through a Lonza Nucleofector TM2b electroporation into nuclei, the specific steps are as follows:

[0197] ① Add medium b prepared in Example 2 to the wells of G-REX6 in advance, 40 mL per well, and then transfer to a cell culture incubator at 37°C and 5% CO2 for preheating for 1 hour;

[0198] ② Prepare the electroporation solution ratio for each well according to the table below:

[0199] ③Take the TIL harvested in step 5) and put it into EP tubes. Add 1×10 7 The cells were centrifuged at 800 g for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of normal saline and the centrifugation step was repeated to wash the cell pellet;

[0200] ④ Add 0.6-1 μL (3-5 μg) of each membrane-anchored IL-7 expression vector prepared in Example 1 and adjusted to a concentration of 5 μg / mL to the electroporation solution prepared in ②, and then let it stand at room temperature for less than 30 minutes (the total volume can be slightly larger than 100 μL at this time);

[0201] ⑤ Resuspend TIL with the plasmid-containing electroporation solution prepared in ④, carefully pipette the cell resuspension into a LONZA 100μL electroporation cup, and place the electroporation cup into the LONZA Nucleofector TM 2b In the electroporation tank, start the electroporation program and select T-020;

[0202] ⑥ After electroporation, carefully remove the electroporation cup, aspirate the cell suspension and transfer it to an EP tube. Add 200 μL of preheated X-VIVO 15 medium to each tube, and then transfer it to the well of G-REX6 in ① containing preheated medium b of Example 2. Repeat the above electroporation operation until 1×10 8 -2×10 8 After all live TIL seed cells have been electroporated, they are cultured at 37°C and 5% CO2. Every two days, half the volume of the old seed cell culture medium is removed and replaced with half the volume of fresh seed cell culture medium until the cell viability recovers to above 90%, and then the cells are ready to proceed with subsequent operations.

[0203] 6) Take the electroporated TIL seed cells in 5) and centrifuge them at 800g for 5 minutes. Resuspend them to 1.0×10 5 / mL~1.0×10 6 / mL, added to the cell culture vessel pre-treated with the coating component, activated at 37℃ 5% CO2 for 2-3 days, then the activated cells were collected by centrifugation and inoculated into G-REX500M culture tanks with pre-warmed expansion medium. The expansion medium in the G-REX500M culture tank is the same as the expansion medium containing all other components in the corresponding solution except the coating component. The volume of expansion medium in each G-REX500M is 5L. The activated seed cells are cultured at 2.0×10 5 / cm 2 ~5.0×10 5 / cm 2 The cells were inoculated at a density of 1.0 × 10 and cultured at 37 °C with 5% CO2. After counting the cells every 4 days, half the volume of the old expansion medium was removed and half the volume of fresh expansion medium was added. The total number of cells in each G-REX500M tank reached 1.0 × 10 10 Afterwards, the cells were split into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to 5 L before continued cultivation. After a total of 10-12 days of culture in the expansion medium of the G-REX500M culture tank, the cells were harvested to obtain TIL product cells expressing membrane-anchored IL-7.

[0204] The following Table 2 shows the tumor tissue sample number, the number of the membrane-anchored IL-7 expressing TIL obtained by culture, the type of cancer, the number of the seed cell culture medium and the expansion culture medium used in the culture process corresponding to Example 2, the culture time of each tissue sample seed cell culture medium and the expansion culture medium, the specific vector name used for electroporation of each tumor tissue sample, and the G-REX500M cell seeding density; Table 3 shows the coating vessel type, coating activation resuspended cell density, and coating activation time of the corresponding tumor tissue sample in step 6).

[0205] Table 2

[0206] Table 3

[0207] Preparation of natural TILs

[0208] The following steps were used to generate natural TILs from T001-T014 tumor tissues:

[0209] 1) Prepare normal saline solution containing a final concentration of 100 U / mL penicillin, 100 μg / mL streptomycin, and 50 μg / mL gentamicin.

[0210] 2) Under a sterile environment in a Class II biosafety cabinet, wash the freshly isolated tumor tissue sample in a 10 cm dish containing 30 mL of the saline prepared in step 1). Then, wash the sample in a fresh 10 cm dish containing 30 mL of the saline prepared in step 1). Repeat this three times.

[0211] 3) Use a sterile surgical blade to remove fat tissue and necrotic tissue, and cut the tumor tissue into pieces with a diameter of 3×3×3mm. 3 Take two G-REX100 culture jars (purchased from Wilsonwolf), place 42 randomly selected tumor tissue blocks in each G-REX100 culture jar, and add one of the above-prepared TIL seed cell culture media a, b, or c as the seed cell culture medium to the culture jar; the excess tumor tissue blocks are frozen in liquid nitrogen using CryoStor10 (purchased from BioLifeSolutions) freezing solution;

[0212] 4) Add 1 L of TIL seed cell culture medium to the G-REX100 culture jar containing the tumor tissue fragments from 3) and culture the tumor tissue fragments at 37°C in 5% CO2. Remove half the volume of the old seed cell culture medium every 4 days and replace with half the volume of fresh seed cell culture medium. Harvest the TIL seed cells by centrifugation on days 11-15, and count the total cell count and viability.

[0213] 5) Take the TIL seed cells in 4) and centrifuge them at 800g for 5 minutes. Resuspend them to 1.0×10 5 / mL~1.0×10 6 / mL, added to the cell culture vessel pre-treated with the coating component, activated at 37℃ 5% CO2 for 2-3 days, then the activated cells were collected by centrifugation and inoculated into G-REX500M culture tanks with pre-warmed expansion medium. The expansion medium in the G-REX500M culture tank is the same as the expansion medium containing all other components in the corresponding solution except the coating component. The volume of expansion medium in each G-REX500M is 5L. The activated seed cells are cultured at 2.0×10 5 / cm 2 ~5.0×10 5 / cm 2 The cells were inoculated at a density of 1.0 × 10 and cultured at 37 °C with 5% CO2. After counting the cells every 4 days, half the volume of the old expansion medium was removed and half the volume of fresh expansion medium was added. The total number of cells in each G-REX500M tank reached 1.0 × 10 10Afterwards, the cells were split into flasks at a ratio of 1:2, and fresh expansion medium was added to each flask to 5 L before continuing to culture. After a total of 10-12 days of culture in the expansion medium of the G-REX500M culture tank, the cells were harvested to obtain natural TIL product cells.

[0214] The seed cell culture medium and expansion medium used in the culture process of each tumor tissue sample, the culture time of the seed cell culture medium and expansion medium of each tissue sample, and the G-REX500M cell seeding density of each tumor tissue sample are the same as those in Table 2; the coating vessel type, coating activation resuspended cell density and coating activation time of the corresponding tumor tissue sample in step 5) are the same as those in Table 3.

[0215] The prepared groups of natural TILs were named T001-TIL, T002-TIL, T003-TIL, T004-TIL, T005-TIL, T006-TIL, T007-TIL, T008-TIL, T009-TIL, T010-TIL, T011-TIL, T012-TIL, T013-TIL, and T014-TIL.

[0216] Example 3 Phenotypic Detection of TILs Expressing Membrane-Anchored IL-7

[0217] 1. Cell survival rate and positive rate of TIL expressing membrane-anchored IL-7

[0218] The cell survival rate of each group was detected by trypan blue staining and cell counting. The results showed that the cell survival rate of each group of TIL expressing membrane-anchored IL-7 and natural TIL prepared in Example 2 was above 95%.

[0219] The BCMA extracellular domain contained in the extracellular region of each cell expressing IL-7 on the membrane surface prepared above was used as a label, and the proportion of cells positive for exogenous gene expression was detected by flow cytometry as follows:

[0220] 1) Collect cells from groups T001-1, T002-2, T003-2, T004-1, T005-3, T006-5, T007-6, T008-8, T009-3, T010-4, T003-7, T011-9, T012-9, T013-9, and T014-9, with 1×10 cells collected from each group. 6 The cells were centrifuged at 800 g for 3 min. Natural T002-TIL that did not express membrane-anchored IL-7 was used as the control cell.

[0221] 2) Discard the supernatant, add physiological saline to resuspend the cells, and centrifuge at 800g for 3 minutes;

[0222] 3) Discard the supernatant, add 100 μL of saline to each tube to resuspend the cells, add 2 μL of BCMA flow cytometry antibody (purchased from Biolegend, catalog number: 357504) to each tube, and incubate at room temperature for 30 minutes;

[0223] 4) Add appropriate amount of physiological saline to each tube, centrifuge at 800g for 3 minutes, wash twice, and discard the supernatant;

[0224] 5) Resuspend in 400 μL of physiological saline and analyze by flow cytometry.

[0225] The results showed that the positive rate of cells in each group is shown in Table 4 below

[0226] Table 4

[0227] The results in Table 4 show that, using BCMA extracellular domain expression as a criterion for TIL samples electroporated with membrane-anchored IL-7, the percentage of positive cells in all samples, except T007-6, which utilizes a flexible linker in its structural design, and T003-7, which utilizes a conventional CD8 transmembrane domain, exceeded 40%. The percentages of positive cells in T003-2, T005-3, and T009-3 exceeded 50%. The positive rates of T007-6 and T003-7 were both below 20%, significantly lower than those of the other samples. T003-2 showed a clear advantage over T003-7.

[0228] The above results indicate that: 1) When the BCMA extracellular region or truncated BCMA extracellular region is connected to the transmembrane region through a flexible linker, its recognition by antibodies may be affected and it may not truly function as an extracellular tag; 2) Compared with conventional transmembrane regions, the cytokine IL-7 anchored on the TIL surface through the GPI anchor region can better stimulate the expansion of positive TILs and greatly increase the proportion of positive TILs.

[0229] 2. Lymphocyte phenotype and cytokine secretion levels

[0230] The harvested TILs expressing membrane-anchored IL-7 and natural TILs from each group were incubated with CD45, CD3, CD4, and CD8 flow cytometry antibodies, and the unbound antibodies were washed and counted on a flow cytometer. The HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) was used to detect the level of IFN-γ secretion according to the method described in the instructions. The results are shown in Table 5 below:

[0231] Table 5

[0232] Example 4 In vitro tumor cell killing function of TIL expressing membrane-anchored IL-7

[0233] 1. Culture of primary tumor cells derived from melanoma tissue

[0234] T002 melanoma tumor tissue was obtained and cut into 3×3×3 mm fragments. The fragments were mixed as evenly as possible and then cultured according to the Materials and Methods described in Robert Suriano et al. Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines J Cancer 2013; 4(5): 371-382 to obtain primary melanoma cells.

[0235] 2. Enhanced cytotoxicity of TIL expressing membrane-anchored IL-7 on target cells in vitro

[0236] Agilent's real-time label-free cell function analyzer (RTCA) was used to detect the in vitro cytotoxicity of T002-2 and T002-TIL obtained in Example 2 against their homologous primary melanoma cells. The specific steps are as follows:

[0237] (1) Zero adjustment: Add 50 μL of DMEM culture medium to each well, place it in the instrument, select step 1, and adjust to zero;

[0238] (2) Target cell plating: The primary melanoma cells obtained from the culture in step 1 were plated at 10 per well. 4 Spread 50 μL of cells on a plate containing detection electrodes and leave it for a few minutes. After the cells are stable, place them in the instrument and start step 2 to culture the cells.

[0239] (3) Adding effector cells: After culturing target cells for 18 h to 24 h, observe the cell index. When the cell index is 2, add effector cells T002-2 and T002-TIL, 50 μL per well, respectively. Count the viable effector cells, set the effector-target ratio to 4:1, and start step 3. After culturing for 60 h, observe the cell proliferation curve.

[0240] The results are shown in Figure 1. Compared with the tumor cell group without effector cells, T002-TIL and T002-2 both had significant killing effects on homologous melanoma cells, and T002-2 expressing membrane-anchored IL-7 had a greater improvement in killing target cells than T002-TIL that did not express exogenous genes, indicating that membrane-anchored IL-7 can significantly enhance the killing ability of TIL against tumor cells it recognizes.

[0241] Example 5 In vitro molecular brake function detection of TIL expressing membrane-anchored IL-7

[0242] Antibody synthesis

[0243] The commercial synthesis of BCMA antibodies was commissioned. The heavy chain sequence of the antibody was SEQ ID NO: 29 of Chinese patent CN103562225B, and the light chain sequence was SEQ ID NO: 31 of the patent. The patent is incorporated into this application by reference in its entirety.

[0244] Antibody-dependent cellular cytotoxicity (ADCC) assay

[0245] NK cells were cultured and expanded from the blood of healthy volunteers using an NK cell culture kit (purchased from Tongli Haiyuan, catalog number AS-01) according to the instructions. T001-1, T002-2, T003-2, T004-1, T005-3, T006-5, T007-6, T008-8, T009-3, T010-4, T003-7, T011-9, T012-9, T013-9, and T014-9 prepared in Example 2 were used as target cells and resuspended in AIM-V medium. T002-TIL and T011-TIL were used as negative control target cells and treated in the same way. The cells were expressed as 1.0×10 4 50 μL of cells / well was added to the wells of a 96-well plate. At the same time, the BCMA antibody synthesized above was also resuspended in AIM-V to a final concentration of 500 μg / mL, and 10 μL was added to the wells of a 96-well plate. The cells were pre-incubated with the target cells in the wells at room temperature for 40 minutes. The NK cells used as effector cells were resuspended in AIM-V medium containing 2% FCS to a concentration of 2.5×10 6 After a 40-minute incubation of target cells with BCMA antibody, 40 μL of the NK cell suspension was added to the target cell-BCMA antibody mixture to achieve an effector-target ratio of 10:1. The final BCMA antibody concentration in the system was 50 μg / mL, and the total volume was 100 μL. The system was gently pipetted to mix thoroughly, and the plate was incubated in a 5% CO2, 37°C incubator for 3 hours. The antibody-dependent cellular cytotoxicity (ADCC) effect of BCMA antibody against each of the above target cells was evaluated using the LDH-Cytotoxicity Colorimetric Assay Kit (Biovision, Cat. No. K313-500).

[0246] Complement-dependent cytotoxicity (CDC) assay

[0247] T001-1, T002-2, T003-2, T004-1, T005-3, T006-5, T007-6, T008-8, T009-3, T010-4, T003-7, T011-9, T012-9, T013-9, T011-9, T012-9, T013-9 and T014-9 prepared in Example 2 were used as target cells and resuspended in AIM-V medium. T002-TIL and T011-TIL were used as negative control target cells and treated in the same manner. 4 Add 50 μL of cells / well to the wells of a 96-well plate. Simultaneously, resuspend the aforementioned BCMA antibody in AIM-V to a final concentration of 500 μg / mL. Then, add 10 μL to the wells of a 96-well plate and incubate with the target cells in the wells at room temperature for 40 minutes. Thaw fresh normal human serum complement (Quidel, Cat. No. A112) and dilute it 3-fold with AIM-V medium. After the 40-minute incubation of target cells with BCMA antibody, add 40 μL of diluted human serum complement to a final concentration of 50 μg / mL. The total volume of the system is 100 μL. Gently pipette the system to mix thoroughly, and incubate the wells in a 5% CO2, 37°C incubator for 3 hours. The complement-dependent cytotoxicity (CDC) effect of complement on each of the above target cells was assessed using the LDH-Cytotoxicity Colorimetric Assay Kit (Biovision, Cat. No. K313-500). According to the LDH-Cytotoxicity Colorimetric Assay Kit instructions, cytotoxicity was calculated using the following formula:

[0248] The low control value is the absorbance value corresponding to the LDH content in the supernatant under the condition that the cells are not lysed at all, and the high control value is the absorbance value of the supernatant under the condition that the cells are completely lysed.

[0249] The ADCC and CDC results are shown in Table 6. After the addition of BCMA antibodies, all cell samples expressing surface IL-7, except T007-6 and T003-7, showed very obvious ADCC and CDC toxicity, which is consistent with the positive rate test results in Table 4 of Example 3. However, natural TILs (T002-TIL and T011-TIL) that do not express surface IL-7 showed no obvious ADCC and CDC toxicity. The ADCC and CDC toxicity of T007-6, which contains a flexible linker in its extracellular structure, were significantly lower than those of other samples, indicating that the addition of BCMA antibodies failed to fully mediate the occurrence of ADCC and CDC effects. This indicates that when the extracellular region of BCMA is connected to the transmembrane region through a flexible linker, its epitope structure may be affected, resulting in the inability of BCMA antibodies to bind normally.

[0250] Table 6

[0251] Example 6 In vivo tumor killing function of CDX model expressing membrane-anchored IL-7 TIL

[0252] Construction of experimental cells

[0253] The SKOV-3 cell line SKOV-3320 stably expressing human CD3 antibody (OKT3) on its membrane surface was constructed as an ovarian cancer target cell line as follows:

[0254] 1. Construction of lentiviral vector expressing membrane surface CD3 antibody

[0255] The structure encoding the membrane-bound CD3 antibody (αCD3TM) includes a CD8 signal peptide, a CD3 scFv, a linker, and a CD8 transmembrane region. Its amino acid sequence is shown in SEQ ID NO:49, and its DNA sequence is shown in SEQ ID NO:50. A DNA sequence containing PmeI and MluI restriction enzyme sites at the 5' and 3' ends, respectively, including SEQ ID NO:50, was commissioned for synthesis. After digestion, it was ligated into the lentiviral vector pWPXL-EGFP expression plasmid (purchased from Addgene) to obtain pWPXL-αCD3TM-EGFP. A DNA sequence encoding a fusion protein (EGFP-puro) containing EGFP, a P2A linker, and a puromycin resistance gene (puro) was commissioned for synthesis. It contained MluI and SpeI restriction enzyme sites at the 5' and 3' ends, respectively, and after digestion, it was ligated into pWPXL-αCD3TM-EGFP to obtain the pWPXL-αCD3TM-EGFP-puro lentiviral expression plasmid.

[0256] 2. Lentivirus Preparation

[0257] The following packaging plasmids psPAX2 and pMD2.G were purchased from Addgene.

[0258] 1) 293T cells were passaged one day before transfection and seeded into 6-well plates at a confluence of 35%;

[0259] 2) One hour before transfection the next day, aspirate the culture medium in the 6-well plate and slowly add 2 mL of Opti-MEM to each well. TM (purchased from ThermoFisher) culture medium;

[0260] 3) Preparation of plasmid transfection solution:

[0261] a)Lipofectamine TM 3000 (purchased from ThermoFisher) dilution: 750 μL Opti-MEM TM Add 24 μL of Lipofectamine TM 3000, mix thoroughly;

[0262] b) DNA dilution: The plasmid addition ratio is pWPXL-αCD3TM-EGFP-puro:pSPAX2:pMD2.G = 4:3:1. The total amount of three plasmids added to each well is 2.5 μg, that is, the amount of three plasmids added to 6 wells is 7.5 μg, 5.625 μg, and 1.875 μg respectively, in 750 μL Opti-MEM TM Add the corresponding volume of plasmid and 30 μL of P3000 TM (purchased from ThermoFisher), mix thoroughly;

[0263] c) Add the DNA dilution to the Lipofectamine TM 3000 dilution, mix well;

[0264] 4) After incubation at room temperature for 13 minutes, 250 μL of DNA-lipid complex was added to each well;

[0265] 5) After 6 hours, the culture medium in the 6-well plate was aspirated and 2 mL of complete culture medium (H-DMEM + 10 v / v% FBS, both purchased from ThermoFisher) was added to each well.

[0266] 6) 48 hours after cell transfection, collect all the cell supernatant into a 15 mL centrifuge tube, centrifuge at 1500 rpm for 10 min, filter through a 0.22 μM filter membrane, and then aliquot into 1.5 mL EP tubes (1.5 mL / tube) and freeze at -80°C.

[0267] 3. Infect target cells with lentivirus

[0268] The target cells were ovarian cancer cell line SKOV3-Luc that integrated and expressed luciferase gene, purchased from BPS Bioscience (Cat#78425).

[0269] 1) SKOV3-Luc was passaged one day before infection and plated into two wells of a 6-well plate at 20% confluence;

[0270] 2) On the day of infection, remove one tube of virus solution from the -80°C freezer and thaw at room temperature;

[0271] 3) Aspirate the culture medium from one well, add 1.5 mL of fresh complete culture medium (IMDM + 15 v / v% FBS + 1× non-essential amino acid NEAA, IMDM and FBS purchased from ThermoFisher, 100× non-essential amino acid purchased from Cyagen) and 1.5 mL of virus solution, then add polybrene to a final concentration of 8 μg / mL. After shaking, place in a 37°C carbon dioxide incubator. Leave the other well untreated.

[0272] 4. Screening of positive mixed clones and stable cell lines

[0273] 1) On the third day after lentiviral infection, the cell confluence in both wells reached over 90%;

[0274] 2) Discard the culture medium and add 3 mL of fresh complete culture medium to each well, followed by puromycin to a final concentration of 3 μg / mL;

[0275] 3) After 48 hours of culture with puromycin, the cell death of the uninfected control cells is close to 100%, and the cell confluence of the infected wells reaches 90%. All infected cells are transferred to a T25 culture flask and continue to be cultured with puromycin to a final concentration of 3 μg / mL;

[0276] 4) After the cell proliferation period stabilized, the cells were expanded and cryopreserved, and the positive rate was detected by flow cytometry. The resulting mixed clone stably transfected cell line was named SKOV3-αCD3TM-EGFP;

[0277] 5) Cytotoxicity assays were performed on host cells (SKOV3-Luc) and a stably transfected mixed cell line (SKOV3-αCD3TM-EGFP) using the xCELLigence RTCA TP real-time label-free cell analyzer (Agilent). T cells derived from healthy human peripheral blood mononuclear cells (PBMCs) served as effector cells. The results showed that T cells exhibited significantly greater cytotoxicity against SKOV3-αCD3TM-EGFP compared to untransformed SKOV3-Luc.

[0278] 5. Monoclonal cell line screening

[0279] 1) Filter the prepared complete culture medium (IMDM + 15 v / v% FBS + 1×NEAA) through a 0.22 μM filter to reduce impurities in the reagent that may affect subsequent single-cell microscopy observations;

[0280] 2) Mix and clone the stably transfected cell line SKOV3-αCD3TM-EGFP and count the cells;

[0281] 3) Plate two 96-well cell culture plates at a plating density of 0.8 cells / well: Use an 8-channel pipette (maximum volume of 50 μL) to add 100 μL of culture medium / well, followed by 50 μL of cell suspension / well (16 cells / mL, prepare 15 mL), and then add 1000 cells to well A1 of the 96-well plate.

[0282] 4) After culturing in a 37°C CO2 incubator for 6 hours, observe the cells under a microscope and take photos of wells containing single cells.

[0283] 5) Wells containing single cells were photographed on day 1, day 2, and day 3 to capture the cell division process;

[0284] 6) Replace the culture medium of the labeled single-cell clones by aspirating 100 μL of culture medium from each well and adding 150 μL of fresh culture medium. Replace the culture medium every 3 days.

[0285] 7) When the confluence of the single cell clones in the well reaches more than 50%, transfer them and expand the culture in sequence from 96-well plates to 24-well plates, then to 6-well plates, then to T25 culture flasks, and finally to T75 culture flasks;

[0286] 8) The amplified monoclonal cells are tested for positive expression by flow cytometry and cryopreserved;

[0287] 9) The xCELLigence RTCA TP real-time label-free cell analyzer was used to test the cell killing ability of different monoclonal cell lines. The killing results showed that the monoclonal cell line numbered 320 was highly responsive to T cell killing and that the cell killing was dependent on the number of cells initially. As the effector-target ratio increased, the killing ability increased. This monoclonal cell line was named SKOV-3320 and used as the tumor target cell in subsequent experiments.

[0288] Effector cells: TIL T014-9 expressing membrane-anchored IL-7, cultured from ovarian cancer tissue T014, was selected as the effector cell.

[0289] experimental animals

[0290] Immunodeficient B-NDG mice (purchased from Biocytogen) were selected as experimental animals for the construction of the CDX model.

[0291] Experimental design and grouping: as shown in Table 7 below

[0292] Table 7 Dosage regimen and grouping of mice

[0293] The TIL cells were T014-9, a TIL product expressing membrane-anchored IL-7, derived from tissue sample T014 prepared in Example 2. Before tail vein injection, the cells were centrifuged and resuspended in PBS. The cell density of group 2 was 2.5×10 7 / mL cell suspension, and the cell density of group 3 was 7.5×10 7 / mL of cell suspension.

[0294] Animal husbandry

[0295] After purchasing the required amount of B-NDG mice, they were raised in an SPF-grade experimental animal room for an adaptation period of 7-10 days.

[0296] Environment: Mice will be housed in clear resin plastic cages in an animal room. Cage bedding will consist of autoclaved sawdust and corncob bedding, which will be changed regularly. The animal room will be equipped with a HEPA air filter and maintained at a temperature between 20-26°C (68-79°F) and a relative humidity of 40-70%. Temperature and humidity will be continuously monitored and recorded. Lighting conditions will consist of 12 hours of fluorescent lighting and 12 hours of no lighting per day.

[0297] Food and drinking water: Experimental mice had unlimited access to special mouse chow (irradiated and sterilized, Shanghai Slake Laboratory Animal Co., Ltd., China) and had unimpeded access to sterile clean drinking water at any time.

[0298] Establishment of subcutaneous ectopic tumor model

[0299] After counting, SKOV-3320 cells were used for tumor inoculation. Each mouse was subcutaneously inoculated with 0.2 mL of tumor cell solution containing 50% matrigel (1E7 / mouse) in the right axilla. The day of inoculation was designated as D0.

[0300] Animal grouping and drug administration

[0301] The average tumor volume reached 100 mm 3 At the same time, 24 animals were selected from 36 animals according to the tumor volume (mean 111 mm 3 ) were randomly divided into groups to ensure that all groups were comparable at baseline. The day of grouping was recorded as P0. During the experiment, the body weight and tumor volume of the animals were measured three times a week, and the clinical symptoms of the animals were observed daily. Tumor volume is expressed in mm 3 The formula is: V = 0.5 × a × b 2, where a and b are the long and short diameters of the tumor, respectively. Tumor fluorescence was measured using a small animal in vivo imaging system 7 weeks after the first administration and at the end of the experiment.

[0302] In vivo imaging

[0303] On the day of the experiment, bioluminescence signals from mice were measured using an IVIS LuminaXR (PerkinElmer). Animals were anesthetized with isoflurane after an intraperitoneal injection of 150 mg / kg sodium fluorescein. Bioluminescence images were acquired 10 minutes after sodium fluorescein injection with a 60-second exposure time. Images were quantified as photons / second using Living Image software.

[0304] result

[0305] The experimental results are shown in Figures 2 and 3. Figure 2 shows the differences in tumor volume between groups. The differences in tumor volume between different groups were analyzed by one-way ANOVA analysis, followed by a Bonferroni post hoc test to observe whether there were significant differences between different groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001. Figure 3 shows the results of fluorescent in vivo imaging of experimental mice in each group on the day the experiment ended. The results shown in Figure 2 show that compared with the PBS group, the 0.5×10 7 The tumor volume of mice in the T014-9 re-infusion dose group was significantly smaller; while the 1.5×10 7 The tumors of mice in the TIL re-infusion group had completely disappeared by D65. Figure 3 shows that at D65, 1.5×10 7 In the T014-9 re-infusion dose group, no tumor cell fluorescence was observed. These results indicate that T014-9 cells have a very significant killing effect on ovarian cancer CDX tissue in mice.

[0306] Example 7 In vivo tumor killing function of PDX models expressing membrane-anchored IL-7 TIL

[0307] T011 cervical cancer tumor tissue was used to construct an immunodeficient mouse PDX tumor model, and the model mice were used to detect the tumor killing effect of TIL-PDX animals. Immunodeficient B-NDG mice (purchased from Biocytogen) were selected as PDX model experimental animals.

[0308] Experimental design and grouping: as shown in Table 8 below

[0309] Table 8 Dosage regimen and grouping of mice

[0310] The TIL cells were the TIL product cells derived from tissue sample T011 prepared in Example 2. Before tail vein injection, the cells were centrifuged and resuspended in PBS to a cell density of 1×10 8 / mL PBS cell suspension.

[0311] Animal husbandry

[0312] After purchasing the required amount of B-NDG mice, they were raised in an SPF-grade experimental animal room for an adaptation period of 7-10 days.

[0313] Environment: Mice will be housed in clear resin plastic cages in an animal room. Cage bedding will consist of autoclaved sawdust and corncob bedding, which will be changed regularly. The animal room will be equipped with a HEPA air filter and maintained at a temperature between 20-26°C (68-79°F) and a relative humidity of 40-70%. Temperature and humidity will be continuously monitored and recorded. Lighting conditions will consist of 12 hours of fluorescent lighting and 12 hours of no lighting per day.

[0314] Food and drinking water: Experimental mice had unlimited access to special mouse chow (irradiated and sterilized, Shanghai Slake Laboratory Animal Co., Ltd., China) and had unimpeded access to sterile clean drinking water at any time.

[0315] Construction of PDX models

[0316] 1) Patient tumor tissue sample processing: Take a portion of T011 tumor tissue, remove necrotic tissue, fat tissue, connective tissue, etc. under sterile conditions, rinse it clean, and then cut it into several 5×5×5mm pieces with a scalpel. 3 The tissue blocks were placed in UW, a transport medium containing tumor samples, and were ready to be used to inoculate B-NDG mice with tumor blocks;

[0317] 2) Tumor Tissue Inoculation: Several B-NDG mice were sampled. After preparatory skin removal from their scapulae, the mice were immobilized using a subcutaneous tumor inoculation fixture. After disinfection with iodine and local anesthesia with lidocaine, the tumor mass described in step 1 was inoculated into the right groin using a PDX model tumor inoculation trocar. The day of inoculation was designated P0. Tumors were measured twice weekly. Tumor volume was calculated using the formula: V = 0.5 × a × b. 2 , where a and b are the long and short diameters of the tumor, respectively;

[0318] 3) PDX tissue passage: Observe the growth of tumor tissue in each inoculated mouse and wait until the tumor tissue volume exceeds 300mm 3 After that, the mice were anesthetized, the tumor was removed, and cut into 5×5×5 mm pieces with a scalpel under sterile conditions. 3 Repeat step 2) after the tissue block is inoculated into the right groin of a new mouse and wait for the next generation of PDX tumor growth;

[0319] 4) Repeat step 3) for 2-3 more generations, then remove PDX tissue from some mice for histological analysis. After confirming that the PDX tissue is still human (not mouse) tissue, continue to inoculate mice with PDX tissue at 1.5 times the number of mice used in the experimental plan in Table 8 (i.e., inoculate 36 mice with PDX tissue blocks). Observe the mice for tumor formation and measure the tumors twice weekly until tumor formation is achieved.

[0320] Animal grouping and drug administration

[0321] When the tumor volume of PDX-inoculated mice reaches 50 mm 3 Around 24 animals with appropriate tumor volumes were selected from the 36 animals and randomly divided into groups (n = 8) based on tumor volume to ensure baseline comparability among all groups. The day of grouping was designated as D0, and drug administration was performed according to the schedule in Table 8. Animal body weights and tumor volumes were measured three times a week during the experiment, and clinical symptoms of the animals were observed daily. Tumor volume is expressed in mm 3 The tumor measurement formula is the same as above.

[0322] The results are shown in Figure 4. One-way ANOVA analysis was performed to examine differences in tumor volume between groups, followed by Bonferroni post hoc testing to determine if there were significant differences between any two groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001. The results showed that by day 40 after administration, tumor volume growth in the T011-TIL group was significantly inhibited compared to the PBS control group. The inhibition of tumor volume growth by T011-9 was even more significant compared to the T011-TIL group. This indicates that T011-derived natural TIL products have a significant killing effect on PDX tissue derived from the same patient in vivo, and that modification of natural TIL with membrane-anchored IL-7 further enhances this effect.

[0323] Example 8 Clinical transfusion of TIL expressing membrane-anchored IL-7

[0324] To validate the clinical safety and efficacy of the membrane-anchored IL-7-expressing TILs described in this application, a clinical trial of TIL transfusion therapy for solid tumors was conducted. This clinical trial has been approved by the Ethics Committee of Shanghai Tenth People's Hospital. For details, please see www.clinicaltrials.gov, registration number: NCT05468307.

[0325] Study plan

[0326] 1. Subject inclusion and exclusion criteria and group allocation method

[0327] Inclusion criteria:

[0328] 1) 18 years old ≤ ≤ 75 years old;

[0329] 2) Patients with gynecological tumors diagnosed as primary / recurrent / metastatic by pathology;

[0330] 3) life expectancy > 3 months;

[0331] 4) Karnofsky score ≥60% or ECOG score 0-2;

[0332] 5) The subject has failed standard treatment or has no standard treatment options;

[0333] 6) The subject must have a tumor area or malignant effusion suitable for biopsy or resection of the tumor to isolate TILs;

[0334] 7) at least one evaluable tumor lesion;

[0335] 8) Hematological and biochemical indicators (within 7 days before enrollment)

[0336] Absolute white blood cell count ≥2.5×109 / L;

[0337] Absolute neutrophil count ≥1.5×109 / L;

[0338] Absolute lymphocyte count ≥0.7×109 / L;

[0339] Platelet count ≥100×109;

[0340] Hemoglobin ≥90 g / L;

[0341] Activated partial thromboplastin time (APTT) ≤ 1.5 × ULN (except for patients who have received anticoagulant therapy within the previous 3 days);

[0342] International normalized ratio (INR) ≤ 1.5 × ULN (except for patients who have received anticoagulant therapy within the previous 3 days);

[0343] Serum creatinine ≤1.5 mg / dL (or ≤132.6 μmol / L), or serum creatinine clearance ≥50 mL / min;

[0344] Serum ALT (alanine aminotransferase) / AST (aspartate aminotransferase) ≤ 3 × ULN (for subjects with liver metastasis ≤ 3 × ULN)

[0345] Total bilirubin ≤1.5×ULN;

[0346] 9) No absolute or relative contraindications to surgery or puncture;

[0347] 10) Subjects of childbearing potential must be willing to start using approved highly effective contraceptive methods at the time of informed consent and continue using them for 1 year after completion of the lymphodepletion regimen;

[0348] 11) Any treatment for malignant tumors, including radiotherapy, chemotherapy, and biological agents, must be stopped 28 days before obtaining TILs;

[0349] 12) Have sufficient understanding and voluntarily sign the informed consent form;

[0350] 13) Ability to adhere to follow-up schedule and other protocol requirements.

[0351] Exclusion criteria:

[0352] 1) Patients who require glucocorticoid treatment, with a daily dose of more than 15 mg of prednisone (or equivalent dose of hormones) or autoimmune diseases requiring immunosuppressive treatment;

[0353] 2) Forced expiratory volume in 1 second (FEV1) <2L, diffusing capacity of the lung for carbon monoxide (DLCO) (corrected) <40%;

[0354] 3) Any of the following definitions of significant cardiovascular abnormalities: New York Heart Association (NYHA) class III or IV congestive heart failure, clinically significant hypotension, clinically uncontrolled hypertension, uncontrolled symptomatic coronary artery disease, or ejection fraction <35%; severe cardiac rhythm and conduction abnormalities, such as ventricular arrhythmias requiring clinical intervention, II-III degree atrioventricular block, etc.;

[0355] 4) Patients have human immunodeficiency virus (HIV) infection or positive HIV antibody test, active hepatitis B or hepatitis C virus infection (HBsAg positive and / or anti-HCV positive), syphilis infection or positive Treponema pallidum antibody;

[0356] 5) Serious physical or mental illness;

[0357] 6) Active systemic infection requiring treatment, or positive blood culture (or imaging evidence of infection);

[0358] 7) Receiving or currently receiving other drugs, or other biological, chemotherapy or radiotherapy within 1 month;

[0359] 8) History of allergic reaction to compounds with similar chemical or biological compositions as cell therapy;

[0360] 9) Patients who have received immunotherapy and experienced irAE grade ≥ 3;

[0361] 10) The adverse reactions of previous anti-tumor treatment have not recovered to CTCAE 5.0 version 1 or lower (excluding toxicities such as alopecia that the researcher judges to be without safety risks);

[0362] 11) Pregnant or lactating women;

[0363] 12) History of organ transplantation, allogeneic stem cell transplantation, and kidney replacement;

[0364] 13) The researcher believes that the subject has a history of other serious systemic diseases or is not suitable for participating in this clinical study for other reasons.

[0365] 2. Efficacy evaluation criteria

[0366] Main efficacy indicators:

[0367] 1) objective response rate (ORR);

[0368] 2) disease control rate (DCR);

[0369] 3) duration of response (DOR);

[0370] 4) progression-free survival (PFS);

[0371] 5) Overall survival (OS).

[0372] Secondary efficacy indicators:

[0373] 1) Clinical efficacy evaluation: complete remission (CR), partial remission (PR), disease control (SD), progressive disease (PD), etc.;

[0374] 2) Changes in quality of life before and after treatment.

[0375] Efficacy was defined as investigator-observable ORR according to RECIST v1.1 criteria.

[0376] 3. Records of adverse events and treatment measures

[0377] 3.1 Definition of adverse events

[0378] Adverse event (AE): An adverse medical event that occurs between the time the subject signs the informed consent and is enrolled in the trial and the last medical follow-up. It may manifest as symptoms, signs, disease, or laboratory test abnormalities, but does not necessarily have a causal relationship with the treatment or trial drug.

[0379] New conditions or worsening of existing conditions were considered AEs. Stable chronic diseases, such as arthritis, that existed before study entry and did not worsen during the study were not considered AEs. Abnormal laboratory test results, clinical symptoms, or signs that were judged by the investigator to be clinically significant were considered AEs.

[0380] Serious adverse event (SAE): refers to an adverse event that occurs during the trial and meets one or more of the following conditions: (1) causes death; (2) is life-threatening, meaning that the patient is at immediate risk of death, not that death may occur if the condition develops seriously in the future; (3) causes hospitalization or prolonged hospitalization; (4) causes permanent or significant functional loss; (5) causes teratogenesis or birth defects; (6) other important medical events.

[0381] 3.2 Criteria for Determining the Severity of Adverse Events

[0382] The severity of all adverse events occurring during the study was evaluated according to NCI-CTCAE version 5.0 and was graded from 1 to 5:

[0383] Grade 1: Mild; asymptomatic or mild; clinical or diagnostic observation only; no treatment required.

[0384] Grade 2: Moderate; minor, local, or noninvasive treatment required; age-appropriate limitation in instrumental activities of daily living.

[0385] Grade 3: Severe or medically important but not immediately life-threatening; resulting in hospitalization or prolonged hospitalization; disabling; limiting personal activities of daily living.

[0386] Grade 4: Life-threatening; urgent treatment required.

[0387] Grade 5: Death related to AE.

[0388] 4. Pretreatment before reinfusion

[0389] Before TIL cell transfusion, patients receive a lymphocyte pretreatment regimen (hydroxychloroquine 600-800 mg × 1 day, cyclophosphamide 20-25 mg / kg / d × 3 days). The researchers will make adjustments to the drugs, dosage, and number of days (dosage frequency) used in the pretreatment regimen based on the actual situation of the patient.

[0390] The day of reinfusion is defined as day 0. Before TIL cell reinfusion, researchers need to perform the following chemotherapy pretreatment on patients (after pretreatment, patients will experience leukopenia and need to pay attention to infection prevention, such as avoiding unnecessary personnel flow in the ward and requiring patients to wear masks):

[0391] - Cyclophosphamide (Cy) 20-25 mg / kg intravenously plus hydroxychloroquine (HCQ) 600-800 mg orally for 5 days

[0392] - Cyclophosphamide 20-25 mg / kg IV for 4 days

[0393] - Cyclophosphamide 20-25 mg / kg IV for 3 days

[0394] -2, -1 days for the patient to rest

[0395] TIL cells were reinfused on day 0.

[0396] 5. TIL transfusion and efficacy evaluation

[0397] After the 4 pre-infusion treatments, the patients were reinfused on day 0. Each patient received a single infusion with a single infusion dose of 1.0×109-5.0×109 TIL cells. The cell density of the infused cell preparation was 1.0×10 7 / mL-5.0×10 7 / mL, and the volume of the infused cell preparation is 100mL. The patient undergoes TIL cell infusion on day 0, and no cytokines are administered after the infusion. The patient needs to be observed in the hospital for 5-8 days after the infusion, and the patient's condition after the infusion is observed and recorded. With the patient's informed consent, peripheral blood is drawn from the patient at different time points to detect the composition and changes of PBMCs, and MRI imaging effects are evaluated 1-6 months after the infusion.

[0398] 6. TIL clinical infusion results

[0399] The comprehensive clinical infusion information and efficacy evaluation results of some subjects who participated in the infusion are shown in Tables 9 and 10 below (the evaluation time ranged from 1 to 6 months after the infusion).

[0400] Table 9 Comprehensive information of patients' TIL clinical transfusion

[0401] The tumor diameter of patient T011 was 2.2 cm before re-infusion. 14 weeks after re-infusion, the tumor lesion could no longer be detected by MRI, and the patient was judged to have achieved complete remission (CR), as shown in Figure 5.

[0402] The tumor diameter of patient T012 was 4.5 cm before re-infusion. 22 weeks after re-infusion, the tumor lesion could no longer be detected by MRI, and was determined to be a partial response (CR), as shown in Figure 6.

[0403] The tumor diameter of patient T013 was 2.1 cm before re-infusion. 13 weeks after re-infusion, the tumor lesion was found to have significantly shrunk by magnetic resonance imaging and was determined to be a partial response (PR), as shown in Figure 7.

[0404] The tumor diameter of patient T014 before re-infusion was 2.6 cm × 2.5 cm. 24 weeks after re-infusion, the primary lesion tumor could no longer be detected by MRI. The primary lesion was judged to be completely remitted (CR), and the comprehensive judgment was partial remission (PR), as shown in Figure 8.

[0405] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

[0406] Part of this article

[0407] Membrane surface CD3 scFv

Claims

1. A modified TIL expressing membrane-anchored IL-7, Preferably, the membrane-anchored IL-7 comprises IL-7 or a functional fragment thereof and a transmembrane region.

2. The modified TIL according to claim 1, characterized in that The transmembrane region is a protein transmembrane domain; preferably, the protein transmembrane domain includes any one selected from the group consisting of a CD8 transmembrane region, a CD28 transmembrane region and an IL-7Rα transmembrane region, or The transmembrane region is a GPI anchor region; preferably, the GPI anchor region includes a membrane anchor region of one or more GPI membrane anchor proteins selected from the group consisting of CD44, CD56, CD73, CD55, Thy1, AchE, IAP, ALPP, CD59, CD14, CD16, CD24, CD28, CD48, CD52, CD58, CD66a, CD66c, CD66d, CD66e, CD67, CD87, CD108, CD157, uPAR, JMH protein, GDNFR, CNTFR, TAG-1, PrP, phosphatidylinositol protein, semaphorin 7, CEA, GFR, Ly6G, transferrin receptor, contactin (F3) and T-cadherin.

3. The modified TIL according to claim 1, characterized in that The membrane-anchored IL-7 also includes a membrane surface tag, Preferably, the membrane surface tag comprises the extracellular domain of a B cell surface antigen or a variant thereof that retains the antibody binding function. More preferably, the membrane surface label is the BCMA extracellular region or a variant thereof that retains the function of binding to an anti-BCMA antibody. More preferably, the variant is a truncation variant, an insertion variant, a deletion variant, a substitution variant or a combination thereof; more preferably, the amino acid sequence of the variant is SEQ ID NO: 23 or SEQ ID NO:

25.

4. The modified TIL according to claim 3, characterized in that The membrane-anchored IL-7 further comprises a linker between the IL-7 and the membrane surface tag; preferably, the linker is a rigid linker or a flexible linker, preferably a rigid linker, and / or The membrane-anchored IL-7 further comprises a hinge region or a linker located at the N-terminus of the transmembrane region; preferably, the hinge region includes but is not limited to: CD4 extracellular hinge region, CD8 extracellular hinge region, CD28 extracellular hinge region, IgG1Fc Hinge region and IgG4Fc hinge region, the linker is a rigid linker or a flexible linker; more preferably, the sequence of the rigid linker is any one or more selected from SEQ ID NO: 9, 11, 15 and 17; the sequence of the flexible linker is SEQ ID NO:

13. The membrane-anchored IL-7 further comprises a signal peptide; preferably, the signal peptide is a CD52 signal peptide or an IL-7 signal peptide.

5. The modified TIL according to any one of claims 1 to 3, characterized in that The membrane-anchored IL-7 comprises: an optional signal peptide, IL-7, an optional linker, a BCMA extracellular region or a variant thereof, an optional hinge region or linker, and a transmembrane region, which are sequentially connected. The signal peptide is a CD52 signal peptide or an IL-7 signal peptide, and the transmembrane region is a CD8 transmembrane region or CD52. Preferably, the membrane-anchored IL-7 comprises: a signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, and a transmembrane region, which are connected in sequence. More preferably, the membrane-anchored IL-7 comprises sequentially linked: CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region, CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD52, CD52 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, hinge region or linker, and CD52, CD52 signal peptide, IL-7, BCMA extracellular region or its variant, hinge region or linker, and CD52, IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region, or IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD52.

6. The modified TIL according to any one of claims 1 to 3, characterized in that The TIL is prepared by a method comprising the following steps: incubating a sample containing TIL with a TIL seed cell culture medium to obtain a TIL cell population, or The TIL is prepared by a method comprising the following steps: (1) incubating a sample containing TIL with a TIL seed cell culture medium to obtain a first TIL cell population, and (2) incubating the first TIL cell population with a TIL cell expansion culture medium to obtain a second TIL cell population.

7. The modified TIL according to claim 6, characterized in that The sample is selected from: ascites, surgical resection of primary lesions, synchronous and metachronous surgical resection of metastatic lesions, puncture samples and body fluids, Preferably, the sample is a tissue containing tumor cells; more preferably, the tumor cells are derived from a tumor selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer and osteosarcoma, Preferably, the body fluid comprises blood, pleural effusion, tissue fluid, lymphatic fluid and / or ascites.

8. The modified TIL according to claim 6, characterized in that The TIL seed cell culture medium comprises the following components: cell culture components, cytokines and immune checkpoint antibodies or antigen-binding fragments thereof, wherein the cytokines include IL-2, the immune checkpoints include: PD-1, LAG-3, TIGIT and / or CTLA-4, the cell culture components are serum culture medium or serum-free culture medium, Preferably, the TIL seed cell culture medium further comprises serum and / or antibiotics. More preferably, the TIL seed cell culture medium comprises any one of the following combinations of components: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α and basal culture medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody and basal culture medium; 4) IL-1β, IL-2, IL-7, G-CSF, GM-CSF, IFN-γ, LAG3 antibody, TNF-α and basal culture medium. 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody and basal culture medium; 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α and basal culture medium; 7) IL-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1 antibody and basal culture medium; 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, C D40 antibody, OX-40 antibody, TIGIT antibody, PD-1 antibody and basal culture medium; 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α and basal culture medium; 10) IL-2, IL-7, IL-12, G-CSF, GM-CSF, IFN-α, IFN-γ, CD28 antibody, CD40 antibody, TIGIT antibody, PD-1 antibody, TNF-α and basal culture medium; 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101 and basal culture medium basal medium; 12) IL-2, IL-7, IL-15, GM-CSF, M-CSF, PD-1 antibody, CNI-1493 and basal medium; 13) IL-2, IL-7, IL-15, CD137 antibody, CD28 antibody, LAG3 antibody, PD-1 antibody, dasatinib and basal medium; 14) IL-2, IL-6, IL-12, G-CSF, M-CSF, IFN-β, IFN-γ, CTLA-4 antibody, PD-1 antibody, dasatinib, LYC-55716, GENE-1858 and basal medium; 15) IL-1α, IL-2, IL-9, IL-15, GM- CSF, CD137 antibody, CD28 antibody, LAG3 antibody, TIGIT antibody, CNI-1493 and basal culture medium; 16) IL-2, IL-7, IL-12, IL-15, IL-21, G-CSF, M-CSF, IFN-γ, CD28 antibody, CD40 antibody, LAG3 antibody, PD-1 antibody, CNI-1493, dasatinib, GNE-1858 and basal culture medium.

9. The modified TIL according to claim 6, characterized in that The TIL cell expansion medium comprises IL-2, IL-7, IL-15 and immune checkpoint antibodies or antigen-binding fragments thereof. Preferably, the immune checkpoint antibodies include PD-1 antibodies. Preferably, the TIL cell expansion medium further comprises one or more of serum, platelets, antibiotics, and a basal medium. More preferably, the TIL cell expansion medium comprises any one of the following combinations of components: 17) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody and GM-CSF; 18) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody, CD137 antibody and GITR antibody; 19) IL-2, IL-7, IL-15, PD-1 antibody, CD3 antibody and CD28 antibody, preferably, the CD3 antibody and CD28 antibody are coupled to a matrix; 2 0) IL-2, IL-7, IL-15, PD-1 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody and GM-CSF, preferably, the CD3 antibody and CD28 antibody are coupled to a matrix; 21) IL-2, IL-7, IL-15, PD-1 antibody, CD3 antibody, CD28 antibody and CD137 antibody, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix; 22) IL-2, IL-7, IL-15, PD-1 antibody, GITR antibody, CD3 antibody, CD28 antibody, TWS119 and CD137 antibody , preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix; 23) IL-2, IL-7, IL-15, PD-1 antibody, TIGIT antibody, CD3 antibody, CD28 antibody, CD137 antibody and GM-CSF, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix; 24) IL-2, IL-7, IL-15, LAG3 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody, CD137 antibody and GITR antibody, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to a matrix. Matrix coupling; 25) IL-2, IL-7, IL-15, PD-1 antibody, TIGIT antibody, CD3 antibody, CD28 antibody, CD137 antibody, CD40 antibody, OX-40 antibody and autologous platelets, preferably, the CD3 antibody, CD137 antibody and CD28 antibody are coupled to the matrix; 26) IL-2, IL-7, IL-15, PD-1 antibody, CTLA-4 antibody, CD3 antibody, CD28 antibody, CD137 antibody, GITR antibody, GM-CSF, TWS119 and allogeneic platelets, preferably, the CD3 antibody and CD28 antibody are coupled to the matrix.

10. The modified TIL according to claim 8 or 9, characterized in that The basal culture medium is selected from any one or more of AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizerTM and FUJIFILM Irvin MHM-C, and / or The serum is selected from human AB serum, subject autologous serum or animal-derived serum.

11. A method for preparing the modified TIL according to any one of claims 1 to 9, comprising introducing the nucleic acid encoding the membrane-anchored IL-7 into the TIL, Preferably, the introduction is by electroporation or liposome nanoparticle introduction. Preferably, the encoding nucleic acid is an expression vector, including a viral vector or a non-viral vector. Preferably, the expression vector comprises a nucleic acid sequence encoding the membrane-anchored IL-7 according to any one of claims 1 to 5, More preferably, the viral vector is a lentiviral vector, and the non-viral vector is a vector based on a transposon system; even more preferably, the non-viral vector is a vector based on the PiggyBac transposition system or the Sleeping Beauty transposition system.

12. The method for preparing the modified TIL according to claim 11, characterized in that: The TIL is prepared by the method for preparing TIL described in any one of claims 6 to 8, Preferably, the method for preparing the modified TIL comprises: 1) incubating a sample containing TIL with a TIL seed cell culture medium to obtain a first TIL cell population; 2) introducing the nucleic acid construct expressing membrane-anchored IL-7 into the first TIL cell population by electroporation or liposome nanoparticles to obtain a second TIL cell population; and 3) incubating the second TIL cell population with a TIL cell expansion culture medium to obtain a third TIL cell population.

13. The modified TIL prepared by the method for preparing the modified TIL according to claim 11 or 12.

14. Use of the modified TIL according to any one of claims 1 to 9 in the preparation of a drug for preventing or treating tumors, Preferably, the tumor is selected from one or more of the following: melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, Bladder cancer, prostate cancer, kidney cancer and osteosarcoma.

15. A method for preventing or treating a tumor, comprising administering the modified TIL according to any one of claims 1 to 9 to a subject in need thereof, Preferably, the tumor is selected from one or more of the following: melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, intestinal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, pelvic poorly differentiated adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer and osteosarcoma.

16. A polypeptide comprising: an optional signal peptide, IL-7, an optional linker, a BCMA extracellular region or a variant thereof, an optional hinge region or a linker, and a transmembrane region connected in sequence, wherein the signal peptide is a CD52 signal peptide or an IL-7 signal peptide, and the transmembrane region is a CD8 transmembrane region or CD52.

17. The polypeptide of claim 16, comprising: CD52 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region, CD52 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, hinge region or linker, and CD52, CD52 signal peptide, IL-7, BCMA extracellular region or its variant, hinge region or linker, and CD52, IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD8 transmembrane region, or IL-7 signal peptide, IL-7, linker, BCMA extracellular region or its variant, and CD52.

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