Tumor-infiltrating lymphocytes expressing membrane-bound cytokines

Abstract

We provide tumor-infiltrating lymphocytes expressing membrane-bound cytokines, specifically modified TILs expressing membrane-tethered IL-7. These TILs can be effectively activated and proliferated, efficiently detected and sorted, and effectively mediate the role of molecular brakes.

Description

[Technical Field]

[0001] The present invention relates to the biotechnology field, and more specifically to tumor-infiltrating lymphocytes expressing membrane-bound cytokines and a method for preparing the same. [Background technology]

[0002] Tumor-infiltrating lymphocyte (TIL) therapy is an emerging immunotherapy that has shown relatively good therapeutic efficacy against solid tumors in its rudimentary form. TILs present within tumors are a highly heterogeneous population, subject to immunosuppression by the tumor microenvironment, resulting in limited tumor-killing effectiveness and a limited survival time in the body. Current TIL therapies involve culturing a population of TIL cells reactivated and amplified from the patient's solid tumor tissue outside the body and reinjecting them into the patient's body to improve targeted killing of solid tumors. However, the immunosuppressive microenvironment within the tumor still suppresses the reinjected TILs, and the activity and survival time of reinjected TILs in the body still need improvement. [Overview of the project] [Problems that the invention aims to solve]

[0003] Modifying unmodified natural TILs with foreign genes to be reinjected is one way to solve the above problems, but currently the types of genetically modified TILs in this field are limited, and there is an urgent need to develop modification technologies that enhance the proliferative capacity and tumor-killing effect of TILs and extend their survival time in the body. [Means for solving the problem]

[0004] A first aspect of the present invention is to first provide a modified TIL which expresses a membrane-tethered cytokine, the cytokine being membrane-tethered IL-7.

[0005] In one or more embodiments, the membrane anchoring IL-7 includes IL-7 or a functional fragment thereof and a transmembrane region. Preferably, IL-7 or a functional fragment thereof is located in the N-terminal direction of the transmembrane region.

[0006] In one or more embodiments, the amino acid sequence of IL-7 is shown as SEQ ID NO:7, and its exemplary coding sequence is shown as SEQ ID NO:8.

[0007] In one or more embodiments, the transmembrane region is a protein transmembrane domain. Preferably, the protein transmembrane domain includes one selected from the CD8 transmembrane region, the CD28 transmembrane region, and the IL-7Rα transmembrane region. Preferably, the CD8 transmembrane region is shown as SEQ ID NO:3, and its exemplary coding sequence is shown as SEQ ID NO:4.

[0008] In one or more embodiments, the transmembrane region is a GPI anchoring region. Preferably, the GPI anchoring region includes a membrane anchoring region of one or more GPI membrane anchoring proteins selected from 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 anchoring region is the membrane anchoring region of one or more GPI membrane anchoring proteins selected from CD52, CD48, CD55, ALPP, and CD90.

[0009] In one or more embodiments, the membrane anchoring IL-7 further includes a membrane surface tag. Preferably, the membrane surface tag is located between the IL-7 and the transmembrane region. Preferably, the membrane anchoring IL-7 includes the IL-7, the membrane surface tag, and the transmembrane region in order from the N-terminus to the C-terminus.

[0010] In one or more embodiments, the membrane surface tag includes a variant thereof that has the function of binding to the extracellular domain of a B cell surface antigen or an antibody. Preferably, the B cell surface antigen is BCMA, and the membrane surface tag is a variant thereof that has the function of binding to the extracellular region of BCMA or an anti-BCMA antibody. Preferably, the variant is a shortened variant, an insertion variant, a deletion variant, a substitution variant, or a combination thereof.

[0011] In one or more embodiments, the BCMA extracellular region is indicated by SEQ ID NO:21.

[0012] In one or more embodiments, the BCMA extracellular region variant is a shortened variant, for example, shown as SEQ ID NO:23.

[0013] In one or more embodiments, the BCMA extracellular region variant is a shortened substitution variant, for example, shown in SEQ ID NO:25.

[0014] In one or more embodiments, the membrane anchoring IL-7 further includes a linker located 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 arrangement of the rigid linkers is one selected from SEQ ID NO: 9, 11, 15, 17.

[0015] In one or more embodiments, the membrane anchoring IL-7 further includes 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). The hinge region includes, but is not limited to, the CD4 extracellular hinge region, the CD8 extracellular hinge region, the CD28 extracellular hinge region, the IgG1Fc hinge region, and the IgG4Fc 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. The linker is a rigid linker or a flexible linker, preferably a rigid linker. Preferably, the sequence of the rigid linker is one selected from SEQ ID NO: 9, 11, 15, and 17.

[0016] Preferably, the linker is located between IL-7 and the film surface tag and / or between the film surface tag and the film penetration region.

[0017] In one or more embodiments, the membrane-attached IL-7 comprises a signal peptide. The signal peptide is located at the N-terminus of the membrane-attached IL-7. Preferably, the signal peptide is a CD52 signal peptide or an IL-7 signal peptide. Preferably, the CD52 signal peptide is shown in SEQ ID NO:1, and its exemplary coding sequence is shown in SEQ ID NO:2. Preferably, the IL-7 signal peptide is shown in SEQ ID NO:5, and its exemplary coding sequence is shown in SEQ ID NO:6.

[0018] In one or more embodiments, the membrane-anchored IL-7 comprises, in order, any signal peptide, IL-7, any linker, a BCMA extracellular region or a variant thereof, any 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.

[0019] In one or more embodiments, the membrane-anchored IL-7 comprises a signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and a transmembrane domain linked in sequence.

[0020] In one or more embodiments, the membrane anchoring IL-7 is connected in sequence. CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD52, CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, CD52 signal peptide, IL-7, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, It contains IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD52.

[0021] In one or more embodiments, the amino acid sequence of the membrane-attached IL-7 is one or more selected from SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, and 47.

[0022] In one or more embodiments, the coding nucleic acid sequence of the membrane-attached IL-7 is one or more of the coding nucleic acid sequences of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, and 47. Preferably, the coding nucleic acid sequence of the membrane-attached IL-7 is one or more selected from SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, and 48.

[0023] In the first group of embodiments of the first aspect, the TIL is prepared by incubating a TIL-containing sample in a TIL seed cell medium to obtain a TIL cell population.

[0024] In one or more embodiments, the sample is selected from ascites, surgically resected primary lesion samples, synchronously and metachronically resected metastatic lesion samples, puncture samples, and body fluids.

[0025] In one or more embodiments, the sample is tissue containing tumor cells. Preferably, the tissue containing tumor cells undergoes pretreatment including fragmentation and / or dissociation of the tumor tissue. The tumor cells are derived from tumors selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, colorectal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, poorly differentiated pelvic 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.

[0026] In one or more embodiments, the body fluid includes blood, pleural fluid, tissue fluid, lymph fluid, and / or ascites.

[0027] In one or more embodiments, the seed cell medium comprises, as components, a cell culture component, a cytokine, and an immune checkpoint antibody or its antigen-binding fragment, wherein the cytokine comprises IL-2, the immune checkpoint comprises PD-1, LAG-3, TIGIT, and / or CTLA-4, and the cell culture component is serum medium or serum-free medium.

[0028] In one or more embodiments, the TIL seed cell medium is: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α, and basal medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α, and basal medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody, and basal medium; 4) IL-1β, IL-2, IL-7, G-CSF, GM-CSF, IFN-γ, LAG3 antibody, PD-1 antibody , TNF-α, and basal medium, 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody, and basal medium, 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α, and basal medium, 7) IL-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1 antibody, and basal medium, 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, CD40 antibody, OX-40 antibody, 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α, and basal 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 medium, 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101, and 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,The preparation comprises one of the following combinations of components of the basal medium: TIGIT antibody, CNI-1493, and basal 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 the components of the basal medium. Preferably, the above combination of components further includes serum and / or antibiotics.

[0029] In the second group of embodiments of the first embodiment, the TIL is prepared by a method comprising the steps of (1) incubating a TIL-containing sample in TIL seed cell medium to obtain a first TIL cell population, and (2) incubating the first TIL cell population in TIL cell amplification medium to obtain a second TIL cell population.

[0030] In one or more embodiments, the sample is selected from ascites, surgically resected primary lesion samples, synchronously and metachronically resected metastatic lesion samples, puncture samples, and body fluids.

[0031] In one or more embodiments, the sample is tissue containing tumor cells. Preferably, the tissue containing tumor cells undergoes pretreatment including fragmentation and / or dissociation of the tumor tissue. The tumor cells are derived from tumors selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, colorectal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, poorly differentiated pelvic 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.

[0032] In one or more embodiments, the body fluid includes blood, pleural fluid, tissue fluid, lymph fluid, and / or ascites.

[0033] In one or more embodiments, step (1) the TIL seed cell medium is: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α, and basal medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α, and basal medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody, and basal medium; 4) IL-1β, IL-2, IL- 7) G-CSF, GM-CSF, IFN-γ, LAG3 antibody, PD-1 antibody, TNF-α, and basal medium, 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody, and basal medium. Basal medium, 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α, and basal medium, 7) IL-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1Antibodies and basal media, 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, CD40 antibody, OX-40 antibody, TIGIT antibody, PD-1 antibody, and basal media, 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α, and basal media, 10) IL-2, IL-7, IL-12, G-CSF, GM-C SF, IFN-α, IFN-γ, CD28 antibody, CD40 antibody, TIGIT antibody, PD-1 antibody, TNF-α, and basal medium, 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101, and 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-C SF, CD137 antibody, CD28 antibody, LAG3 antibody, TIGIT antibody, CNI-1493, and basal 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 one combination of components of basal medium. Preferably, the above combination of components further includes serum and / or antibiotics.

[0034] In one or more embodiments, the TIL cell amplification medium in step (2) comprises IL-2, IL-7, IL-15, and an immune checkpoint antibody or its antigen-binding fragment, preferably the immune checkpoint antibody comprises a PD-1 antibody.

[0035] In one or more embodiments, the TIL cell amplification medium further comprises platelets or platelets, wherein the platelets are heteroplatins or target autologous platelets.

[0036] In one or more embodiments, the TIL cell amplification medium in step (2) is 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 the matrix. 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, G 1) ITR antibody, CD3 antibody, CD28 antibody, TWS119, and CD137 antibody, preferably the CD3 antibody, CD137 antibody, and CD28 antibody are coupled to the 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 the matrix, 24) IL-2, IL-7, IL-15, LAG3 antibody, IL-21, IL-1 2) CD3 antibody, CD28 antibody, CD137 antibody, and GITR antibody, preferably the CD3 antibody, CD137 antibody, and CD28 antibody are coupled to the matrix, 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,The matrix comprises CTLA-4 antibody, CD3 antibody, CD28 antibody, CD137 antibody, GITR antibody, GM-CSF, TWS119, and heterozoamyl platelets, preferably the CD3 antibody and CD28 antibody comprising any one combination of components of those coupled to the matrix.

[0037] In one or more embodiments, the TIL cell amplification medium further comprises serum and / or platelets, and a basal medium.

[0038] In one or more embodiments, the basal medium in the seed cell medium and amplification medium is one or more selected from AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizer™, and FUJIFILM Irvin MHM-C.

[0039] In one or more embodiments, the serum is selected from human AB serum, the subject's own serum, or animal-derived serum.

[0040] The present invention further provides a method for preparing a modified TIL according to any one embodiment herein, comprising the step of introducing the coding nucleic acid sequence of the membrane-anchored IL-7 into the TIL.

[0041] In one or more embodiments, the TIL does not exhibit the membrane anchoring IL-7 before introduction.

[0042] In one or more embodiments, a TIL that does not express the membrane-attached IL-7 is an unmodified TIL.

[0043] In one or more embodiments, the unmodified TIL is prepared by a method for preparing TIL described in any one of the first or second group embodiments of the first aspect of the present invention.

[0044] In one or more embodiments, the method includes the step of introducing a nucleic acid construct expressing the membrane-anchored IL-7, such as an expression vector or mRNA, into the TIL. Preferably, the expression vector is a viral vector or a non-viral vector. Preferably, the expression vector is an embedded vector or a non-embedded vector. Preferably, the viral vector is a lentiviral vector.

[0045] In one or more embodiments, the nonviral vector is a transposon-based vector. Preferably, the nonviral vector is a vector based on the PiggyBac transposon system or the Sleeping Beauty transposon system. Preferably, the nonviral vector is a pKB20 or pNB328 vector.

[0046] In one or more embodiments, the expression vector comprises a membrane-attached IL-7 coding nucleic acid sequence described in any one embodiment of the first aspect of the present invention. Preferably, the expression vector is a pKB20 expression vector comprising a membrane-attached IL-7 coding nucleic acid sequence described in any one embodiment of the first aspect of the present invention.

[0047] In one or more embodiments, the method includes the step of introducing the nucleic acid construct into an unmodified TIL by electroporation or lipid nanoparticles (LNPs).

[0048] In one or more embodiments, the method comprises: step 1) incubating a TIL-containing sample in a TIL seed cell medium described in any one of the second group embodiments of the first embodiment of the present invention to obtain a first TIL cell population; step 2) introducing a nucleic acid construct expressing the membrane-attached IL-7 into the first TIL cell population by electroporation or lipid nanoparticles to obtain a second TIL cell population; and step 3) incubating the second TIL cell population in a TIL cell amplification medium described in any one of the second group embodiments of the first embodiment of the present invention to obtain a third TIL cell population. Other features of the method are as described in the second group embodiments of the first embodiment of the present invention.

[0049] The present invention further provides modified TIL prepared by a modified TIL preparation method described in any one embodiment herein.

[0050] The present invention further provides the use of modified TILs described in any one embodiment herein in the preparation of pharmaceuticals for the prevention or treatment of tumors.

[0051] The present invention further provides a method for preventing or treating a tumor, comprising the step of administering a modified TIL as described in any one embodiment herein to a subject requiring such treatment.

[0052] In one or more embodiments, the tumor is one or more selected from melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, colorectal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, poorly differentiated pelvic adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma.

[0053] The present invention further provides a polypeptide comprising any signal peptide, IL-7, any linker, a BCMA extracellular region or a variant thereof, any 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.

[0054] In one or more embodiments, the polypeptides are linked in order. CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, CD52 signal peptide, IL-7, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, It contains IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD52. [Effects of the Invention]

[0055] The advantages of the present invention are as follows:

[0056] This invention effectively activates and proliferates TILs by anchoring the cytokine IL-7 to the TIL surface using the GPI anchoring protein, significantly improving its tumor-killing function. Furthermore, it significantly improves the expression intensity of membrane-surface IL-7 anchored by GPI compared to conventional immobilization using amino acid transmembrane domains. In addition, by introducing the wild-type or mutant sequence of BCMA, a low molecular weight protein not present on the T cell surface, into the structure of membrane-anchored IL-7 and using it as a detection and selection tag, this invention not only effectively detects and selects membrane-anchored IL-7-positive TILs, but also effectively mediates the ADCC and CDC effects in vivo under the presence of antibodies, timely killing TILs expressing membrane-anchored IL-7, including the BCMA extracellular domain, acting as a molecular brake, and significantly improving the safety of clinical application of membrane-anchored IL-7-expressing TILs. [Brief explanation of the drawing]

[0057] [Figure 1] These are the RTCA killing curves for T002-TIL cells and T002-2 cells against homologous melanoma cells. [Figure 2] This is a curve showing the change in tumor volume in an SKOV-3320CDX mouse model that was killed in vivo by T014-9 cells. [Figure 3] This is the in vivo fluorescence imaging result of an SKOV-3320CDX mouse model that was killed by T014-9 cells in vivo. [Figure 4] This is a curve showing the change in tumor volume in a syngeneic PDX mouse model that was killed in vivo by T011-TIL and T011-9 cells. [Figure 5] This is an evaluation of tumor lesion images in T011 patients before and after TIL reinfusion treatment. [Figure 6] This is an evaluation of tumor lesion images in T012 patients before and after TIL reinfusion treatment. [Figure 7] This is an evaluation of tumor lesion images in T013 patients before and after TIL reinfusion treatment. [Figure 8] This is an evaluation of tumor lesion images in T014 patients before and after TIL reinfusion treatment. [Modes for carrying out the invention]

[0058] The inventors have discovered that by anchoring the cytokine IL-7 and a portion of BCMA to the surface of TILs, TILs can be effectively activated and proliferated, and membrane-anchored IL-7-positive TILs can be detected and selected, and the role of molecular brakes can be effectively mediated.

[0059] Definition of Terms

[0060] In this application, the term "interferon" typically refers to a family of highly species-specific proteins that inhibit viral replication and cell proliferation and modulate immune responses. Typical preferred interferons include, but are not limited to, recombinant interferon α-2b, recombinant interferon α-2a, recombinant interferon α-2C, interferon α-n1, consensus α-interferon, or interferon α-n3.

[0061] In this application, the term "colony-stimulating factor" usually refers to a secreted glycoprotein that activates intracellular signaling pathways by binding to receptor proteins on the surface of hematopoietic stem cells, thereby promoting cell proliferation and differentiation into specific types of blood cells (usually leukocytes; see erythropoietin for erythrocyte formation). These may be artificially synthesized and administered exogenously.

[0062] In this application, the term "antigen-binding fragment" usually refers to a part of an antibody molecule, containing amino acids responsible for specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope," as mentioned above. As mentioned above, the antigen-binding domain typically includes the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), but does not necessarily include both. An Fd fragment, for example, has two VH regions and usually retains part of the antigen-binding function of a complete antigen-binding domain. Examples of antibody antigen-binding fragments include (1) Fab fragments, which are monovalent fragments having VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab')2 fragments, which are bivalent fragments having two Fab fragments linked by disulfide crosslinks in the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains of a single antibody arm; (5) dAb fragments having a VH domain (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); (6) isolated complementarity-determining regions (CDRs); and (7) single-stranded Fv (scFv) derived from, for example, scFV libraries.The two domains of the Fv fragment, VL and VH, are encoded by independent genes, but can be linked together in a recombinant manner by a synthetic linker, which is prepared so that the VL and VH regions pair to form a single monovalent single protein chain (called single-chain Fv (scFv)) (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 common techniques known to those skilled in the art and are evaluated for function in the same manner as complete antibodies. In this application, the term “antigen-binding fragment” further includes ligands that specifically bind to antigens, particularly cell surface receptor molecular antigens. Ligands are typically polypeptides or compounds that bind to receptor proteins (e.g., the aforementioned antigen) with high affinity and specificity to elicit a functional response. For example, the ligands include the ligand CD40L for CD40, the ligand CD137L for CD137, the ligand CD80 for CD28, and / or the ligand OX-40L for OX-40.

[0063] In this application, the term “immune checkpoint” typically refers to several molecules on the surface of CD4+ and CD8+ T cells. These molecules can be effectively used as “brakes” for downregulating, modulating, or inhibiting the antitumor immune response. These inhibitory molecules can be inhibited by inhibition at the DNA, RNA, or protein level.

[0064] In this application, the term "activating antibody" usually refers to an antibody against receptors such as TCRs and / or costimulatory receptors on the surface of immune cells such as T cells, which, by binding to TCRs and / or costimulatory receptors, can open the downstream activation signaling pathways of these receptors and significantly improve the activation and proliferation levels of the immune cells.

[0065] In this application, the term "costimulatory receptor" refers to a receptor typically expressed on the surface of immune cells, such as T cells, that is dependent on the response of an activated immune response. Assuming the presence of an antigen-presenting cell and the provision of a primary signal, the costimulatory receptor opens the costimulatory signal by binding to a corresponding activated antibody, ligand, or antigen-binding fragment, and further fully activates immune effector cells. Activation of the costimulatory receptor-mediated signaling pathway typically plays a crucial role in the effective development of an immune response. Common costimulatory receptors include, but are not limited to, CD28, CD40, CD137, and OX-40.

[0066] In this application, the term "tumor death factor TNF" usually refers to the TNF protein, but not the TNF ligand superfamily. The Genbank accession number for human TNFα is NP_000585.2. The term "TNFα" includes the precursor form of TNFα, pro-TNFα, full-length TNFα, and any form of TNFα produced by intracellular processes. The term further includes native and non-native variants of TNFα, such as splicing variants, allele variants, and isotypes. TNFα can bind to two receptors: TNFR1 (type 1 TNF receptor; CD120a; p55 / 60) and TNFR2 (type 2 TNF receptor; CD120b; p75 / 80). TNFα plays a role as a pro-inflammatory cytokine, for example, in neuroinflammation.

[0067] In this application, the term "tumor-infiltrating lymphocytes" usually refers to infiltrating lymphocytes isolated from tumor tissue. Immunohistochemical staining has revealed that tumor-infiltrating lymphocytes may include CD3+ T cells, CD20+, CD79α+ B cells, plasma cells, and CD56+ NK cells, but may also include T cells, small amounts of B cells, NK cells, macrophages, and dendritic cells. These tumor-infiltrating lymphocytes can produce a highly efficient and specific antitumor-killing effect through direct cytotoxic T lymphocytes and secreted cytokines. In this application, these tumor-infiltrating lymphocytes may include mononuclear leukocytes that leave the bloodstream and migrate to the tumor. These tumor-infiltrating lymphocytes may be selected from the group consisting of T cells, B cells, innate killer cells, and innate killer T cells.

[0068] In this application, the term "seed cells" usually refers to tumor-infiltrating lymphocytes cultured from tumor tissue using any suitable medium, including the medium of this application. These cells may be a starting cell population for further expansion culture, or a cell population for a terminal use, such as a cell population for reinjection therapy in a subject. The seed cells can be obtained from tumor tissue over any different culture time, and the culture time of the seed cells is not limited to a specific culture time but can be determined according to the subsequent use of the seed cells.

[0069] In this application, the term "IL-15" usually refers to a pro-inflammatory cytokine that acts as an effective growth, survival, and activation factor for T cells (especially intestinal intraepithelial lymphocytes (IELs)) and innate killer (NK) cells, and is also called "IL-15 antigen" and "interleukin-15". Increased IL-15 expression has been observed in several 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 driving factor of lymphoma formation in RCD.

[0070] In this application, the term "IL-2" refers to a protein that is normally produced from normal peripheral blood lymphocytes and derived from lymphoid factors present in the body at low concentrations. Morgan et al. (1976) Science 193:1007-1008 initially described IL-2 and called it T cell growth factor because it can induce the proliferation of stimulated T lymphocytes. This term also includes proteins that have been modified after IL-2 expression (e.g., glycosylation, acetylation, phosphorylation, etc.).

[0071] In this application, the term "IL-7" usually refers to the protein encoded by the IL-7 gene. IL-7 is a hematopoietic growth factor secreted from matrix cells in the bone marrow and thymus. It can be produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells. IL-7 can be involved in the action of T cells and B cells.

[0072] In this application, the term "PD-1" usually refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on previously activated T cells in the body and binds to two ligands, PD-L1 and PD-L2. The complete hPD-1 sequence is described in GenBank accession number U64863.

[0073] In this application, the term "LYC-55716" usually refers to one of the RORγ activators with CAS number 2055536-64-4.

[0074] In this application, the term "GNE-1858" usually refers to the HPK1 inhibitor with CAS number T11438.

[0075] In this application, the term "TWS119" usually refers to a GSK-3 inhibitor with CAS number 601514-19-6.

[0076] In this application, the term "RRX-001" usually refers to an epigenetic modulator having radiosensitizing activity. RRRX-001 downregulates the CD47 / SIRPα axis and can repolarize TAMs and other immunosuppressive cells in the tumor microenvironment to an immunostimulatory phenotype. Its CAS number is 925206-65-1.

[0077] In this application, the term "CNI-1493" typically refers to a compound with CAS number 164301-53 that can inhibit the production of inflammatory cytokines (including TNF).

[0078] In this application, the term "anchoring" means that a part of interest is fixed to a substance by intermolecular interactions (chemical bonds, van der Waals forces, and hydrogen bonds). Membrane-anchored cytokines refer to cytokines that are fixed to the cell membrane.

[0079] In this specification, the term "inducing TILs" refers to the process of isolating TIL cells from animal tumors. By amplifying TIL cells to a certain number outside the body and then reinjecting them into the animal's body, it is possible to achieve an effect of controlling tumor growth and metastasis.

[0080] The present invention is suitable for isolating TILs from any tumor tissue, including tumor tissue obtained by open surgery or minimally invasive surgery. Exemplary tumor tissues include, for example, tumor tissue from cancers such as 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, and osteosarcoma.

[0081] Details of the invention

[0082] The present invention first provides a modified TIL which expresses membrane-anchored IL-7. Membrane-anchored IL-7 is a polypeptide comprising one or more IL-7 or its functional fragments and transmembrane regions, and optionally also comprising a membrane surface tag. The sequence of IL-7 is as shown in SEQ ID NO:7. The functional fragment of IL-7 has a common meaning in the art, and its sequence will be known to those skilled in the art.

[0083] The inventors have found that the expression intensity of membrane-surface IL-7 anchored by GPI is significantly improved, but conventional protein transmembrane domains can also achieve a certain effect. The transmembrane region may be the transmembrane region of the membrane-surface tag, or the transmembrane region of another transmembrane protein, as long as it can anchor the membrane-anchored IL-7 to the surface of the cell membrane without adversely affecting its activity and function. Therefore, those skilled in the art can select an appropriate transmembrane region as needed (for example, depending on the structure or composition of the membrane). The transmembrane region includes, but is not limited to, any one of the following transmembrane regions: 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-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40. Exemplary transmembrane regions include the CD28 transmembrane region, the CD8 transmembrane region, and the IL-7R transmembrane region. The transmembrane region and its adjacent structures (e.g., membrane surface tags) are connected via hinge regions or linkers. Therefore, the polypeptide may be a membrane surface protein or a transmembrane protein.

[0084] Preferably, the transmembrane region is a GPI anchoring region and may include the membrane anchoring region of one or more GPI membrane anchoring proteins selected from 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 anchoring region is the membrane anchoring region of one or more GPI membrane anchoring proteins selected from CD52, CD48, CD55, ALPP, and CD90. The amino acid sequence of the GPI anchoring region of CD52 is shown in SEQ ID NO:29, and its exemplary coding sequence is shown in SEQ ID NO:30.

[0085] The membrane surface tag is located at the N-terminus or C-terminus of 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 multiple embodiments of IL-7, 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, first IL-7-membrane surface tag-second IL-7-transmembrane region, or first IL-7-second IL-7-membrane surface tag-transmembrane region. This can be inferred by analogy. Preferably, the IL-7 is located at the N-terminus of the membrane surface tag.

[0086] In embodiments of multiple membrane surface tags, the multiple membrane surface tags may be arranged in series or dispersed at any extracellular location of membrane surface cytokines (for example, the N-terminus and C-terminus of any IL-7), provided that they are located at the N-terminus of the transmembrane region and do not affect the function of the membrane surface tags and membrane surface cytokines.

[0087] The N-terminus of the transmembrane region (for example, between the membrane surface tag and the transmembrane region) may contain a hinge region or a linker, or 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. The hinge region includes, but is not limited to, the CD4 extracellular hinge region, the CD8 extracellular hinge region, the CD28 extracellular hinge region, the IgG1Fc hinge region, and the IgG4Fc 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, and its exemplary coding sequence is shown in SEQ ID NO:20. The linkers are rigid linkers or flexible linkers, and their sequences are shown in SEQ ID NO: 9, 11, 13, 15, and 17, with exemplary code sequences shown in SEQ ID NO: 10, 12, 14, 16, or 18, respectively. Preferably, the sequence of the rigid linkers is one selected from SEQ ID NO: 9, 11, 15, and 17.

[0088] In embodiments containing a film surface tag, a linker may be provided between IL-7 and the film surface tag. In one or more embodiments, the linker is a rigid linker or a flexible linker, preferably a rigid linker. Preferably, the arrangement of the rigid linkers is one selected from SEQ ID NO: 9, 11, 15, 17.

[0089] In some embodiments, linkers are provided between IL-7 and the film surface tag, and between the film surface tag and the film penetration region.

[0090] In this specification, a membrane surface tag (or membrane surface tag) includes a variant thereof that retains the function of binding to the BCMA extracellular domain or an anti-BCMA antibody. The BCMA extracellular domain is shown in SEQ ID NO:21, and its exemplary coding sequence is shown in SEQ ID NO:22. The variant may be a shortened variant, insertion variant, deletion variant, 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 may be any anti-BCMA antibody known to those skilled in the art. Exemplary examples include the truncated variant of the BCMA extracellular domain shown in SEQ ID NO:23, its exemplary coding sequence shown in SEQ ID NO:24; the substituted variant of the BCMA extracellular domain shown in SEQ ID NO:25, its exemplary coding sequence shown in SEQ ID NO:26; and the truncated substituted variant of the BCMA extracellular domain shown in SEQ ID NO:27, its exemplary coding sequence shown in SEQ ID NO:28.

[0091] The membrane-bound IL-7 may further contain a signal peptide. Those skilled in the art can select a suitable signal peptide in a desired expression form. The signal peptide can be cleaved during the secretion of the polypeptide. Exemplarily, the signal peptide is located at the N-terminus of the membrane-bound IL-7. Preferably, the signal peptide is a CD52 signal peptide or an IL-7 signal peptide. Preferably, the CD52 signal peptide is shown in SEQ ID NO:1, and its exemplary coding sequence is shown in SEQ ID NO:2. Preferably, the IL-7 signal peptide is shown in SEQ ID NO:5, and its exemplary coding sequence is shown in SEQ ID NO:6.

[0092] In some embodiments, the present invention provides a polypeptide or a TIL expressing such polypeptide, wherein the polypeptide is membrane-anchored IL-7 and comprises, in sequence, an arbitrary signal peptide, IL-7, an arbitrary linker, a BCMA extracellular region or a variant thereof, an arbitrary 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, in sequence, 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, in order, (1) a CD52 signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, and a CD8 transmembrane region; (2) a CD52 signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, and a CD52; (3) a CD52 signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, a hinge region or linker, and a CD52; (4) a CD52 signal peptide, IL-7, a BCMA extracellular region or a variant thereof, a hinge region or linker, and a CD52; (5) an IL-7 signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, and a CD8 transmembrane region; or (6) an IL-7 signal peptide, IL-7, a linker, a BCMA extracellular region or a variant thereof, and a CD52. In an exemplary embodiment, the amino acid sequence of the membrane-anchored IL-7 is one or more selected from SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, and 45. The exemplary coding nucleic acid sequence is one or more selected from SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, and 46.

[0093] The present invention further includes fragments, derivatives, and analogues of the polypeptide. As used herein, the terms “fragment,” “derivative,” and “analog” refer to polypeptides that substantially retain the same biological function or activity as the polypeptide. Fragments, derivatives, or analogues of a polypeptide or domain may be (i) polypeptides or domains in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, such substituted amino acid residues may or may not be encoded in the genetic code; (ii) polypeptides or domains having substituents on one or more amino acid residues; (iii) polypeptides formed by the fusion of a mature polypeptide or domain with another compound (e.g., a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol); or (iv) polypeptides formed by the fusion of an additional amino acid sequence with the polypeptide or domain sequence (e.g., a lead sequence or secretion sequence, or a sequence or proprotein sequence for purifying the polypeptide, or a fusion protein formed with an antigen IgG fragment). As taught herein, these fragments, derivatives, and analogues are well known to those skilled in the art.

[0094] The term “variant” or “mutant” refers to a peptide or polypeptide whose amino acid sequence has been altered by the insertion, deletion, or substitution of one or more amino acids compared to a reference sequence, but which retains at least one biological activity. A variant described in any one embodiment herein has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the reference sequence (the 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 includes an amino acid sequence that retains the biological activity of the reference sequence (e.g., as a chimeric antigen receptor, surface cytokine expression protein, signal-converting receptor). Sequence identity between two aligned sequences can be calculated, for example, using NCBI’s BLASTp. A variant also includes an amino acid sequence that has one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of a reference sequence, while retaining the biological activity of the reference sequence. The number of mutations is typically 1 to 10 or less, for example, 1 to 8, 1 to 5, or 1 to 3. Substitutions are preferably conservative substitutions. For example, in this art, conservative substitutions by amino acids with similar or closely related properties generally do not alter the function of a protein or polypeptide."Amino acids with similar or identical properties" include, for example, families of amino acid residues with similar side chains, which include 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 nonpolar 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, in the polypeptides of the present invention, substituting one or more sites with other amino acid residues from the same side chain class does not substantially affect their activity.

[0095] Shortened variants of polypeptides or domains may also be used in the present invention, and such variants only need to retain substantially the same biological function or activity as the polypeptide or domain. For example, a shortened variant of the BCMA extracellular domain that retains the reactivity of the BCMA extracellular domain with antigen antibodies may be used.

[0096] Polypeptides described herein may be modified polypeptides. Modifications (generally those that do not alter the primary structure) include chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, for example, polypeptides produced by glycosylation modification during or in a further processing step of the polypeptide. This modification can be achieved by exposing the polypeptide to an enzyme that glycosylates (e.g., mammalian glycosylation enzymes or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (e.g., tyrosine phosphate, serine phosphate, threonine phosphate). Polypeptides whose anti-protein hydrolysis properties are improved or whose solubility is optimized by modification are also included.

[0097] Nucleic acid molecules encoding the polypeptide or domain described herein are also within the scope of this specification. Nucleic acid molecules may be in DNA or RNA form. Nucleic acid molecules described herein include sequences modified by codon optimization, provided that the amino acid sequence encoded by the nucleic acid molecule remains unchanged. Codon-optimized sequences can exhibit more suitable expression for specific biological species. Methods for codon-optimizing nucleic acid molecular sequences are well known in this art. The coding region sequence encoding a mature polypeptide may be a degenerate mutant. As used herein, "degenerate mutant" means a nucleic acid sequence that encodes an amino acid sequence or domain fragment encoded by the nucleic acid sequence shown in the sequence listing, but is different from the corresponding nucleic acid sequence.

[0098] Nucleic acid molecules encoding mature polypeptides include coding sequences that encode only mature polypeptides, coding sequences of mature polypeptides and various additional coding sequences, and coding sequences of mature polypeptides (and any additional coding sequences) and non-coding sequences. The term "nucleic acid molecule encoding a polypeptide" may include any nucleic acid molecule that encodes such polypeptide, or it may include any nucleic acid molecule that further includes additional coding and / or non-coding sequences.

[0099] The nucleic acid molecules of the present invention may be coding sequences of polypeptides or domains, or expression cassettes of polypeptides or domains. In this specification, a coding sequence refers to a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., a CAR, a single-chain antibody, a hinge region, a transmembrane region, an intracellular signaling region, or a cytokine protein). The boundaries of a coding sequence are typically determined by a ribosome binding site immediately upstream of the 5' open reading frame of the mRNA (in the case of prokaryotic cells) and a transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. The coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences. In this specification, an expression cassette refers to a complete set of elements necessary to express a gene of interest, including a promoter, a gene coding sequence, and a PolyA tail signaling sequence. In some embodiments, the coding sequence or expression cassette is integrated into the cellular genome. Thus, in these embodiments, the T cell genome described herein stably incorporates the expression cassette encoding the polypeptide described herein.

[0100] The present invention also relates to variants of the nucleic acid molecule encoding polypeptides or polypeptide fragments, analogs, and derivatives having the same amino acid sequence as the present invention. These polynucleotide variants may be naturally occurring allelic variants or unnaturally occurring variants. These nucleotide variants include substitutional variants, deletion variants, and insertion variants. As is known in the art, allelic variants are polynucleotide substitutional forms that may involve the substitution, deletion, or insertion of one or more nucleotides but do not substantially alter the function of the encoded polypeptide.

[0101] The present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, 85%, 90%, or 95% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the aforementioned polynucleotides of the present invention under stringent conditions (or strict conditions). In this specification, “stringent conditions” means (1) hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 × SSC, 0.1% SDS, 60°C, or (2) addition of a denaturant during hybridization, e.g., 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, or (3) hybridization occurs only when the two sequences are at least 90%, preferably 95% or more, more preferably 95% identical.

[0102] The present invention also relates to a complementary sequence of the sequence or a nucleic acid fragment hybridized with the sequence. As used herein, the length of a “nucleic acid fragment” includes at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragment can be used in nucleic acid amplification techniques such as PCR to determine and / or isolate polynucleotides encoding a desired polypeptide or domain. As used herein, “hybridization” primarily refers to the pairing of nucleic acid sequences under strict conditions. An exemplary strict condition is hybridization in a solution of 0.1 × SSPE (or 0.1 × SSC) in 0.1% SDS at 65°C, followed by washing the membrane.

[0103] In one embodiment, the nucleic acid molecule is a nucleic acid construct containing coding sequences for polypeptides described herein and one or more regulatory sequences operably linked to these sequences. The polynucleotides of the present invention can be manipulated in various ways to ensure polypeptide expression. Depending on the differences or requirements of the expression vector, the nucleic acid construct may be manipulated before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0104] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operably ligated to a sequence encoding the protein to be expressed. The promoter may be any nucleotide sequence exhibiting transcriptional activity in a selected host cell, and may include mutant, truncated, and heterozygous promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell. The regulatory sequence may be a suitable transcriptional terminator sequence that is recognized by the host cell and terminates transcription. The terminator sequence is operably ligated to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in a selected host cell may be used herein. The regulatory sequence may also be a suitable read sequence, which is an untranslated region of mRNA important for translation by the host cell. The read sequence is operably ligated to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in a selected host cell may be used in the present invention.

[0105] In one embodiment, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombination vector. Specifically, the coding sequences of the polypeptides herein can be cloned into many types of vectors, and such vectors include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors may be used to provide the coding sequences of the polypeptides of the present invention. Expression vectors can be provided to cells in vector form. Expression of the polynucleotides of the present invention is usually achieved by operably linking the coding sequences of the present invention to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and incorporation into eukaryotic cells. A typical cloning vector includes a transcriptional and translational terminator, an initiation sequence, and a promoter that regulate the expression of the desired nucleic acid sequence. The vector may be a virus, and viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Vector technology is well known in this art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other manuals for virology and molecular biology. Homologous recombination vectors are used to incorporate the expression cassettes described herein into the host genome.

[0106] Typically, a suitable vector includes a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers.

[0107] Suitable promoters include, but are not limited to, early cytomegalovirus (CMV) promoter sequences, elongation factor-1α (EF-1α), Simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus early promoter, Rous sarcoma virus promoter, and human gene promoters (e.g., actin promoter, myosin promoter, heme promoter, and creatine kinase promoter, etc.).

[0108] To evaluate polypeptide expression, the expression vector introduced into cells may contain selectable marker genes to identify and select expressing cells from a cell population seeking transfection or infection via a viral vector. The selectable marker can be loaded onto another DNA molecule and used in the cotransfection process. Both sides of the selectable marker may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0109] The polynucleotides described herein are typically obtained by PCR amplification. Specifically, primers are designed according to the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the relevant sequences can be obtained by amplification using a commercially available cDNA library or a cDNA library prepared by a common method known to those skilled in the art as a template. If the sequence is long, it is common to perform two or more PCR amplifications and then ligate the fragments amplified each time in the correct order. Alternatively, the nucleic acid molecules described herein may be synthesized directly.

[0110] Methods for introducing nucleic acid molecules into cells and expressing genes in cells are known in the art. Nucleic acid molecules, such as vectors or mRNA, can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means. Physical means include calcium phosphate precipitation, lipid nanoparticle transfection, particle bombardment, microinjection, and electroporation. Biological means include the use of DNA and RNA vectors. Chemical means include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0111] As described herein, host cells express the polypeptides described herein or contain the nucleic acid molecules described herein. The host cells include not only the final target cell (e.g., TIL) to provide the sequence encoding the protein of the present invention or the vector described herein, but also various cells used in the production process of the target cell (e.g., E. coli cells). In one embodiment, this specification provides a TIL that stably expresses the polypeptide (membrane-attached IL-7) described herein. Typically, before introducing membrane-attached IL-7, the TIL is a TIL that does not express membrane-attached IL-7, or the TIL is an unmodified TIL.

[0112] TILs suitable for the present invention may be various types of TILs of various origins, for example, derived from tissue containing tumor cells. Typically, the tissue containing tumor cells undergoes pretreatment including fragmentation and / or dissociation of the tumor tissue. The diameter of the fragmented and / or dissociated tumor tissue is about 1 mm to about 10 mm, and may be, 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 tumors selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, colorectal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, poorly differentiated pelvic adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, cranial glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the tissue containing the tumor cells is the tumor tissue or body fluid of the cancer subject. The body fluid includes blood, pleural fluid, interstitial fluid, lymph fluid, and / or ascites. For example, the body fluid may include ascites (e.g., ascites from a gastric cancer patient or ascites from a breast cancer patient) and / or pleural fluid (e.g., pleural fluid from a patient with endometrial stromal sarcoma).

[0113] The method for producing TILs is not limited, but in a preferred embodiment, TILs are obtained using the method described in PCT / CN 2021 / 133059, which is incorporated in whole into this application. In an exemplary embodiment, the TILs are prepared by the following method, which involves incubating a TIL-containing sample in TIL seed cell medium to obtain a TIL cell population.

[0114] The TIL seed cell mediums described herein include any TIL seed cell medium known in the art, e.g., TIL seed cell mediums described in PCT / CN2021 / 096585 or PCT / CN2021 / 133059, which are incorporated herein by reference in their entirety. In specific embodiments, the TIL seed cell medium comprises, as components, cell culture components, cytokines, and immune checkpoint antibodies or their antigen-binding fragments, 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 or serum-free media. In one or more embodiments, the concentration of IL-2 is about 3000 IU / mL or less. The cytokines may further 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. For example, the concentration of IL-7 is approximately 200 to 1000 U / mL, the concentration of IL-15 is approximately 200 to 500 U / mL, the concentration of TNFα is approximately 10 to 100 pg / mL, and the concentration of GM-CSF is approximately 200 to 5000 U / mL.The concentration of G-CSF is approximately 300 U / mL to approximately 1000 U / mL, the concentration of M-CSF is approximately 300 U / mL to approximately 800 U / mL, the concentration of IFN-γ is approximately 10 to approximately 1000 ng / mL, the concentration of IFN-γ is approximately 300 to approximately 1000 U / mL, the concentration of IFN-α is approximately 500 U / mL to approximately 1000 U / mL, the concentration of IFN-β is approximately 200 U / mL to approximately 500 U / mL, the concentration of IL-4 is approximately 200 to approximately 500 U / mL, and the IL The concentration of IL-1α is approximately 200 to 500 U / mL, the concentration of IL-1β is approximately 200 to 500 U / mL, the concentration of IL-6 is approximately 200 to 500 U / mL, the concentration of IL-9 is approximately 200 to 500 U / mL, the concentration of IL-18 is approximately 200 to 500 U / mL, the concentration of IL-12 is approximately 200 to 1000 U / mL, the concentration of IL-21 is approximately 200 to 500 U / mL, and the concentration of IL-10 is approximately 200 to 500 U / mL.

[0115] The immune checkpoint antibody or its antigen-binding fragment in the TIL seed cell medium may be a human PD-1 antibody or its antigen-binding fragment at a concentration of approximately 1 to 100 μg / mL, a LAG-3 antibody or its antigen-binding fragment at a concentration of approximately 3 to 10 μg / mL, a TIGIT antibody or its antigen-binding fragment at a concentration of approximately 1 to 25 μg / mL, or a CTLA-4 antibody or its antigen-binding fragment at a concentration of approximately 1 to 100 μg / mL.

[0116] The TIL seed cell medium may contain a costimulatory receptor antibody or its antigen-binding fragment, such as a CD40 antibody or its antigen-binding fragment, an OX-40 antibody or its antigen-binding fragment, a CD137 antibody or its antigen-binding fragment, and / or a CD28 antibody or its antigen-binding fragment. The concentration of the CD137 antibody or its antigen-binding fragment is approximately 1 to approximately 100 μg / mL, the concentration of the CD28 antibody or its antigen-binding fragment is approximately 1 to approximately 10 μg / mL, the concentration of the CD40 antibody or its antigen-binding fragment is approximately 5 to approximately 10 μg / mL, and the concentration of the OX-40 antibody or its antigen-binding fragment is approximately 3 to approximately 10 μg / mL.

[0117] The seed cell medium may also contain M2 macrophage inhibitors, such as RRx001 and / or CNI-1493. The concentration of the M2 macrophage inhibitor is approximately 0.1 to 100 μM. The seed cell medium may also contain regulatory T cell inhibitors such as CAL-101, dasatinib, imatinib, and / or panobinostat. The concentration of the regulatory T cell inhibitor is approximately 0.1 to 100 μM. The seed cell medium may further contain bone marrow-derived suppressor cell (MDSC) inhibitors such as AG490, decitabine, sunitinib, and / or BBI608. The concentration of the bone marrow-derived suppressor cell inhibitor is approximately 0.1 to 100 μg / mL. The seed cell medium may further contain 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 medium may also contain a T cell differentiation inhibitor such as TWS119. The concentration of the T cell differentiation inhibitor is about 1 to about 10 μM.

[0118] In specific embodiments, the TIL seed cell medium is: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α, and basal medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α, and basal medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody, and basal medium; 4) IL-1β, IL-2, IL-7, G-CSF, GM-CSF, IFN-γ, LAG3 antibody, PD-1 antibody, TNF-α, and basal medium, 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody, and basal medium, 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α, and basal medium, 7) IL-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1 antibody, and basal medium, 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, CD40 antibody, OX-40 antibody, TIGIT antibody , PD-1 antibody, and basal medium, 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α, and basal 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 medium, 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101, and basal medium, 12) IL-2, IL-7, IL-15, GM-C SF, 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 medium, 16) comprising any one combination of 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 components of basal medium. Preferably, the combination of components further comprises serum and / or antibiotics. The serum concentration is about 1 to about 10% (v / v). The antibiotic comprises penicillin and / or streptomycin (e.g., PS biantibiotic). The antibiotic concentration is about 1 to about 200 U / mL.

[0119] In other embodiments, the TIL is prepared by a method comprising the steps of (1) incubating a TIL-containing sample in TIL seed cell medium to obtain a first TIL cell population, and (2) incubating the first TIL cell population in TIL cell amplification medium to obtain a second TIL cell population. The TIL seed cell medium is as described above.

[0120] The TIL cell amplification medium may be any medium for growing TIL cells in the art, for example, the culture composition for amplifying TIL cells and the TIL cell amplification medium containing the culture composition described in PCT / CN2021 / 133059, the entire literature of which is incorporated herein by reference. In a specific embodiment, the TIL cell amplification medium comprises IL-2, IL-7, IL-15, an immune checkpoint antibody or its antigen-binding fragment, and a basal medium. The amplification medium further comprises one, two, three, or four of the following: IL-21, IL-12, GM-CSF, and a small molecule that increases the proportion of memory T cells. Preferably, the amplification medium further comprises IL-21 and IL-12, or IL-21, IL-12, and GM-CSF, or GM-CSF and a small molecule that increases the proportion of memory T cells. Preferably, the small molecule that increases the proportion of memory T cells is TWS119. The aforementioned immune checkpoint antibody includes one or more selected from PD-1 antibody, CTLA-4 antibody, LAG3 antibody, TIM3 antibody, TIGIT antibody, and BTLA antibody.

[0121] The TIL cell amplification medium may further contain autologous platelets or heterologous platelets. The TIL cell amplification medium may further contain one or more selected from CD3 antibody, CD28 antibody, CD137 antibody, OX40 antibody, GITR antibody, ICOS antibody, CD206 antibody, and CD40 antibody. Preferably, the culture composition further contains one, two, three, or four selected from CD3 antibody, CD28 antibody, CD137 antibody, and GITR antibody. For example, the amplification medium further contains (1) CD3 antibody and CD28 antibody, or (2) CD3 antibody, CD28 antibody, CD137 antibody, and GITR antibody.

[0122] In one or more embodiments, the TIL cell amplification medium is 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, wherein preferably the CD3 antibody and CD28 antibody are coupled to the matrix. 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 being 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 being coupled to the matrix; 22) IL-2, IL-7, IL-15, PD-1 antibody, GITR 1) Antibodies, CD3 antibody, CD28 antibody, TWS119, and CD137 antibody, preferably the CD3 antibody, CD137 antibody, and CD28 antibody are coupled to the 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 the 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 the matrix, 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,The matrix comprises CTLA-4 antibody, CD3 antibody, CD28 antibody, CD137 antibody, GITR antibody, GM-CSF, TWS119, and heterozoamyl platelets, preferably the CD3 antibody and CD28 antibody comprising any one combination of components of those coupled to the matrix.

[0123] In one or more embodiments, the TIL cell amplification medium comprises a basal medium and one component selected from the following, 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 equivalent concentrations 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 equivalent concentrations of these components; (3) 200-6000 (4) 200-6000 U / 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 equivalent concentrations of the above components, (4) 200-6000 (5) 200-6000 U / 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, 0.5-10 μg / mL of CD28 antibody, and 200-5000 U / mL of GM-CSF, or equivalent concentrations of the above 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 of CD137 antibody, and 1-100 μg / mL of GITR antibody, or equivalent concentrations of these components, (6) 200-6000IU / 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, 0.5-10 μg / mL of CD3 antibody, 1-10 μg / mL of CD28 antibody, 1-100 μg / mL of CD137 antibody, and 1-100 μg / mL of GITR antibody, or equivalent concentrations 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 concentrations 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, matrix-coupled CD3 antibody, matrix-coupled CD28 antibody, 1-100 μg / mL of CD137 antibody, and 1-100 μg / mL of GITR antibody, or equivalent concentrations of these components, (9) 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, matrix-coupled CD3 antibody, matrix-coupled CD28 antibody, 1-100 μg / mL CD137 antibody, and 1-100 μg / mL GITR antibody, or equivalent concentrations of these components, (10) 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-coupled CD3 antibody, matrix-coupled CD28 antibody, and matrix-coupled CD137 antibody, or equivalent concentrations of these components, (11) 200-6000IU / mL IL-2, 5-100 ng / mL IL-7, 5-100 ng / mL IL-15, 1-100 μg / mL PD-1 antibody, matrix-coupled CD3 antibody, matrix-coupled CD28 antibody, matrix-coupled CD137 antibody, 1-100 μg / mL GITR antibody, and 1-500 μM TWS119, or equivalent concentrations of these components, (12) 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-coupled CD3 antibody, matrix-coupled CD28 antibody, matrix-coupled CD137 antibody, 1-100 μg / mL of TIGIT antibody, and 200-5000 U / mL of GM-CSF, or equivalent concentrations 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 concentrations of these components, (14) 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, matrix-coupled CD3 antibody, matrix-coupled CD28 antibody, matrix-coupled CD137 antibody, 1-100 μg / mL TIGIT antibody, 1-100 μg / mL CD40 antibody, 1-100 μg / mL OX-40 antibody, and 1 × 10⁶ 8 ~5×10 8(15) Autologous platelets in 1 / mL or equivalent proportions of these components, (15) IL-2 in 200-6000 IU / mL, IL-7 in 5-100 ng / mL, IL-15 in 5-100 ng / mL, PD-1 antibody in 1-100 μg / mL, CTLA-4 antibody in 1-100 μg / mL, matrix-coupled CD3 antibody, matrix-coupled CD28 antibody, CD137 antibody in 1-100 μg / mL, GITR antibody in 1-100 μg / mL, GM-CSF in 200-5000 U / mL, and 1 × 10 8 ~5×10 8 / mL of heteroplasmic platelets, or equivalent concentrations of these components.

[0124] The antibodies in the culture medium described herein may be antibodies of any species or recombinant antibodies, and are preferably monoclonal antibodies. These antibodies are known in the art and include, for example, the PD-1 antibody TEVIMBRA, the PD-1 antibody Sintilimab, the PD-1 antibody Toripalimab, the PD-1 antibody Camrelizumab, the CD3 antibody ab86883, the CD28 antibody MAB342, the GITR antibody shown in SEQ ID NO: 104 and SEQ ID NO: 105 in CN103951753B, and the CD137 antibody shown in SEQ ID NO: 10 and SEQ ID NO: 11 in CN111182919A. These patent documents or their entirety are incorporated herein by reference. One or more antibodies in the TIL cell seed medium and / or amplification medium described herein may be coupled to a matrix such as a microsphere, well plate, dish, cell culture flask, or cell culture bag.

[0125] The serum described herein may be any serum used in the culture of animal cells (e.g., TIL cells) known in the art, such as human AB serum, subject autologous serum, or animal-derived serum. The serum concentration is 1–10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or in the range of any two of the above values.

[0126] The platelets described in this specification may be platelets derived from the subject's blood or platelets derived from allogeneic blood. The density of the platelets is 0.5×10 8 / mL to 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 a range between any two of the above values.

[0127] The "basal medium" described in this specification is a medium used for any TIL cell culture known in the art, including but not limited to any one or more of AIM-V, X-VIVO, DMEM, RPMI1640, OpTmizerTM, and FUJIFILM Irvin MHM-C. The components and contents of these media are known in the art, and their origins are as described in the examples.

[0128] The concentration at which TIL (unelectroporated or electroporated) is inoculated into the TIL cell amplification medium is not limited and can be determined by those skilled in the art. In a preferred embodiment, TIL is inoculated into the TIL cell amplification medium at a concentration of 2.0×10 5 to 5.0×10 5 cells / mL, for example approximately 2.0×10 5 , approximately 2.5×10 5 , approximately 3.0×10 5 , approximately 4.5×10 5 cells / mL or a concentration within the range between any two values.

[0129] The “culture” or “incubation” time described herein may be approximately 3 to 20 days. For example, the culture or incubation time may be approximately 3 to 15 days, for example, approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or any two of these values. During the TIL cell culture period (e.g., the period of incubation of TIL cells in TIL cell amplification medium), the corresponding medium may be changed once every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0130] For example, the culture or incubation temperature may be about 30 to 42°C. For example, it may 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.

[0131] For example, the CO2 concentration of the culture or incubation is about 1% to 10%, and may be, for example, 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%.

[0132] The present invention includes a method for preparing the modified TIL and the modified TIL obtained thereby, the method comprising the step of introducing the membrane-anchored IL-7 or its coding nucleic acid molecule as described herein into the TTIL obtained by the culture method described above. In some embodiments, the introduction is achieved by electroporation. Cells into which the coding sequence has been introduced (e.g., by electroporation) may be activated (or inactivated) before subsequent operations (e.g., culture). Typically, activation is performed by incubating the electroporated cells in TIL cell amplification medium for at least one day. The antibody in the TIL cell amplification medium can be coated on the surface of a container or suspended in the medium. The incubation is preferably 1 to 10 days, for example 1 to 5 days. The incubation conditions may be determined by those skilled in the art, for example, 37°C and 5% CO2. In some embodiments, the concentration in which the electroporated cells are resuspended in the TIL cell amplification medium during activation incubation is 1.0 × 10⁻⁶ 5 ~1.0×10 6 pieces / 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 It is either particles / mL or within a range between any two values.

[0133] Accordingly, in one or more embodiments, the present invention provides a modified TIL which expresses the membrane-tethered IL-7 described herein, and the modified TIL is prepared by a method comprising: step 1) incubating a TIL-containing sample in the TIL seed cell medium described herein to obtain a first TIL cell population; step 2) introducing a nucleic acid molecule encoding the membrane-tethered IL-7 (e.g., an expression vector) into the first TIL cell population (e.g., by electroporation) to obtain a second TIL cell population that stably expresses the membrane-tethered IL-7; and optional step 3) activating the electroporated cells.

[0134] In one or more embodiments, the present invention provides a modified TIL which expresses the membrane-tethered IL-7 described herein, and the modified TIL is prepared by a method comprising: step 1) incubating a TIL-containing sample in the TIL seed cell medium described herein to obtain a first TIL cell population; step 2) incubating the first TIL cell population in the TIL cell amplification medium described herein to obtain a second TIL cell population; step 3) introducing the nucleic acid molecule of the membrane-tethered IL-7 (e.g., an expression vector) into the second TIL cell population (e.g., by electroporation) to obtain a third TIL cell population that stably expresses the membrane-tethered IL-7; and optional step 4) activating the electroporated cells.

[0135] In one or more embodiments, the present invention provides a modified TIL which expresses the membrane-tethered IL-7 described herein, and the modified TIL is prepared by a method comprising: step 1) incubating a TIL-containing sample in the TIL seed cell medium described herein to obtain a first TIL cell population; step 2) introducing a nucleic acid molecule encoding the membrane-tethered IL-7 (e.g., an expression vector) into the first TIL cell population (e.g., by electroporation) to obtain a second TIL cell population; optional step 3) activating the electroporated cells; and step 4) incubating the second TIL cell population in the TIL cell amplification medium described herein to obtain a third TIL cell population that stably expresses the membrane-tethered IL-7.

[0136] In some embodiments of preparing modified TILs as described herein, the TIL seed cell medium is one or more selected from (a), (b), and (c) below: TIL seed cell medium (a): IL-2 (3000 U / mL), IL-7 (50 ng / mL), IL-15 (50 ng / mL), IFN-γ (1000 U / mL), anti-CD137 activated monoclonal antibody (5 μg / mL), anti-CD40 activated monoclonal antibody (5 μg / mL), anti-OX-40 activated monoclonal antibody (5 μg / mL), anti-TIGIT blocking monoclonal antibody (5 μg / mL), anti-PD-1 blocking monoclonal antibody (15 μg / mL), PS biantibiotic (100 U / mL), and X-VIVO to be replenished 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 activated monoclonal antibody (3 μg / mL), anti-CD28 activated monoclonal antibody (3 μg / mL), anti-PD-1 blocking monoclonal antibody (3 μg / mL), TNF-α (10 pg / mL), and X-VIVO to be replenished to final volume. 15 Basal Medium; TIL Seed Cell Medium (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 replenished to final volume.Alternatively, or further, in some embodiments for preparing modified TILs according to this specification, the TIL cell amplification medium is one or more selected from (d) and (e) below: TIL cell amplification 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 activated monoclonal antibody, anti-CD28 activated monoclonal antibody, 5 v / v% human AB serum, and X-VIVO 15 basal medium replenished to final volume. The anti-CD3 activated monoclonal antibody and anti-CD28 activated monoclonal antibody are coated onto the surface of the culture vessel. TIL cell amplification medium (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 activated monoclonal antibody, anti-CD28 activated monoclonal antibody, anti-CD137 activated monoclonal antibody, 5 v / v% human AB serum, and X-VIVO 15 basal medium replenished to final volume. The anti-CD3 activated monoclonal antibody, anti-CD137 activated monoclonal antibody, and anti-CD28 activated monoclonal antibody are coated onto the surface of the culture vessel. In specific embodiments for preparing modified TILs as described herein, the following combinations of TIL seed cell medium and TIL cell amplification medium are used: (a) and (d), (a) and (e), (b) and (d), (b) and (e), (c) and (d), or (c) and (e).

[0137] In a specific embodiment of a modified TIL or a method for preparing it, the TIL-containing sample is derived from gastric cancer tissue, melanoma, ovarian cancer tissue, cervical cancer tissue, intestinal cancer tissue, lung cancer tissue, osteosarcoma, or recurrent cervical cancer bladder metastasis tumor tissue, and the membrane-tethered IL-7 is linked in order to: (i) CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain; (ii) CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD52; (iii) CD (iv) CD52 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, hinge region or linker, and CD52, (v)(vi) IL-7 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, hinge region or linker, and CD52, or (vii) IL-7 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, and CD8 transmembrane region, or (vii) IL-7 signal peptide, IL-7, linker, BCMA extracellular region or variant thereof, and CD52. The seed cell medium for preparing TIL is the TIL seed cell medium (a), (b) or (c) described above, with a culture time of approximately 11 to 15 days in the seed cell medium, the TIL cell amplification medium is the TIL cell amplification medium (d) or (e) described above, with a culture time of approximately 11 to 15 days in the amplification medium, and the cell inoculation density of the amplification medium is 2.0 × 10⁶ 5 ~4.5×10 5 Preferably, the sequence of membrane-attached IL-7 is represented by one or more of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, and 45.

[0138] In an embodiment of the first set of modified TILs or a method for preparing them, the TIL-containing sample is derived from gastric cancer tissue, the membrane-tethered IL-7 comprises, in order, a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TILs is the TIL seed cell medium (a), the culture time in the seed cell medium is approximately 11 days, the TIL cell amplification medium is the TIL cell amplification medium (e), the culture time in the amplification medium is approximately 12 days, and the cell inoculation density in the amplification medium is 2.5 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:31.

[0139] In an embodiment of the second set of modified TILs or a method for preparing them, the TIL-containing sample is derived from melanoma, the membrane-tethered IL-7 comprises, in order, a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TILs is the TIL seed cell medium (b) above, the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (d) above, the culture time in the amplification medium is approximately 12 days, and the cell inoculation density in the amplification medium is 2.5 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:33.

[0140] In an embodiment of the third set of modified TILs or a method for preparing them, the TIL-containing sample is derived from ovarian cancer tissue, the membrane-tethered IL-7 comprises, in order, a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TILs is the TIL seed cell medium (a) described above, the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (e) described above, the culture time in the amplification medium is approximately 12 days, and the cell inoculation density of the amplification medium is 2.0 × 10⁻⁶ 5Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:33.

[0141] In the fourth embodiment of the modified TIL or method for preparing it, the TIL-containing sample is derived from cervical cancer tissue, the membrane-tethered IL-7 comprises a sequentially linked CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TIL is the TIL seed cell medium (a) described above, the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (e) described above, the culture time in the amplification medium is approximately 12 days, and the cell inoculation density of the amplification medium is 2.5 × 10⁻⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:31.

[0142] In the fifth embodiment of the modified TIL or method for preparing it, the TIL-containing sample is derived from ovarian cancer tissue, the membrane-tethered IL-7 comprises a sequentially linked CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TIL is the TIL seed cell medium (a) described above, the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (e) described above, the culture time in the amplification medium is approximately 11 days, and the cell inoculation density of the amplification medium is 2.5 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:35.

[0143] In the sixth embodiment of the modified TIL or method for preparing it, the TIL-containing sample is derived from intestinal cancer tissue, the membrane-tethered IL-7 comprises a sequentially linked CD52 signal peptide, IL-7, the BCMA extracellular domain or its variant, a linker, and CD52, the seed cell medium for preparing the TIL is the TIL seed cell medium (a), the culture time in the seed cell medium is approximately 13 days, the TIL cell amplification medium is the TIL cell amplification medium (e), the culture time in the amplification medium is approximately 11 days, and the cell inoculation density in the amplification medium is 2.5 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:39.

[0144] In the seventh embodiment of the modified TIL or method for preparing it, the TIL-containing sample is derived from lung cancer tissue, the membrane-tethered IL-7 comprises a sequentially linked CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TIL is the TIL seed cell medium (a) described above, the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (e) described above, the culture time in the amplification medium is approximately 12 days, and the cell inoculation density of the amplification medium is 3.0 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:41.

[0145] In the eighth embodiment of the modified TIL or method for preparing it, the TIL-containing sample is derived from osteosarcoma tissue, the membrane-tethered IL-7 comprises sequentially linked IL-7 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TIL is the TIL seed cell medium (a) described above, the culture time in the seed cell medium is approximately 15 days, the TIL cell amplification medium is the TIL cell amplification medium (e) described above, the culture time in the amplification medium is approximately 15 days, and the cell inoculation density of the amplification medium is 2.5 × 10⁶ 5Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:45.

[0146] In the ninth embodiment of the modified TIL or method for its preparation, the TIL-containing sample is derived from recurrent cervical cancer bladder metastasis tumor tissue, the membrane-tethered IL-7 comprises, in order, a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or variant thereof, and CD52, the seed cell medium for preparing the TIL is the TIL seed cell medium (a) described above, the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (e) described above, the culture time in the amplification medium is approximately 12 days, and the cell inoculation density of the amplification medium is 2.5 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:35.

[0147] In the 10th embodiment of the modified TIL or method for preparing the TIL, the TIL-containing sample is derived from ovarian cancer tissue, the membrane-bound IL-7 comprises, in order, a CD52 signal peptide, IL-7, a BCMA extracellular domain or a variant thereof, a CD8 extracellular hinge domain, and CD52, the seed cell medium for preparing the TIL is the TIL seed cell medium (a) described above, the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (e) described above, the culture time in the amplification medium is approximately 12 days, and the cell inoculation density of the amplification medium is 4.5 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:37.

[0148] In the eleventh embodiment of the modified TIL or method for preparing the TIL, the TIL-containing sample is derived from ovarian cancer tissue, the membrane-tethered IL-7 comprises, in order, a CD52 signal peptide, IL-7, a linker, a BCMA extracellular domain or its variant, and a CD8 transmembrane domain, the seed cell medium for preparing the TIL is the TIL seed cell medium (a), the culture time in the seed cell medium is approximately 12 days, the TIL cell amplification medium is the TIL cell amplification medium (e), the culture time in the amplification medium is approximately 12 days, and the cell inoculation density of the amplification medium is 2.0 × 10⁶ 5 Preferably, the sequence of membrane-attached IL-7 is shown in SEQ ID NO:43.

[0149] The TIL cell population prepared by the method of the present invention may be used in the preparation of pharmaceuticals for cancer treatment. Accordingly, this specification further provides pharmaceutical compositions containing the modified TILs and pharmaceutically acceptable adjuvants described herein. In this specification, pharmaceutically acceptable adjuvants refer to carriers, diluents and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, and include, but are not limited to, pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers and preservatives. More specifically, suitable pharmaceutically acceptable adjuvants may be those commonly used in the art for TIL cell reinfusion (e.g., intravenous infusion).

[0150] Typically, pharmaceutical compositions contain a therapeutically effective dose of modified TILs. A therapeutically effective dose refers to a dose capable of treating, preventing, reducing, and / or alleviating a subject's disease or condition. The therapeutically effective dose may be determined according to factors such as the patient's age, sex, the disease and its severity, and the patient's other health conditions. In this specification, "subject" or "patient" generally refers to a mammal, particularly a human.

[0151] The present invention further includes a cell therapy comprising the step of administering the modified TIL to a subject, wherein the TIL is genetically modified to express membrane-tethered IL-7 as described herein. The administered cells can kill tumor cells in the recipient. The immune response mediated by the TIL cells may also be part of a step in adoptive immunotherapy.

[0152] In this specification, the diseases for which treatment with the pharmaceutical compositions described herein is suitable are related to modified TILs. The diseases described herein include solid tumors and hematological malignancies, specifically solid 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, and leukemias and lymphomas such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0153] The modified TILs of the present invention can be administered alone or as a pharmaceutical composition in a manner suitable for treating (or preventing) a disease. The dosage and frequency of administration will be determined by various factors, including the patient's condition, the type and severity of the patient's disease, and other factors. Administration of the composition can be carried out by any convenient method, including by spray, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodular, intraspinal, intramuscular, intravenous, or intraperitoneal injection. In one embodiment, the polypeptide, cells, or pharmaceutical composition of the present invention is administered to a patient by intradermal or subcutaneous injection (e.g., intravenous injection). Alternatively, the polypeptide, cells, or pharmaceutical composition can be injected directly into a tumor, lymph node, or infected site.

[0154] In some embodiments of the present invention, the modified TIL 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 immunosuppressants. For example, it may be used in combination with radiotherapy or chemotherapeutic agents known in the art for the treatment of tumor antigen-mediated diseases.

[0155] In one embodiment, this specification further provides a kit containing a nucleic acid construct, such as a vector, as described herein. The kit may also include a variety of reagents suitable for transfecting cells with the nucleic acid construct, and optionally instructions for those skilled in the art to transfect cells with the nucleic acid construct.

[0156] In some embodiments, the present invention further provides cell cryopreservation formulations comprising modified TILs and a cryopreservation solution. The cryopreservation solution herein may be a tissue cryopreservation solution or a cell cryopreservation solution capable of maintaining the viability of the TIL cells of the present invention under low-temperature conditions, for example, dry ice freezing or liquid nitrogen freezing. Preferably, the cryopreservation solution may be a serum-free cryopreservation solution that can be directly reinjected into an organism without removal after thawing the cell cryopreservation formulation from low temperatures and does not cause apparent toxicity or side effects to the organism.

[0157] The present invention will be described below with specific examples. Please understand that these examples are merely illustrative and are not intended to limit the scope of the present invention. Unless otherwise specified, the methods and materials used in the examples are conventional methods and materials in the art. [Examples]

[0158] Examples

[0159] Example 1: Construction of an expression vector for membrane-bound IL-7

[0160] The coding sequences of membrane anchoring IL-7, which anchors to each membrane surface in Table 1, were synthesized.

[0161] [Table 1]

[0162] The pKB20 vector was constructed by the method described in Example 1 on page 21 of PCT application WO2022078310A1. Using the method described in the said example, a pKB20 vector 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 was constructed and named 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 Escherichia coli (DH5c), and after proper sequencing, the plasmids were extracted and purified using Qiagen's plasmid purification kit to obtain high-quality plasmids for each recombinant expression vector.

[0163] Example 2 Preparation of TIL expressing membrane-attached IL-7

[0164] The components and numbers of the TIL seed cell medium and TIL cell amplification medium used were as follows:

[0165] TIL seed cell medium: (a) IL-2 (3000 U / mL), IL-7 (50 ng / mL), IL-15 (50 ng / mL), IFN-γ (1000 U / mL), anti-CD137 activated monoclonal antibody (5 μg / mL), anti-CD40 activated monoclonal antibody (5 μg / mL), anti-OX-40 activated monoclonal antibody (5 μg / mL), anti-TIGIT blocking monoclonal antibody (5 μg / mL), anti-PD-1 blocking monoclonal antibody (15 μg / mL), PS biantibiotic (100 U / mL), and X-VIVO 15 basal medium replenished to final volume. (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 activated monoclonal antibody (3 μg / mL), anti-CD28 activated 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 replenished to final volume. (c) IL-2 (3000 U / mL), IL-7 (20 ng / mL), IL-15 (30 ng / mL), anti-CD137 activated antibody (12 μg / mL), anti-CD28 activated 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 refilled to final volume.

[0166] TIL cell amplification 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 activated monoclonal antibody, anti-CD28 activated monoclonal antibody, 5 v / v% human AB serum, and X-VIVO 15 basal medium replenished to final volume. The anti-CD3 activated monoclonal antibody and anti-CD28 activated monoclonal antibody were coated onto the surface of the culture vessel.

[0167] (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 activated monoclonal antibody, anti-CD28 activated monoclonal antibody, anti-CD137 activated monoclonal antibody, 5 v / v% human AB serum, and X-VIVO 15 basal medium replenished to final volume. The anti-CD3 activated monoclonal antibody, anti-CD137 activated monoclonal antibody, and anti-CD28 activated monoclonal antibody were coated onto the surface of the culture vessel.

[0168] Processing of solid tumor samples and preparation of membrane-tethered IL-7 expressing TILs: 1) A physiological saline solution containing penicillin at a final concentration of 100 U / mL, streptomycin at 100 μg / mL, and gentamicin at 50 μg / mL was prepared and set aside for use.

[0169] 2) Under sterile conditions in a Class II biological safety cabinet, freshly isolated tumor tissue samples from tumor patients were washed in a 10 cm culture dish containing 30 mL of the saline solution prepared in step 1). The samples were then transferred to a new 10 cm dish containing 30 mL of the saline solution prepared in step 1, and the washing process was repeated three times.

[0170] 3) Using a sterile scalpel, remove the fatty tissue and necrotic tissue, and cut the tumor tissue to a diameter of 3 x 3 x 3 mm. 3 The tumor tissue was cut into small pieces, and two G-REX100 culture jars (purchased from Wilsonwolf) were prepared. 42 randomly selected tumor tissue pieces were placed in each G-REX100 culture jar, and one of the TIL seed cell media a, b, or c prepared as described above was added to the culture jar as seed cell medium. The remaining tumor tissue pieces were cryopreserved using a liquid nitrogen programmable freezer with CryoStor10 (purchased from BioLifeSolutions) cryopreservation solution.

[0171] 4)3) In a G-REX100 culture jar containing tumor tissue fragments, 1 L of TIL seed cell medium was added, and the tumor tissue fragments were cultured at 37°C and 5% CO2. Every 4 days, half of the old seed cell medium was removed and half of the new seed cell medium was added. On days 11-15, TIL seed cells were collected by centrifugation, and the total cell count and viability were statistically recorded.

[0172] 5)4) Based on the detected viability of the TIL seed cells, 1 × 10 8 ~2×10 8 A TIL seed cell population containing individual living cells was collected and used with a Lonza Nucleofector. TM Using a 2b electroporator, the vector expressing membrane-attached IL-7 prepared in Example 1 was electroporated into the nucleus, and the specific steps were as follows.

[0173] [1] The medium b prepared in Example 2 was added to each well of the G-REX6 in 40 mL in advance, and then transferred to a cell culture incubator at 37°C and 5% CO2 and preheated for 1 hour.

[0174] [2] The formulation of the electroporation solution for one use in each well was prepared according to the table below.

[0175] [Table 2]

[0176] [3] Transfer the TIL collected in step 5) to an EP tube, and put 1 × 10 in each EP tube. 7 The cells were added, centrifuged at 800g for 5 minutes, the supernatant was discarded, the cells were resuspended in 500μL of physiological saline, and the centrifugation was repeated to wash the cell pellet.

[0177] [4] Each of the membrane-tethered IL-7 expressing vectors prepared in Example 1, adjusted to a concentration of 5 μg / mL, was added to the electroporation solution prepared in [2] in amounts of 0.6 to 1 μL (3 to 5 μg) and left at room temperature for no more than 30 minutes (the total volume at this time may be slightly greater than 100 μL).

[0178] [5] Resuspend the TIL in the electroporation solution containing the plasmid prepared in step [4], carefully transfer the cell resuspension to a LONZA 100 μL electroporation cuvette, and transfer the electroporation cuvette to a LONZA Nucleofector TM The 2b electroporation tank was placed in place, and the electroporation program was started. The electroporation program selected was T-020.

[0179] [6] Once electroporation is complete, carefully remove the electroporation cuvette, aspirate the cell suspension and transfer it to an EP tube, add 200 μL of preheated X-VIVO 15 medium to each tube, and transfer to the wells of G-REX6 in [1] containing the preheated medium b of Example 2, 1 × 10 8 ~2×10 8 The electroporation procedure was repeated until all of the live TIL seed cells were electroporated, and the cells were incubated at 37°C and 5% CO2. Every two days, half of the old seed cell medium was removed and half of the new seed cell medium was added until the cell viability recovered to over 90%, and preparations were made to continue the next procedure.

[0180] 6)5) 800g of TIL seed cells electroporated were centrifuged for 5 minutes, and 1.0 × 10⁶ of cells were added to TIL cell expansion medium containing all components of the solution except the coating components. 5 / mL~1.0×10 6The solution was resuspended to a volume of 1 / mL and added to cell culture vessels pre-treated with the coating component. Activation was carried out at 37°C and 5% CO2 for 2-3 days. Afterward, the activated cells were collected by centrifugation and inoculated into a G-REX500M culture tank containing preheated expansion medium. The expansion medium in the G-REX500M culture tank also contained all components of the solution except the coating component. The volume of expansion medium per G-REX500M was 5L. The activated seed cells were 2.0 × 10⁶. 5 / cm 2 ~5.0×10 5 / cm 2 Inoculate at the specified density, culture at 37°C and 5% CO2, count the number of cells every 4 days, remove half of the old expansion medium, and replenish with half the amount of new expansion medium, until the total number of cells in each G-REX500M tank reaches 1.0 × 10⁶. 10 Once the required amount was reached, the cells were divided into bottles in a 1:2 ratio, and each bottle was replenished with new expanding medium to 5L, and the culture was continued. After culturing for a total of 10-12 days in the expanding medium of the G-REX500M culture tank, the cells were harvested to obtain TIL product cells expressing membrane-tethered IL-7.

[0181] Table 2 below shows the tumor tissue sample number, the number of the membrane-tethered IL-7-expressing TIL obtained by culture, the type of cancer, the numbers of the seed cell medium and expansion medium used in the culture process corresponding to Example 2, the culture time of the seed cell medium and expansion medium for each tissue sample, the specific vector name used for electroporation of each tumor tissue sample, and the G-REX500M cell inoculation density. Table 3 shows the type of coating container, the coating activation resuspension cell density, and the coating activation time for the corresponding tumor tissue sample in step 6).

[0182] [Table 3]

[0183] [Table 4]

[0184] Preparation of natural TIL

[0185] In the next step, natural TILs were prepared from T001-T014 tumor tissue.

[0186] 1) A physiological saline solution containing penicillin at a final concentration of 100 U / mL, streptomycin at 100 μg / mL, and gentamicin at 50 μg / mL was prepared and set aside for use.

[0187] 2) Under sterile conditions in a Class II biological safety cabinet, freshly isolated tumor tissue samples from tumor patients were washed in a 10 cm culture dish containing 30 mL of the saline solution prepared in step 1). The samples were then transferred to a new 10 cm dish containing 30 mL of the saline solution prepared in step 1, and the washing process was repeated three times.

[0188] 3) Using a sterile scalpel, remove the fatty tissue and necrotic tissue, and cut the tumor tissue to a diameter of 3 x 3 x 3 mm. 3 The tumor tissue was cut into small pieces, and two G-REX100 culture jars (purchased from Wilsonwolf) were prepared. 42 randomly selected tumor tissue pieces were placed in each G-REX100 culture jar, and one of the TIL seed cell media a, b, or c prepared as described above was added to the culture jar as seed cell medium. The remaining tumor tissue pieces were cryopreserved using a liquid nitrogen programmable freezer with CryoStor10 (purchased from BioLifeSolutions) cryopreservation solution.

[0189] 4)3) In a G-REX100 culture jar containing tumor tissue fragments, 1 L of TIL seed cell medium was added, and the tumor tissue fragments were cultured at 37°C and 5% CO2. Every 4 days, half of the old seed cell medium was removed and half of the new seed cell medium was added. On days 11-15, TIL seed cells were collected by centrifugation, and the total cell count and viability were statistically recorded.

[0190] 5)4) 800g of TIL seed cells were centrifuged for 5 minutes, and 1.0 × 10⁶ of the TIL cell expansion medium containing all components of the solution except the coating component was added. 5 / mL~1.0×10 6 The solution was resuspended to a concentration of 2.0 × 10⁶ / mL, added to cell culture vessels pre-treated with the coating component, and activated at 37°C and 5% CO₂ for 2-3 days. The activated cells were then collected by centrifugation and inoculated into a G-REX500M culture tank containing preheated expansion medium. The expansion medium in the G-REX500M culture tank contained all components of the solution except the coating component. The expansion medium volume per G-REX500M was 5L. The activated seed cells were 2.0 × 10⁶. 5 / cm 2 ~5.0×10 5 / cm 2 Inoculate at the specified density, culture at 37°C and 5% CO2, count the number of cells every 4 days, remove half of the old expansion medium, and replenish with half the amount of new expansion medium, until the total number of cells in each G-REX500M tank reaches 1.0 × 10⁶. 10 Once the required amount was reached, the cells were divided into bottles in a 1:2 ratio, and each bottle was replenished with new expanding medium to 5L, and the culture was continued. After culturing for a total of 10-12 days in the expanding medium of the G-REX500M culture tank, the cells were harvested to obtain natural TIL product cells.

[0191] The seed cell medium and expansion medium used in the culture process of each tumor tissue sample, the culture time of the seed cell medium and expansion medium for each tissue sample, and the G-REX500M cell inoculation density for each tumor tissue sample are all the same as those in Table 2. The type of coating container, the coating activation resuspension cell density, and the coating activation time for the corresponding tumor tissue sample in step 5) are all the same as those in Table 3.

[0192] The natural TILs prepared for each group 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, respectively.

[0193] Example 3: Detection of the phenotype of TIL expressing membrane-attached IL-7

[0194] 1. Cell viability and the positivity rate of membrane-tethered IL-7 expressing TILs Cell viability in each group was detected by trypan blue staining and cell counting. The results showed that the cell viability of membrane-attached IL-7-expressing TILs and native TILs prepared in each group of Example 2 was all above 95%.

[0195] Using the BCMA extracellular domain contained in the extracellular region of each cell expressing the membrane surface IL-7 prepared above as a tag, the percentage of cells positive for exogenous gene expression was detected by flow cytometry as follows.

[0196] 1) From the cells of each group, 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, 1 × 10⁶ cells per group. 6 Individual cells were collected and centrifuged at 800g for 3 minutes. Native T002-TIL cells that do not express membrane-tethered IL-7 were used as control cells.

[0197] 2) Discard the supernatant, add physiological saline to resuspend the cells, and centrifuge at 800g for 3 minutes.

[0198] 3) Discard the supernatant, add 100 μL of physiological 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.

[0199] 4) Add an appropriate amount of physiological saline to each tube, centrifuge at 800g for 3 minutes, wash twice, and discard the supernatant.

[0200] 5) The sample was resuspended in 400 μL of physiological saline and analyzed using a flow cytometer. The results show the positivity rates of cells in each group in Table 4 below.

[0201] [Table 5]

[0202] The results in Table 4 show that, for each TIL sample electroporated with membrane-tethered IL-7, using BCMA extracellular domain expression positivity as the criterion, all samples except T007-6, which used a flexible linker in its structural design, and T003-7, which used a conventional CD8 transmembrane domain structure, had a positive cell percentage of 40% or higher. In T003-2, T005-3, and T009-3, the positive cell percentage exceeded 50%. On the other hand, the positive rates for T007-6 and T003-7 were both less than 20%, significantly lower than the positive rates of the other samples. Among these, T003-2 has a clear advantage over T003-7.

[0203] The results above indicate that 1) when the BCMA extracellular region or shortened BCMA extracellular region used as an extracellular tag is linked to the transmembrane region via a flexible linker, antibody recognition may be affected, and it may not truly function as an extracellular tag; and 2) compared to conventional transmembrane regions, cytokine IL-7 anchored to the TIL surface by the GPI anchoring region can more effectively stimulate the proliferation of positive TILs and significantly increase the proportion of positive TILs.

[0204] 2. Lymphocyte phenotype and cytokine secretion levels TILs from each group expressing membrane-bound IL-7, as well as native TILs, were incubated with CD45, CD3, CD4, and CD8 flow cytometry antibodies. After washing away unbound antibodies, the cell count was measured using a flow cytometer. IFN-γ secretion levels were measured using the HTRF IFN-γ detection kit (Cisbio Human IFN gamma kit, catalog number: 62HIFNGPET) according to the instructions provided, and the results are shown in Table 5 below.

[0205] [Table 6]

[0206] Example 4: Extracorporeal tumor cell killing function of TIL expressing membrane-tethered IL-7

[0207] 1. Culture of primary tumor cells derived from melanoma tissue Tumor tissue from T002 melanoma was cut into 3×3×3 mm pieces, these pieces were mixed as uniformly as possible, and cultured according to the method described in the "Materials and Methods" section of Robert Suriano et al.'s Ex Vivo Derived Primary Melanoma Cells: Implications for Immunotherapeutic Vaccines J Cancer 2013;4(5):371-382 to obtain primary melanoma cells.

[0208] 2. Improvement of in vitro killing function of TILs expressing membrane-tethered IL-7 against target cells. Using Agilent's Real-Time Label-Free Cell Function Analyzer (RTCA), T002-2 and T002-TIL obtained in Example 2 were detected, and their homologous in vitro killing activity against primary melanoma cells was detected. The specific steps are as follows:

[0209] (1) Zero adjustment: 50 μl of DMEM culture medium was added to each well, placed in the instrument, selected step 1, and the zero was adjusted.

[0210] (2) Plating of target cells: The primary melanoma cells obtained in step 1 are plated in 10 wells. 4 The cells were plated in 50 μL of solution onto a plate containing the detection electrode, left for several minutes to stabilize, then placed in the instrument to begin step 2 and culture the cells.

[0211] (3) Addition of effector cells: After culturing target cells for 18-24 hours, the cell index was observed. When the cell index was 2, 50 μL / well each of effector cells T002-2 and T002-TIL were added, the number of viable effector cells was counted, the ratio of effector cells to target cells was set to 4:1, and step 3 was started. After culturing for 60 hours, the cell proliferation curve was observed.

[0212] The results are shown in Figure 1. Compared to the tumor cell group without effector cells, both T002-TIL and T002-2 showed a significant killing effect against homologous melanoma cells. However, T002-2, which expresses membrane-tethered IL-7, showed a much greater killing effect on target cells than T002-TIL, which does not express the exogenous gene. This indicates that membrane-tethered IL-7 can significantly enhance the killing ability of TILs against tumor cells they recognize.

[0213] Example 5: Detection of the in vitro molecular braking function of TIL expressing membrane-attached IL-7

[0214] Antibody synthesis

[0215] Commercial synthesis of BCMA antibodies has been commissioned, and the heavy chain sequence of the antibody is SEQ ID NO: 29 of Chinese Patent CN103562225B, and the light chain sequence is SEQ ID NO: 31 of the same patent, which is incorporated in its entirety by reference.

[0216] Measurement of antibody-dependent cytotoxicity (ADCC)

[0217] Using an NK cell culture kit (purchased from Doritsu Kaigen Co., Ltd., catalog number AS-01), NK cells were obtained by culturing and growing cells from the blood of healthy volunteers 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 resuspended in AIM-V medium, and T002-TIL and T011-TIL were treated similarly as negative control target cells. 1.0 × 10⁶ cells were placed in the wells of a 96-well plate. 4 Cells were added at a rate of 50 μL per well. 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 each well of a 96-well plate. The antibody was pre-incubated at room temperature for 40 minutes along with the target cells in the wells. The NK cells used as effector cells were incubated in 2% FCS-containing AIM-V medium at a rate of 2.5 × 10⁶. 6 The NK cell suspension was resuspended in 1 / mL, and the target cells were incubated with BCMA antibody for 40 minutes. Then, 40 μL of the NK cell suspension was added to the mixture of target cells and BCMA antibody to achieve an effector cell-to-target cell ratio of 10:1. At the same time, the final concentration of BCMA antibody in the system was 50 μg / mL, and the total volume of the system was 100 μL. The system was gently pipetted to ensure uniformity, and the well plate was placed in an incubator at 5% CO2 and 37°C for 3 hours. To evaluate the antibody-dependent cytotoxicity (ADCC) effect of BCMA antibody on each of the target cells, the LDH lactate dehydrogenase-cytotoxicity detection analysis kit (LDH-Cytotoxicity Colorimetric Assay Kit, Biovision, catalog number K313-500) was used.

[0218] Measurement of complement-dependent cytotoxicity (CDC)

[0219] 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 resuspended in AIM-V medium as target cells, and the same treatment was performed with T002-TIL and T011-TIL as negative control target cells. 1.0 × 10⁶ cells were placed in the wells of a 96-well plate. 4 Cells were added at a rate of 50 μL per well. Simultaneously, the BCMA antibody synthesized above was resuspended in AIM-V to a final concentration of 500 μg / mL, and 10 μL was added to each well of a 96-well plate. The system was incubated with the target cells in the wells at room temperature for 40 minutes. Freshly thawed normal human serum complement (Quidel, catalog number A112) was diluted three-fold in AIM-V medium. After incubating the target cells and BCMA antibody for 40 minutes, 40 μL of the diluted human serum complement was added to achieve a final BCMA antibody concentration of 50 μg / mL and a total system volume of 100 μL. The system was gently pipetted to ensure uniform mixing, and the well plate was placed in a 5% CO2, 37°C incubator and incubated for 3 hours. To evaluate the complement-dependent cytotoxicity (CDC) effect of complement on each of the above target cells, the LDH-Cytotoxicity Colorimetric Assay Kit (Biovision, catalog number K313-500) was used. According to the instructions for the LDH-Cytotoxicity Colorimetric Assay Kit, the formula for calculating cytotoxicity is as follows:

[0220]

number

[0221] Here, the low control value is the absorbance value corresponding to the LDH content in the supernatant under conditions where the cells are not completely lysed, and the high control value is the absorbance value of the supernatant under conditions where the cells are completely lysed.

[0222] The results for ADCC and CDC are shown in Table 6. After the addition of the BCMA antibody, all cell samples expressing membrane-surface IL-7, except for T007-6 and T003-7, showed clear ADCC and CDC toxicity, consistent with the detection results of the positive rates in Table 4 of Example 3. Natural TILs that do not express membrane-surface IL-7 (T002-TIL and T011-TIL) did not show clear ADCC and CDC toxicity. The ADCC and CDC toxicity of T007-6, which contains a flexible linker in its extracellular structure, was significantly lower than that of the other samples, indicating that the addition of the BCMA antibody did not completely mediate the occurrence of the ADCC and CDC effect. This suggests that when the extracellular region of BCMA is linked to the transmembrane region via a flexible linker, its epitope structure may be affected, potentially preventing it from binding properly to the BCMA antibody.

[0223] [Table 7]

[0224] Example 6: In vivo tumor-killing function of a CDX model of a membrane-tethered IL-7 expressing TIL.

[0225] Construction of experimental cells The SKOV-3 cell line SKOV-3320, which stably expresses human CD3 antibody (OKT3) on its membrane surface, was constructed as an ovarian cancer target cell line as follows.

[0226] 1. Construction of a lentiviral vector expressing membrane surface CD3 antibody. The structure encoding the membrane surface CD3 antibody (αCD3™) includes a CD8 signal peptide, CD3scFv, 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. We commissioned the synthesis of a DNA sequence containing SEQ ID NO:50, with PmeI at the 5' end and MluI restriction enzyme sites at the 3' end. After enzymatic digestion, it was ligated to the lentiviral vector pWPXL-EGFP expression plasmid (purchased from addgene) to obtain pWPXL-αCD3™-EGFP. We also commissioned the synthesis of a DNA sequence encoding a fusion protein of EGFP, a P2A linker, and a puromycin resistance gene (puro) (EGFP-puro), with MluI at the 5' end and SpeI restriction enzyme sites at the 3' end. After enzymatic digestion, it was ligated to pWPXL-αCD3™-EGFP to obtain the pWPXL-αCD3™-EGFP-puro lentiviral expression plasmid.

[0227] 2. Preparation of lentiviruses The following packaging plasmids, psPAX2 and pMD2.G, were both purchased from Addgene.

[0228] 1) The day before transfection, 293T cells were passaged and inoculated into 6-well plates with 35% cell confluence. 2) One hour before transfection the following day, aspirate the culture medium from the 6-well plate and add Opti-MEM to each well. TM (Purchased from ThermoFisher) 2 mL of culture medium was slowly added.

[0229] 3) Preparation of plasmid transfection solution: a) Lipofectamine TM Dilution of 3000 (purchased from ThermoFisher): 750 μL of Opti-MEM TM Add 24 μL of Lipofectamine TM Add 3000 and mix well.

[0230] b) DNA Dilution: The addition ratio of plasmids is pWPXL-αCD3TM-EGFP-puro:pSPAX2:pMD2.G = 4:3:1. A total of 2.5 μg of the three types of plasmids was added to each well. That is, the addition amounts of the three types of plasmids per 6 wells were 7.5 μg, 5.625 μg, and 1.875 μg, respectively. The corresponding amounts of plasmids and P3000 TM (purchased from ThermoFisher) 30 μL was added to 750 μL of Opti-MEM TM and mixed well. c) The DNA dilution solution was added to the Lipofectamine TM 3000 dilution solution and mixed well.

[0231] 4) After incubating at room temperature for 13 minutes, 250 μL of the DNA-lipid complex was dropped into each well.

[0232] 5) After 6 hours, the medium in the 6-well plate was aspirated, and 2 mL of complete medium (H-DMEM + 10% FBS, purchased from ThermoFisher) was added to each well.

[0233] 6) 48 hours after cell transfection, all the cell supernatants were collected into 15 mL centrifuge tubes, centrifuged at 1500 rpm for 10 minutes, filtered through a 0.22 μM filter membrane, dispensed into 1.5 mL EP tubes (1.5 mL / tube), and frozen in a -80°C refrigerator.

[0234] 3. Infection of Target Cells with Lentivirus The target cells are the ovarian cancer cell line SKOV3-Luc (Cat#78425) that incorporates and expresses the luciferase gene, purchased from BPS Bioscience.

[0235] 1) One day before infection, SKOV3-Luc cells were passaged and inoculated into two wells of a 6-well plate at 20% confluence.

[0236] 2) On the day of infection, one tube of the virus solution was taken out from the -80°C refrigerator and thawed at room temperature.

[0237] 3) Aspirate and remove the medium from one well, add 1.5 mL of fresh complete medium (IMDM + 15 v / v% FBS + 1× non-essential amino acids NEAA, IMDM and FBS are purchased from ThermoFisher, 100× non-essential amino acids are purchased from Cyagen) and 1.5 mL of virus solution, add polybrene to a final concentration of 8 μg / mL, shake well, place in a 37°C, CO2 incubator, and leave the other well as it is.

[0238] 4. Screening of cell lines stably transfected with positive mixed clones 1) On the 3rd day after lentivirus infection, the cell confluence in both wells reached over 90%.

[0239] 2) Discard the medium, add 3 mL of fresh complete medium to each well, and then add puromycin to a final concentration of 3 μg / mL.

[0240] 3) After culturing for 48 hours with puromycin added, the virus-uninfected control cells were almost 100% dead, and the cell confluence in the infected wells reached 90%. Transfer all the infected cells to a T25 culture flask, add puromycin to a final concentration of 3 μg / mL, and continue the culture.

[0241] 4) After the cell growth period became stable, the cells were proliferated, cryopreserved, the positive rate was measured by flow cytometry, and the finally obtained cell line stably transfected with the mixed clone was named SKOV3-αCD3TM-EGFP.

[0242] 5) The cytotoxic activity of the cell line SKOV3-αCD3TM-EGFP, which was stably transfected with host cells SKOV3-Luc and mixed clones, was detected using the xCELLigence RTCA TP real-time label-free cell analyzer (Agilent). T cells derived from healthy human peripheral blood PBMCs were used as effector cells. The results showed that T cells exhibited significantly stronger cytotoxic activity against SKOV3-αCD3TM-EGFP compared to unmodified SKOV3-Luc.

[0243] 5. Screening of monoclonal cell lines 1) The prepared complete medium (IMDM + 15v / v%FBS + 1×NEAA) was filtered through a 0.22 μM filter membrane to reduce the influence of impurities in the reagents on subsequent single-cell microscopy observations.

[0244] 2) The number of cells in the SKOV3-αCD3TM-EGFP cell line, which was stably transfected with mixed clones, was counted.

[0245] 3) Two 96-well cell culture plates were plated at a plating density of 0.8 cells / well. Using an 8-channel pipette (maximum volume 50 μL), 100 μL of culture medium was added to each well, followed by 50 μL of cell suspension (16 cells / mL, 15 mL prepared) in each well. Then, 1000 cells were added to well A1 of the 96-well plate.

[0246] 4) After culturing in a 37°C CO2 incubator for 6 hours, the cells were observed under a microscope, and photographs were taken of the wells containing single cells.

[0247] 5) Wells containing single cells were photographed on day 1, day 2, and day 3 to record the process of cell division.

[0248] 6) The medium for the marked single-cell clones was changed, 100 μL of medium was aspirated and removed from each well, and 150 μL of fresh medium was replenished, after which the medium was changed every 3 days.

[0249] 7) Once the confluence of single-cell clones in the wells reached 50% or more, the cells were transferred sequentially from the 96-well plate to the 24-well plate, then to the 6-well plate, then to the T25 culture flask, and finally to the T75 culture flask for expansion culture.

[0250] 8) The proliferated monoclonal cells were tested for positive expression by flow cytometry and then cryopreserved.

[0251] 9) Using the xCELLigence RTCA TP real-time label-free cell analyzer, the cytotoxic activity of various monoclonal cell lines was detected. The results showed that monoclonal cell line number 320 was highly responsive to T cell killing, and that its cytotoxic activity was initially dependent on the number of cells. As the ratio of effector cells to target cells increased, the cytotoxic activity also increased. This monoclonal cell line was named SKOV-3320 and was subsequently used as a tumor target cell in experiments.

[0252] Effector cells: TIL T014-9, which expresses membrane-tethered IL-7 cultured from ovarian cancer tissue T014, was selected as effector cells.

[0253] Laboratory animals Immunodeficient B-NDG mice (purchased from Biocytogen) were selected as experimental animals for constructing the CDX model.

[0254] Experimental design and grouping: As shown in Table 7 below.

[0255] [Table 8]

[0256] The TIL cells are T014-9, which are membrane-tethered IL-7 expressing TIL product cells derived from tissue sample T014 prepared in Example 2. The cells were centrifuged and resuspended in PBS before tail vein injection, and group 2 had a cell density of 2.5 × 10⁶. 7For Group 3, a cell suspension of 7.5×10 7 / mL was prepared.

[0257] Animal breeding After purchasing the required number of B-NDG mice, they were bred in an SPF-grade experimental animal room for a 7- to 10-day acclimation period.

[0258] Environment: The mice were housed in transparent resin plastic cages in the animal room. The bedding in the cages was autoclaved sawdust and corn cob bedding, which was replaced regularly. The animal room was equipped with a HEPA filter, and the temperature was maintained at 20-26°C (68-79°F), and the relative humidity was maintained at 40-70%. The temperature and humidity were continuously observed and recorded. The lighting conditions were 12 hours of fluorescent light on and 12 hours off per day.

[0259] Food and drinking water: The experimental mice were constantly provided with mouse-specific feed (sterilized by radiation, Shanghai Slack Experimental Animal Co., Ltd., China) and sterilized clean drinking water without limit.

[0260] Establishment of subcutaneous xenograft tumor model

[0261] SKOV-3 320 cells were counted and used for tumor inoculation. In the right axilla of each mouse, 0.2 mL (1E7 / mouse) of a tumor cell solution containing 50% Matrigel was subcutaneously inoculated, and the inoculation day was designated as D0.

[0262] Animal grouping and administration

[0263] When the average volume of the tumors reached 100 mm 3 To ensure that all groups were comparable at the baseline, 24 animals were selected from 36 animals according to the tumor volume (average 111 mm 3 ) and randomly grouped. The grouping day 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. The tumor volume was V = 0.5×a×b2 Using the formula, mm 3 The values ​​are expressed in units, where a and b are the long and short diameters of the tumor, respectively. Tumor fluorescence values ​​were measured using a small animal in vivo imaging system 7 weeks after the initial administration and at the end of the experiment.

[0264] Bioimaging

[0265] On the final day of the experiment, bioluminescence signals from mice were detected using IVIS Lumina XR (Perkin Elmer). First, 150 mg / kg of fluorescein sodium was intraperitoneally injected into the animals, and they were anesthetized with isoflurane. Ten minutes after injecting luciferin sodium into the animals, bioluminescence images were collected with an exposure time of 60 seconds. Images were quantified as photons / second using Living Image software.

[0266] result

[0267] The experimental results are shown in Figures 2 and 3. Figure 2 shows the difference in tumor volume between the groups. The difference in tumor volume between the groups was analyzed by one-way ANOVA analysis, and then a Bonferroni post hoc test was performed to verify whether there was a significant difference between the groups. *: p<0.05; **: p<0.01; ***: p<0.001. Figure 3 shows the fluorescence bioimaging results of the experimental mice in each group on the day the experiment ended. The results shown in Figure 2 are 0.5 × 10⁻⁶ compared to the PBS group. 7 The tumor volume of mice in the T014-9 reinjection dose group was significantly smaller, at 1.5 × 10⁻⁶. 7 Figure 3 shows that the tumors in the TIL reinjection dose group of mice were completely gone by D65. 7 This indicates that no fluorescence was observed in tumor cells in the T014-9 reinjection dose group per mouse. The above results demonstrate that T014-9 cells have a very significant killing effect against ovarian cancer CDX tissue in mice.

[0268] Example 7: In vivo tumor-killing function of a PDX model of a membrane-tethered IL-7 expressing TIL.

[0269] Using T011 cervical cancer tumor tissue, we constructed an immunodeficient mouse PDX tumor model. Using this model mouse, we verified the in vivo tumor-killing effect of TIL-PDX in animals. Immunodeficient B-NDG mice (purchased from Biocytogen) were selected as the PDX model experimental animals.

[0270] Experimental design and grouping: As shown in Table 8 below.

[0271] [Table 9]

[0272] The TIL cells are TIL product cells derived from the tissue sample T011 prepared in Example 2. Before administration by tail vein injection, the cells were centrifuged and resuspended in PBS, with a cell density of 1 × 10⁶. 8 A PBS cell suspension of 1 / mL was prepared.

[0273] Animal Husbandry

[0274] After purchasing the required number of B-NDG mice, they were reared in an SPF-grade laboratory animal room for a 7-10 day acclimatization period.

[0275] Environment: Mice were housed in transparent plastic cages within the animal room. The cage bedding consisted of autoclaved sawdust and corn cobs, which were replaced regularly. The animal room was equipped with a HEPA filter, and the temperature was maintained at 20-26°C (68-79°F) and the relative humidity at 40-70%. Temperature and humidity were continuously observed and recorded. Lighting conditions consisted of 12 hours of fluorescent lighting on and 12 hours of lighting off per day.

[0276] Food and drinking water: Laboratory mice were constantly provided with unlimited supply of specialized mouse feed (sterilized by radiation, Shanghai Slice Laboratory Animals Co., Ltd., China) and sterile, clean drinking water.

[0277] Building a PDX Model

[0278] 1) Processing of patient tumor tissue samples: A portion of the T011 tumor tissue is taken, necrotic tissue, adipose tissue, and connective tissue are removed under sterile conditions, and after washing, a 5×5×5mm section is cut using a scalpel. 3 The tissue was cut into pieces, and the pieces were immersed in UW, a preservation solution for tumor sample transport, in preparation for inoculation into B-NDG mice.

[0279] 2) Inoculation of tumor tissue samples: Several B-NDG mice were selected, and after pre-treatment of the mouse scapular skin, the mice were fixed using a subcutaneous tumor inoculation fixation device, disinfected with iodine, and after local anesthesia with lidocaine, the tumor mass prepared in 1) was inoculated into the right inguinal region using a PDX model tumor mass inoculation trocar. The inoculation day was designated as P0, the tumor was measured twice a week, and the tumor volume was V = 0.5 × a × b 2 It is calculated using the following formula, where a and b are the longest and shortest diameters of the tumor, respectively.

[0280] 3) Passaging of PDX tissue: Observe the growth of tumor tissue in each inoculated mouse, and when the volume of tumor tissue reaches 300 mm³ 3 If the size exceeds 5mm, the mouse is anesthetized, the tumor mass is removed, and then a scalpel is used to cut it into 5x5x5mm pieces under sterile conditions. 3 The tissue was divided into sections, and step 2) was repeated. These sections were then inoculated into the right groin of new mice, and the growth of the next generation of PDX tumors was awaited.

[0281] 4) Repeat step 3), continue subculturing for 2 - 3 generations, then collect PDX tissues from some mice, perform pathological analysis of tissue sections. If it is confirmed that the PDX tissue is of human origin (not of mouse origin), continue inoculating the PDX tissue into 1.5 times the number of mice used in the experimental plan in Table 8 (that is, inoculate PDX tissue pieces into 36 mice), observe the presence or absence of tumor formation in the mice, and measure the tumors twice a week until tumor formation is achieved.

[0282] Grouping and administration of animals

[0283] When the tumor volume of the mice inoculated with PDX reached approximately 50 mm 3 At this time, select 24 animals with appropriate tumor volumes from the 36 animals, and randomly group them according to tumor volume (n = 8) to ensure that all groups are comparable at the baseline. Record the day of grouping as D0, and perform administration according to the plan in Table 8. 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. The tumor volume was expressed in mm 3 and the tumor measurement formula was the same as above.

[0284] The results are shown in Figure 4. The differences in tumor volume between each group were analyzed by one-way ANOVA analysis, and then the Bonferroni post hoc test was used to verify whether there were significant differences between each group. *: p < 0.05; **: p < 0.01; ***: p < 0.001. According to the experimental results, until the 40th day after administration, the increase in tumor volume of the mice in the T011-TIL group was significantly suppressed compared with the tumor-bearing mice in the PBS injection control group. On the other hand, the suppression of tumor volume increase by T011-9 was more significant than that of the T011-TIL group. The natural TIL product cells derived from T011 have a very obvious killing effect on the same tumor PDX tissue of the patient from which they are derived in vivo, indicating that this effect may be further enhanced after membrane tethered IL-7 modification of natural TIL.

[0285] Example 8 Clinical reinjection of TIL expressing membrane-tethered IL-7

[0286] To verify the clinical safety and efficacy of membrane-tethered IL-7 expressing TILs as described in this application, a clinical trial of TIL reinjection therapy for solid tumors was conducted. This clinical trial was approved by the Ethics Committee of Shanghai Tenth People's Hospital. For details, please see www.clinicaltrials.gov (registration number: NCT05468307).

[0287] Research plan 1. Criteria for selecting and excluding subjects and methods for grouping subjects

[0288] Selection criteria: 1) 18 years old ≤ Age ≤ 75 years old 2) Patients with gynecological tumors diagnosed pathologically as primary, recurrent, or metastatic. 3) Life expectancy > 3 months 4) Karnofsky ≥ 60% or ECOG score 0-2 points 5) The subject has failed to receive standard treatment or has no standard treatment options available. 6) The subject must have a tumor area or malignant fluid suitable for aspiration or excision biopsy to isolate the TIL. 7) There is at least one evaluable tumor lesion. 8) Hematological and biochemical indicators (within 7 days prior to participation) Absolute white blood cell count ≥ 2.5 × 10⁻⁶ 9 / L Absolute neutrophil count ≥ 1.5 × 10 9 / L Absolute lymphocyte count ≥ 0.7 × 10 9 / L Platelet count ≥ 100 × 10 9 Hemoglobin ≥ 90 g / L Activated partial thromboplastin time (APTT) ≤ 1.5 × ULN (excluding cases where anticoagulation therapy was received within the past 3 days) International normalized ratio (INR) ≤ 1.5 × ULN (excluding cases where anticoagulation therapy was received within the past 3 days) Serum creatinine ≤ 1.5 mg / dL (or ≤ 132.6 μmol / L), or serum creatinine clearance ≥ 50 mL / min. Serum ALT (alanine aminotransferase) / AST (aspartate aminotransferase) ≤ 3 × ULN (≤ 3 × ULN in subjects with liver metastases) Total bilirubin ≤ 1.5 × ULN 9) There are no absolute or relative contraindications to surgery or puncture. 10) Subjects who may become pregnant must be willing to begin using an approved, highly effective method of contraception at the time of informed consent and must continue using it for one year after completion of lymph node dissection therapy. 11) All treatments for malignant tumors, including radiotherapy, chemotherapy, and biological agents, must be discontinued 28 days before TIL occurs. 12) Fully understand informed consent and sign it voluntarily. 13) Ability to comply with follow-up schedules and other protocol requirements. Exclusion criteria: 1) The patient has an autoimmune disease requiring glucocorticoid therapy, with daily administration of 15 mg or more of prednisone (or an equivalent dose of a hormone), or immunosuppressive therapy. 2) Forced expiratory volume per second (FEV1) < 2L, Carbon monoxide pulmonary diffusion capacity (DLCO) (corrected) < 40% 3) Any one of the following definitions of a major cardiovascular abnormality: congestive heart failure of New York Heart Association (NYHA) class III or IV, clinically significant hypotension, clinically uncontrolled hypertension, uncontrolled symptomatic coronary artery disease, or severe cardiac rhythm and conduction abnormalities such as ejection fraction <35%, ventricular arrhythmias requiring clinical intervention, or second- or third-degree atrioventricular block. 4) The patient is infected with human immunodeficiency virus (HIV) or tests positive for HIV antibodies, has active hepatitis B or hepatitis C virus infection (HBsAg positive and / or anti-HCV positive), has syphilis infection or tests positive for Treponema pallidum antibodies. 5) Severe physical or mental illness 6) Active systemic infection requiring treatment, or positive blood culture (or imaging evidence of infection) 7) You have received or are currently receiving other medications, other biological therapies, chemotherapy, or radiation therapy within the past month. 8) A history of allergic reactions to compounds with a similar chemical or biological composition to cell therapy. 9) The patient has received immunotherapy and has developed an irAE of grade 3 or higher. 10) Side effects from previous antitumor treatments have not decreased to CTCAE 5.0 version 1 or lower (excluding toxicities such as alopecia that the principal investigator has determined to pose no safety risk). 11) Pregnant or breastfeeding women 12) History of organ transplantation, allogeneic stem cell transplantation, or renal replacement therapy 13) The researchers determined that the subject had a history of other serious systemic diseases or was otherwise unsuitable to participate in this clinical study.

[0289] 2. Criteria for evaluating effectiveness Primary efficacy endpoint: 1) Objective response rate (ORR) 2) Disease control rate (DCR) 3) Duration of response (DOR) 4) Progression-free survival (PFS) 5) Overall survival (OS)

[0290] Secondary efficacy endpoints 1) Evaluation of clinical efficacy: complete response (CR), partial response (PR), disease control (SD), disease progression (PD), etc. 2) Changes in quality of life before and after treatment Efficacy was defined as an ORR observable by the investigator, according to the RECIST v1.1 criteria.

[0291] 3. Recording of adverse events and treatment measures 3.1 Definition of Adverse Events Adverse events (AEs): Unfavorable medical events that occur between the time a subject signs informed consent and enrolls in a study, and the final medical follow-up. These may manifest as symptoms, signs, illness, or abnormal clinical laboratory values, but are not necessarily causally related to the treatment or investigational drug.

[0292] New symptoms or worsening of existing symptoms were considered adverse events (AEs). Stable chronic conditions, such as arthritis, that existed before participation in the study and did not worsen during the study were not considered AEs. Abnormal laboratory test results, clinical symptoms, or signs that the researchers deemed clinically significant were considered AEs.

[0293] Serious adverse events (SAEs) refer to adverse events that occur during an experiment and meet one or more of the following conditions: (1) cause death; (2) life-threatening, meaning there is a risk of immediate death but not necessarily death due to future complications; (3) cause hospitalization or prolonged hospitalization; (4) cause permanent or significant loss of function; (5) cause teratogenicity or birth defects; and (6) other significant medical events.

[0294] 3.2 Criteria for determining the severity of adverse events The severity of all adverse events that occurred during the study was assessed according to NCI-CTCAE version 5.0 and graded from 1 to 5. Grade 1: Mild; asymptomatic or mild; seen only clinically or diagnostically, no treatment required. Grade 2: Moderate; requires minor, local, or non-invasive treatment; age-appropriate limitations in daily living activities. Grade 3: Severe or medically significant, but not immediately life-threatening; may result in hospitalization or extended hospital stay, physical disability, or limitation of personal activities of daily living. Grade 4: Life-threatening; requires emergency treatment. Grade 5: Death related to adverse events (AEs).

[0295] 4. Pretreatment before reinjection Before reinfusion of TIL cells, patients received a lymphocyte pretreatment regimen (hydroxychloroquine 600 - 800 mg once a day, cyclophosphamide 20 - 25 mg / kg / day for 3 days), and the researchers adjusted the drugs, dosages, and number of days of administration (administration frequency) used in the pretreatment regimen based on the actual situation of the patients. The day of reinfusion was defined as day 0. Before TIL cell reinfusion, the researchers needed to perform the following chemotherapy pretreatment on the patients (after pretreatment, patients experience leukopenia, so it is necessary to avoid unnecessary people coming and going in the ward and let the patients wear masks, etc., pay attention to preventing infection). - On day -5, intravenous injection of cyclophosphamide (Cy) 20 - 25 mg / kg + oral administration of hydroxychloroquine (HCQ) 600 - 800 mg - On day -4, intravenous injection of cyclophosphamide 20 - 25 mg / kg - On day -3, intravenous injection of cyclophosphamide 20 - 25 mg / kg - On days -2 and -1, the patients rested On day 0, TIL cells were reinfused.

[0296] 5. TIL reinfusion and efficacy evaluation After the completion of the above - mentioned 4 - time pretreatment before reinfusion, patients received reinfusion on day 0. Each patient received a single - dose injection of a cell preparation for reinfusion with 1.0×10 9 ~5.0×10 9 TIL cells, cell density 1.0×10 7 / mL~5.0×10 7 / mL, in a volume of 100 mL. Patients received TIL cell reinfusion on day 0, and no cytokines were administered after injection. After reinfusion, patients were observed in the hospital for 5 - 8 days to observe and record their conditions. With the consent of the patients, peripheral blood of the patients was collected at different time points to detect the composition and changes of PBMC, and the MRI image effect was evaluated 1 - 6 months after reinfusion.

[0297] 6. TIL clinical reinfusion results Tables 9 and 10 below show comprehensive clinical reinfusion information and efficacy evaluation results for some subjects who participated in the reinfusion (evaluation period ranges from 1 to 6 months after reinfusion).

[0298] [Table 10]

[0299] Patient T011's tumor diameter before reinfusion was 2.2 cm. Fourteen weeks after reinfusion, the tumor lesion was no longer detectable by magnetic resonance imaging, and a complete response (CR) was determined, as shown in Figure 5.

[0300] Patient T012's tumor diameter before reinfusion was 4.5 cm. 22 weeks after reinfusion, the tumor lesion was no longer detectable by magnetic resonance imaging, and a partial response (CR) was determined, as shown in Figure 6.

[0301] Patient T013's tumor diameter before reinjection was 2.1 cm. Thirteen weeks after reinjection, magnetic resonance imaging confirmed a significant reduction in the tumor lesion, and as shown in Figure 7, it was determined to be a partial response (PR).

[0302] Patient T014's tumor size before reinjection was 2.6 cm × 2.5 cm. 24 weeks after reinjection, the primary tumor was no longer detectable by magnetic resonance imaging, and as shown in Figure 8, the primary tumor was judged to have achieved a complete response (CR), with an overall assessment of a partial response (PR).

[0303] While specific embodiments of the present invention have been described in detail, various modifications and substitutions can be made to those details based on all the disclosed teachings, as will be understood by those skilled in the art, and all such modifications fall within the scope of the present invention. The entire scope of the present invention is given by the appended claims and their equivalents.

[0304] A part of the sequence of this specification mbIL-7-1 SEQ ID NO:31 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHAEAAAKEAAAKAMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0305] mbIL-7-2 SEQ ID NO:33 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH RADAAPTVSMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0306] mbIL-7-3 SEQ ID NO:35 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHRTVAAPSVFMLQMAGQCSQNEYFDSLLHACIPCDLRCSSNTPPLTCQRYCNASVTNSVKGTNAGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0307] mbIL-7-4 SEQ ID NO:37 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHPSPLFPGPSKPMLQMAGQCSQNEYFDSLLHACIPCDLRCSSNTPPLTCQRYCNAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0308] mbIL-7-5 SEQ ID NO:39 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHMLQMAGQCSQNEYFDSLLHACIPCDLRCSSNTPPLTCQRYCNASVTNSVKGTNARTVAAPSVFGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0309] mbIL-7-6 SEQ ID NO:41 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHGGGGSGGGGMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0310] mbIL-7-7 SEQ ID NO:43 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHRADAAPTVSMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAIYIWAPLAGTCGVLLLSLVITLYC

[0311] mbIL-7-8 SEQ ID NO:45 MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHRADAAPTVSMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNAGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0312] mbIL-7-9 SEQ ID NO:47 MKRFLFLLLTISLLVMVQIQTGLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHPSPLFPGPSKPMLQMAGQCSQNEYFDSLLHACIPCDLRCSSNTPPLTCQRYCNAGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

[0313] Membrane surface CD3 scFv SEQ ID NO:49 MALPVTALLLPLALLLHAARP QVQLQESGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRRADAAPTVSIYIWAPLAGTCGVLLLSLVITLYC

Claims

1. A modified TIL expressing membrane-attached IL-7, Preferably, the membrane anchoring IL-7 is a modified TIL comprising IL-7 or its functional fragment and a transmembrane region.

2. The transmembrane region is a protein transmembrane domain, and preferably the protein transmembrane domain includes one selected from the CD8 transmembrane region, the CD28 transmembrane region, and the IL-7Rα transmembrane region, or The transmembrane region is a GPI anchoring region, and preferably the GPI anchoring region includes the membrane anchoring region of one or more GPI membrane anchoring proteins selected from 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. The modified TIL according to feature 1.

3. The aforementioned membrane anchoring IL-7 further includes a membrane surface tag, Preferably, the membrane surface tag includes a variant thereof that retains the function of binding to the extracellular domain of a B cell surface antigen or to an antibody. More preferably, the membrane surface tag is a variant thereof that retains the function of binding to the extracellular domain of BCMA or an anti-BCMA antibody. More preferably, the variant is a shortened variant, an insertion variant, a deletion variant, a substitution variant, or a combination thereof, and more preferably, the amino acid sequence of the variant is SEQ ID NO: 23 or SEQ ID NO:

25. The modified TIL according to feature 1.

4. The membrane anchoring IL-7 further includes a linker located between the IL-7 and the membrane surface tag, preferably the linker being a rigid linker or a flexible linker, preferably a rigid linker, and / or The membrane anchoring IL-7 further includes a hinge region or linker located at the N-terminus of the transmembrane region, preferably the hinge region includes, but is not limited to, a CD4 extracellular hinge region, a CD8 extracellular hinge region, a CD28 extracellular hinge region, an IgG1Fc hinge region, and an IgG4Fc hinge region, and the linker is a rigid linker or a flexible linker, more preferably the arrangement of the rigid linker is one or more selected from SEQ ID NO: 9, 11, 15, and 17, and the arrangement of the flexible linker is SEQ ID NO:

13. The membrane-bound IL-7 further comprises a signal peptide, preferably a CD52 signal peptide or an IL-7 signal peptide. The modified TIL according to feature 3.

5. The membrane-anchored IL-7 comprises, in order, any signal peptide, IL-7, any linker, a BCMA extracellular region or a variant thereof, any 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 a CD52 transmembrane region. Preferably, the membrane-attached IL-7 comprises a signal peptide, IL-7, a linker, a BCMA extracellular domain or a variant thereof, and a transmembrane domain linked in sequence. More preferably, the membrane anchoring IL-7 is connected in sequence. CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD52, CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, CD52 signal peptide, IL-7, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, or Includes IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD52 A modified TIL according to any one of claims 1 to 3.

6. The TILs are prepared by a method comprising the step of incubating a TIL-containing sample in TIL seed cell medium to obtain a TIL cell population, or The TILs are prepared by a method comprising the steps of (1) incubating a TIL-containing sample in TIL seed cell medium to obtain a first TIL cell population, and (2) incubating the first TIL cell population in TIL cell amplification medium to obtain a second TIL cell population. A modified TIL according to any one of claims 1 to 3.

7. The aforementioned samples are selected from ascites, surgically resected primary tumor samples, synchronously and metachronically resected metastatic tumor samples, puncture samples, and body fluids. Preferably, the sample is tissue containing tumor cells, and more preferably, the tumor cells are derived from tumors selected from the group consisting of melanoma, glioma, thyroid tumor, gastric cancer, lung cancer, gastrointestinal stromal tumor, colorectal cancer, liver cancer, cervical cancer, ovarian cancer, breast cancer, endometrial stromal sarcoma, poorly differentiated pelvic adenocarcinoma, gallbladder cancer, bile duct cancer, head and neck cancer, colorectal cancer, cranial glioma, pancreatic cancer, bladder cancer, prostate cancer, kidney cancer, and osteosarcoma. Preferably, the body fluids include blood, pleural fluid, tissue fluid, lymph fluid, and / or ascites. The modified TIL according to feature 6.

8. The seed cell medium comprises, as components, cell culture components, cytokines, and immune checkpoint antibodies or their antigen-binding fragments, 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 medium or serum-free medium. Preferably, the TIL seed cell medium further comprises serum and / or antibiotics. Preferably, the TIL seed cell medium is: 1) IL-2, IL-6, IL-21, IFN-γ, TIGIT antibody, PD-1 antibody, TNF-α, and basal medium; 2) IL-2, IL-4, IL-10, IL-21, CD137 antibody, LAG3 antibody, PD-1 antibody, TNF-α, and basal medium; 3) IL-2, IL-7, IL-12, IL-21, CD137 antibody, CD28 antibody, PD-1 antibody, and basal medium; 4) IL-1β, IL-2, IL-7, G-CSF, GM-CSF, IFN-γ, LAG3 antibody, PD-1 antibody, TNF-α, and basal medium. 5) IL-2, IL-4, IL-12, GM-CSF, M-CSF, IFN-β, IFN-γ, TIGIT antibody, CTLA-4 antibody, and basal medium, 6) IL-2, IL-7, IL-15, GM-CSF, CD137 antibody, PD-1 antibody, TNF-α, and basal medium, 7) I L-2, IL-4, IL-10, IL-15, G-CSF, M-CSF, CD28 antibody, OX-40 antibody, PD-1 antibody, and basal medium, 8) IL-2, IL-7, IL-15, IFN-γ, CD137 antibody, CD40 antibody, OX-40 antibody, TIGIT antibody, PD-1 antibody, and basal medium, 9) IL-2, IL-7, IL-15, GM-CSF, IFN-γ, CD137 antibody, CD28 antibody, PD-1 antibody, TNF-α, and basal 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 medium, 11) IL-2, IL-7, IL-15, GM-CSF, PD-1 antibody, RRx-001, CAL-101, and 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 medium, 16) comprising any one combination of 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 components of the basal medium, The modified TIL according to feature 6.

9. The TIL cell amplification medium comprises IL-2, IL-7, IL-15, and an immune checkpoint antibody or its antigen-binding fragment, preferably the immune checkpoint antibody comprises a PD-1 antibody. Preferably, the TIL cell amplification medium further comprises one or more of serum, platelets, and antibiotics, and a basal medium. More preferably, the TIL cell amplification medium is 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 the matrix, 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 1) Antibodies, TWS119, and CD137 antibody, preferably the CD3 antibody, CD137 antibody, and CD28 antibody are coupled to the 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 the matrix, 24) IL-2, IL-7, IL-15, LAG3 antibody, IL-21, IL-12, CD3 antibody, CD28 antibody, 25) CD137 antibody and GITR antibody, preferably the CD3 antibody, CD137 antibody and CD28 antibody are coupled to the matrix, 26) 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, 27) IL-2, IL-7, IL-15, PD-1 antibody, CTLA-4 antibody, CD3 antibody,CD28 antibody, CD137 antibody, GITR antibody, GM-CSF, TWS119, and heterochromia, preferably the CD3 antibody and CD28 antibody include any one combination of components of those coupled to the matrix. The modified TIL according to feature 6.

10. The basal culture medium is one or more selected from AIM-V, X-VIVO, DMEM, RPMI1640, OpTMizer™, and FUJIFILM Irvin MHM-C, and / or The serum is selected from human AB serum, the subject's own serum, or animal-derived serum. The modified TIL according to claim 8 or 9.

11. This includes introducing the coding nucleic acid of the membrane-attached IL-7 into the TIL, Preferably, the introduction is by electroporation or by lipid nanoparticles. Preferably, the coding nucleic acid is an expression vector comprising a viral vector or a non-viral vector. Preferably, the expression vector comprises a nucleic acid sequence encoding membrane-attached IL-7 as described in any one of claims 1 to 5. More preferably, the viral vector is a lentiviral vector, the nonviral vector is a transposon-based vector, and even more preferably, the nonviral vector is a vector based on the PiggyBac transposon system or the Sleeping Beauty transposon system. A method for preparing modified TIL according to any one of claims 1 to 9.

12. 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 TILs includes: step 1) incubating a TIL-containing sample in TIL seed cell medium to obtain a first TIL cell population; step 2) introducing a nucleic acid construct expressing the membrane-attached IL-7 into the first TIL cell population by electroporation or lipid nanoparticles to obtain a second TIL cell population; and step 3) incubating the second TIL cell population in TIL cell amplification medium to obtain a third TIL cell population. The method for preparing modified TIL according to feature 11.

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

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

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

16. A polypeptide comprising any signal peptide linked in order, IL-7, any linker, a BCMA extracellular domain or a variant thereof, any 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.

17. The polypeptides are linked in order. CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, CD52 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, CD52 signal peptide, IL-7, BCMA extracellular domain or variant thereof, hinge domain or linker, and CD52, IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD8 transmembrane domain, or Includes IL-7 signal peptide, IL-7, linker, BCMA extracellular domain or variant thereof, and CD52 The polypeptide according to claim 16.

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