Fusion protein and application thereof

Abstract

A fusion protein comprising a cytokine binding extracellular domain and a signal transduction intracellular domain; the cytokine binding extracellular domain is selected from an IL-10 receptor extracellular domain, an IL-4 receptor extracellular domain, an IL-13 receptor extracellular domain, an IL-6 receptor extracellular domain or a variant of any receptor extracellular domain; the signal transduction intracellular domain is an IL-9 receptor intracellular domain or a variant thereof. The fusion protein can block the inhibition effect of immunosuppressive cytokines on immune effector cells, convert the stimulation of the extracellular immunosuppressive cytokines into the activation of IL-9 signal pathways, reverse the influence of tumor microenvironment and play the role of immunostimulation / activation, so that the immunosuppressive effect of the immunosuppressive cytokines on the immune effector cells is achieved. The proliferation capacity and durability of adoptively transferred immune effector cells in vivo are improved, the depletion of the immune effector cells is inhibited, and the survival function of the immune effector cells is maintained, so that the effect of killing tumor cells is improved.

Description

A fusion protein and its application Technical Field

[0001] The present invention belongs to the technical field of cell therapy, and in particular relates to a fusion protein and an application thereof. Background Art

[0002] Tumor immunotherapy is a treatment approach that controls and eliminates tumors by reinitiating and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. Tumor immunotherapy encompasses treatments such as monoclonal antibody-based immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapies, and small molecule inhibitors. In recent years, tumor immunotherapy has rapidly advanced, becoming the fourth most popular cancer treatment technology after surgery, radiotherapy, and chemotherapy. While immune checkpoint inhibitors, such as anti-PD-1 monoclonal antibodies, have been approved for multiple cancer indications and continue to challenge current standard treatment options, cell therapy has taken a significant lead in the field of hematological malignancies. In tumor immunotherapy, cell therapy involves the use of immune cells, induced by cytokines, to generate large numbers of highly active anti-tumor cells ex vivo. These cells are then adoptively transferred into the patient to restore the patient's immune system and attack tumor cells. Currently, seven CAR-T therapy-based drugs have been approved for hematological malignancies, establishing the importance of cell therapy in the treatment of various hematological malignancies.

[0003] Unfortunately, while cell therapy has significant anti-tumor activity in patients with blood cancers, its efficacy in patients with solid tumors is very limited. Therefore, how to improve the proliferation and sustained killing capacity of adoptively transferred immune effector cells against solid tumor cells has become a technical problem that needs to be solved urgently by those skilled in the art.

[0004] Summary of the Invention

[0005] In view of this, the present invention provides a fusion protein and its application, which are used to solve the problem that adoptively transferred immune effector cells have poor killing ability against solid tumor cells.

[0006] In a first aspect, the present invention provides a fusion protein comprising a cytokine binding extracellular domain and a signal transduction intracellular domain;

[0007] The cytokine binding extracellular domain is selected from the group consisting of IL-10 receptor extracellular domain, IL-4 receptor extracellular domain, IL-13 receptor extracellular domain, IL-6 receptor extracellular domain, or variants of any of the above receptor extracellular domains;

[0008] The signal transduction intracellular domain is an IL-9 receptor intracellular domain or a variant thereof.

[0009] Studies have shown that solid tumor growth may be associated with suppression of the immune response. Many different mechanisms contribute to immune evasion, yet many types of cancer have exploited the regulatory effects of cytokines to downregulate appropriate immune responses aimed at destroying cancer cells. These cytokines achieve immune evasion by secreting immunosuppressive cytokines, which recruit regulatory immune cells to tumors and directly inhibit cytotoxic Th1 cells and / or repolarize cytotoxic Th1 cells to an ineffective Th2 phenotype. Evidence suggests that immunosuppressive cytokines secreted by cancer cells or the surrounding tumor stroma include at least IL-10, IL-4, IL-13, and IL-6. The IL-9 receptor is naturally expressed by mast cells, memory B cells, innate lymphoid cells, and hematopoietic progenitor cells and is a less studied member of the γc cytokine receptor family.

[0010] The fusion protein of the present invention comprises a cytokine binding extracellular domain and a signal transduction intracellular domain; the cytokine binding extracellular domain is selected from the IL-10 receptor extracellular domain, the IL-4 receptor extracellular domain, the IL-13 receptor extracellular domain, the IL-6 receptor extracellular domain or a variant of any of the above receptor extracellular domains; the signal transduction intracellular domain is the IL-9 receptor intracellular domain or a variant thereof. The present invention has discovered that the combination of the cytokine-binding extracellular domain and the signal transduction intracellular domain in the above-mentioned fusion protein can block the inhibitory effect of immunosuppressive cytokines on immune effector cells. The principle is: considering that the immunosuppressive cytokines secreted by cancer cells or surrounding tumor stroma include at least IL-10, IL-4, IL-13 and IL-6, the fusion protein of the present invention designs the cytokine-binding extracellular domain to be an IL-10 receptor extracellular domain, an IL-4 receptor extracellular domain, an IL-13 receptor extracellular domain, an IL-6 receptor extracellular domain or variants thereof, thereby converting the stimulation of extracellular immunosuppressive cytokines into activation of the IL-9 signaling pathway. Experimental results show that converting the stimulation of extracellular immunosuppressive cytokines into activation of the IL-9 signaling pathway can reverse the effects of the tumor microenvironment, exert an immunostimulatory / activating effect, enhance the proliferation ability and persistence of adoptively transferred immune effector cells in vivo, inhibit the exhaustion of immune effector cells, and maintain the survival of immune effector cells, thereby enhancing the effect of killing tumor cells.

[0011] In some embodiments, the fusion protein further comprises a first transmembrane domain, and the cytokine binding extracellular domain, the first transmembrane domain, and the signal transduction intracellular domain are connected in sequence directly or through a linker;

[0012] The cytokine binding extracellular domain is used for binding to IL-10, IL-4, IL-13 and / or IL-6, and the signal transduction intracellular domain is used for IL-9 receptor intracellular signal transduction.

[0013] It can be understood that by connecting through a linker, the linker can be designed to have no tendency to form an ordered secondary structure that may interfere with the function of the cytokine binding extracellular domain and the signal transduction intracellular domain, to have minimal hydrophobicity or charged properties that may interact with the functional protein domain and / or to provide spatial separation of the two cytokine binding extracellular domains and signal transduction intracellular domains, while direct connection has the advantage of making the fusion protein low in immunogenicity. The specific setting can be based on the specific application requirements.

[0014] Preferably, the cytokine binding extracellular domain is an IL-4 receptor extracellular domain or a variant thereof. Experiments have confirmed that selecting the IL-4 receptor extracellular domain as the cytokine binding extracellular domain is more effective in enhancing the function of the fusion protein than selecting fusion proteins with other cytokine binding extracellular domains.

[0015] Furthermore, since IL-4 is generally present in the microenvironment of solid tumors, including but not limited to lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, head and neck cancer, urinary tract malignancies, endometrial cancer, cervical cancer, osteosarcoma, chondrosarcoma, Ewing's sarcoma, thyroid cancer, hepatoblastoma, and Wilms' tumor, etc. Therefore, limiting the cytokine-binding extracellular domain of the fusion protein to the IL-4 receptor extracellular domain or a variant thereof can have significant advantages against solid tumors.

[0016] In some embodiments, the nucleotide sequence encoding the extracellular domain of the IL-4 receptor is the sequence shown in SEQ ID NO: 2, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO: 2.

[0017] In some embodiments, the nucleotide sequence encoding the intracellular domain of the IL-9 receptor is the sequence shown in SEQ ID NO: 4, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO: 4.

[0018] In some embodiments, the first transmembrane domain is the transmembrane domain of the IL-4 receptor or the transmembrane domain of the IL-9 receptor.

[0019] In some embodiments, the nucleotide sequence encoding the first transmembrane domain is the sequence shown in SEQ ID NO: 3 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO: 3.

[0020] In some embodiments, the nucleotide sequence encoding the fusion protein is the sequence shown in SEQ ID NO. 8, 19, 20 or 21, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO. 8, 19, 20 or 21.

[0021] In the present invention, the IL-4 receptor can bind to one or more of IL-4, mutant IL-4, IL-13 and mutant IL-13.

[0022] Preferably, the mutant IL-4 is selected from one or more of a KFR variant, a KF variant and an RGA variant.

[0023] In some embodiments, the fusion protein further comprises a signal peptide. It is understood that the signal peptide can be designed according to conventional techniques based on recognition or localization requirements.

[0024] In some embodiments, the nucleotide sequence encoding the signal peptide is the sequence shown in SEQ ID NO: 1 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO: 1.

[0025] In a second aspect, the present invention provides a nucleic acid molecule encoding the fusion protein described in the first aspect.

[0026] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule described in the second aspect.

[0027] In a fourth aspect, the present invention provides a host cell comprising the vector described in the third aspect. It is understood that the host cell refers to a prokaryotic or eukaryotic cell, such as Escherichia coli, Pseudomonas, and Salmonella, that can be used as a model industrial cell in bioengineering to express a specific protein (e.g., the fusion protein of the present invention) in large quantities.

[0028] In a fifth aspect, the present invention provides an immune effector cell, which expresses the fusion protein described in the first aspect of claim 1, or comprises the nucleic acid molecule described in the second aspect, or comprises the vector described in the third aspect. It is understood that the above-mentioned immune effector cell refers to an immune cell that can participate in the clearance of foreign antigens and exercise effector functions in an immune response, and can be used in cell therapy as autologous or allogeneic cells for cellular immunotherapy.

[0029] In some embodiments, the immune effector cells are selected from one or more of T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells, and stem cell-derived immune effector cells. More preferably, the immune effector cells are selected from T cells, NK cells, or NKT cells. More preferably, the immune effector cells are NKT cells.

[0030] In some embodiments, the immune effector cells are autologous cells and / or allogeneic cells relative to the individual. Autologous cells are preferably autologous T cells, autologous NK cells, or autologous NKT cells; allogeneic cells are preferably allogeneic T cells, allogeneic NK cells, or allogeneic NKT cells, more preferably allogeneic NKT cells.

[0031] NKT cells are a non-MHC-restricted T cell lineage that shares morphological and functional characteristics with both T and NK cells. They are divided into type I and type II NKT cells. Type I NKT cells (iNKT cells) are the predominant subtype of human NKT cells. They express Va-24 and Ja-18 chains in humans and can transform into mature iNKT cells by recognizing glycolipid antigens presented by CDId molecules. α-Galcer is one of the classic ligands for iNKT cells, which helps activate iNKT cells to produce a large number of immunomodulatory factors and tumor-killing cytokines, such as perforin and granzymes. iNKT cells have their own advantages in anti-tumor activities, such as their ability to directly lyse tumor cells, inhibit suppressor cells in the tumor microenvironment (TME), and promote tumor-targeted immune memory.

[0032] It is understandable that the selection of immune effector cells can be set according to specific requirements. However, the present invention found that when the fusion protein of the IL-4 receptor extracellular domain and the IL-9 receptor intracellular domain is expressed in iNKT cells, the killing performance of iNKT cells against IL-4 secreting tumor cells is significantly improved. The tumor inhibition effect of iNKT cells in IL4-HepG2 liver cancer cell-loaded subcutaneous transplanted tumor model mice is also significantly improved. The IL4-9R fusion protein can greatly improve the effect of iNKT cells in killing tumor cells.

[0033] In some embodiments, the fusion protein is expressed constitutively or inducibly. It is understood that when the fusion protein is constitutively expressed, its expression in immune effector cells is designed to be sustained and unaffected by environmental factors, with the advantages of long-lasting expression and stable expression levels. When the fusion protein is inducibly expressed, its expression in immune effector cells is designed to require induction, such as compound-induced expression or conditional promoter-induced expression, with the advantage of controllable expression. The selection can be made based on specific application requirements.

[0034] In some embodiments, the immune effector cells further express an exogenous receptor comprising a targeting extracellular domain that specifically binds to a tumor antigen, a second transmembrane domain, and a targeting intracellular domain.

[0035] In some embodiments, the exogenous receptor is selected from one or more of a chimeric antigen receptor, a modified T cell antigen receptor, a T cell fusion protein, and a T cell antigen coupler. It is understood that the exogenous receptor is a receptor that can specifically bind to a tumor antigen, and its specific modification and fusion method can be adjusted according to different tumor antigens.

[0036] In some embodiments, the exogenous receptor is a chimeric antigen receptor, wherein:

[0037] The targeting extracellular domain comprises: an antibody, an antibody fragment, or a natural ligand of the corresponding antigen, or a combination thereof; and / or

[0038] The second transmembrane domain comprises a transmembrane domain selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, L FA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C; and / or

[0039] The targeting intracellular domain comprises: a primary signaling domain and / or a co-stimulatory signaling domain, wherein:

[0040] (1) the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, or DAP12, or a combination thereof;

[0041] (2) The co-stimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, and CD11 a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46 or NKG2D, or a combination thereof.

[0042] Furthermore, the chimeric antigen receptor is selected from one or more of the following:

[0043] (i) the targeting extracellular domain is selected from an antibody or a fragment thereof that specifically binds to a tumor antigen, the second transmembrane domain is selected from CD28 or the transmembrane domain of CD8, and the targeting intracellular domain is selected from the costimulatory signaling domain of CD28 and the CD3ζ chain;

[0044] (ii) the targeting extracellular domain is selected from an antibody or a fragment thereof that specifically binds to a tumor antigen, the second transmembrane domain is selected from the transmembrane domain of CD28 or CD8, and the targeting intracellular domain is selected from the costimulatory signaling domain of CD137 and the CD3ζ chain;

[0045] (iii) the targeting extracellular domain is selected from an antibody or a fragment thereof that specifically binds to a tumor antigen, the second transmembrane domain is selected from the transmembrane domain of CD28 or CD8, and the targeting intracellular domain is selected from the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137 and the CD3ζ chain.

[0046] It is understandable that when the aforementioned intracellular targeting domain is selected from a variety of structural domains or protein chains, it can be designed and connected according to the conventional methods in the art based on its functional characteristics.

[0047] In some embodiments, the tumor antigen is selected from one or more of the following:

[0048] TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostaglandinase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART 1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1.

[0049] In some embodiments, the tumor antigen is a solid tumor antigen.

[0050] In some embodiments, the solid tumor antigen is selected from B7, CAIX, CD123, CD133, CD171, CD171 / L1-CAM, CEA, Claudin 18.2, cMet, CS1, CSPG4, Dectin1, EGFR, EGFRvIII, EphA2, ERBB receptor, ErbB T4, ERBB2, FAP, folate receptor 1, FITC, FSH, GD2, GPC3, HA-1H / HLA-A2, HER2, IL-11Ra, IL13 receptor α2, IL13R, IL13Rα2 (zetakine), Kappa, LewisY, mesothelin, MUC1, NKG2D, NY-ESO-1, PSMA, ROR-1, TRAIL-receptor 1 or VEGFR2; more preferably, the solid tumor antigen is GPC3.

[0051] In some embodiments, the solid tumor is selected from one or more of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system tumor, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma and squamous cell carcinoma; more preferably, the solid tumor is selected from liver cancer, lung cancer, kidney cancer and / or squamous cell carcinoma.

[0052] In some embodiments, the nucleotide sequence encoding the targeted extracellular domain is the sequence shown in SEQ ID NO: 22 or SEQ ID NO: 29, or the nucleotide sequence encoding the targeted extracellular domain is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 22 or SEQ ID NO: 29;

[0053] The amino acid sequence targeting the extracellular domain is the sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33, or the amino acid sequence targeting the extracellular domain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33.

[0054] In some embodiments, the chimeric antigen receptor encoding nucleotide sequence is the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 30, or the chimeric antigen receptor encoding nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 30;

[0055] The amino acid sequence of the chimeric antigen receptor is the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 34, or the amino acid sequence of the chimeric antigen receptor is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 34.

[0056] In some embodiments, the chimeric antigen receptor and the fusion protein are encoded by the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:31, or by a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:9 or SEQ ID NO:31.

[0057] In a specific embodiment, the antigen specifically bound by the targeting extracellular domain of the exogenous receptor may also be a pathogenic microorganism antigen, which includes viruses, bacteria, fungi, protozoa and / or parasites; more preferably, the pathogenic microorganism is a virus; or more preferably, the pathogenic microorganism is selected from cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and influenza virus.

[0058] In a sixth aspect, the present invention provides a pharmaceutical composition comprising one or more of the fusion protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, and the immune effector cell described in any one of the fifth aspect of the present invention.

[0059] In the seventh aspect, the present invention provides the use of the fusion protein described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention and / or the immune effector cell described in any one of the fifth aspect of the present invention in the preparation of immunotherapy drugs.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The fusion protein of the present invention comprises a cytokine binding extracellular domain and a signal transduction intracellular domain, wherein the cytokine binding extracellular domain is selected from the IL-10 receptor extracellular domain, the IL-4 receptor extracellular domain, the IL-13 receptor extracellular domain, the IL-6 receptor extracellular domain or a variant of the extracellular domain of any of the above receptors; the signal transduction intracellular domain is an IL-9 receptor intracellular domain or a variant thereof. The fusion protein of the present invention can block the inhibitory effect of immunosuppressive cytokines on immune effector cells, and convert the stimulation of immunosuppressive cytokines such as IL-10, IL-4, IL-13 and IL-6 secreted by extracellular cancer cells or surrounding tumor matrix into activation of the IL-9 signaling pathway, reverse the influence of the tumor microenvironment, exert immunostimulatory / activating effects, improve the proliferation capacity and persistence of adoptively transferred immune effector cells in vivo, inhibit the exhaustion of immune effector cells, and maintain the function of immune effector cell survival, thereby improving the effect of killing tumor cells.

[0062] Furthermore, expressing the fusion protein in which the cytokine binding extracellular domain of the present invention is the IL-4 receptor extracellular domain or its variant in immune effector cells can enable the immune effector cells to convert the negative cytokine signal of the inhibitory cytokine IL4 into an IL9 immunostimulatory / activation signal, thereby enhancing the anti-tumor effect of the immune effector cells in solid tumors and improving the further application of cell therapy in solid tumors.

[0063] In addition, the present invention innovatively expresses a fusion protein of the IL-4 receptor extracellular domain and the IL-9 receptor intracellular domain in iNKT cells, and finds that the killing ability of iNKT cells against IL-4-secreting tumor cells is significantly improved. The tumor inhibition effect of iNKT cells in IL4-HepG2 liver cancer cell-loaded subcutaneous transplanted tumor model mice is also significantly improved. The IL4-9R fusion protein can greatly enhance the effect of iNKT cells in killing tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0065] FIG1 is a graph showing the transduction efficiency of immune effector cells expressing chimeric antigen receptors in Example 1 of the present invention;

[0066] FIG2 is a graph showing the results of detecting the killing efficiency of immune effector cells against IL-4 secreting tumor cells in Example 2 of the present invention;

[0067] FIG3 is a graph showing the cytokine secretion levels detected by co-incubation of immune effector cells with IL-4 secreting tumor cells in Example 3 of the present invention;

[0068] FIG4 is a graph showing changes in tumor volume in mice bearing subcutaneous transplanted tumor models of IL4-HepG2 liver cancer cells in Example 4;

[0069] FIG5 is a graph showing the results of detecting the killing efficiency of immune effector cells against IL-4 secreting tumor cells in Example 5. DETAILED DESCRIPTION

[0070] The embodiment of the present invention fuses the immunosuppressive cytokine receptor and the IL-9 receptor to obtain a fusion protein, so that when the immune effector cells bind to the suppressive cytokines IL-10, IL-4, IL-13 and IL-6, the intracellular signals of the immune effector cells can be converted into IL9 immunostimulatory / activation signals, thereby resisting the suppressive cytokines present in the tumor microenvironment and improving the expansion and anti-tumor activity of the immune effector cells.

[0071] The vectors of the present invention, such as the exemplary bicistronic retroviral vector, encode the fusion protein. The vector can be any type of vector, including but not limited to lentiviral vectors, retroviral vectors, adenoviral vectors, plasmids, or adeno-associated viral vectors.

[0072] The immune effector cells of the embodiment of the present invention can be primary T cells, naturally occurring tumor antigen-specific cytotoxic T lymphocytes, NK cells and / or NKT cells, etc., preferably tumor-specific T cells and / or NKT cells. Through fusion proteins, tumor-specific T cells or NKT cells are made to resist negative cytokine signals present in the tumor microenvironment and reverse the effects of the tumor microenvironment. The immune effector cells of the embodiment of the present invention can be used in an autologous or allogeneic environment.

[0073] In an embodiment of the present invention, the functional portion of the immunosuppressive cytokine receptor can be linked to the functional portion of the IL-9 receptor via a linker. The linker linking the functional portion of the immunosuppressive cytokine receptor and the functional portion of the IL-9 receptor can be designed to: (1) allow the functional portion of the immunosuppressive cytokine receptor and the functional portion of the IL-9 receptor to fold and function independently of each other; (2) not have a tendency to form an ordered secondary structure that may interfere with the function of the functional portion of the immunosuppressive cytokine receptor and the functional portion of the IL-9 receptor; (3) have minimal hydrophobicity or charged properties that may interact with functional protein domains; and / or (4) provide spatial separation of the functional portion of the immunosuppressive cytokine receptor and the functional portion of the IL-9 receptor.

[0074] In an embodiment of the present invention, the linker comprises a peptide, the sequence of which can be set according to the spatial conformational requirements or functional requirements of the protein to be adjusted or improved, such as GGGGSGGGGSGGGGS, etc. The linker can be conjugated to the functional portion of the immunosuppressive cytokine receptor and / or the functional portion of IL-9R using recombinant DNA technology.

[0075] In embodiments of the present invention, the fusion protein may include one or more linkers and other functional moieties as needed. These may include binding regions, such as avidin or epitopes, or tags such as polyhistidine tags that can be used to purify and process the fusion protein, as well as other linkers. In addition, detectable markers may be incorporated into the fusion protein to facilitate monitoring of the transport of the fusion protein through the body or cells. Markers include radionuclides, enzymes, fluorophores, chromophores, and similar markers.

[0076] It will be appreciated by those skilled in the art that the biological activity of the coded protein can be changed in many ways.For example, PCR can be used to produce a change in the DNA sequence encoding the fusion protein.These changes in the DNA encoding the fusion protein can be used to optimize the codon preference in the host for expressing the protein, or can comprise other sequence variations that can be convenient to express.

[0077] In order to further understand the technical solutions provided in this article, the definitions of the methods and / or terms in this article are further explained below.

[0078] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0079] The term "tumor microenvironment" refers to the tissues, cells, molecules, and blood vessels that surround and support tumor cells. A tumor's microenvironment is dynamic, and tumors can change their microenvironment, which can affect how tumors grow and spread.

[0080] The term "receptor" includes native receptor proteins as well as variant receptor proteins. As used herein, the sequence of a "native" or "wild-type" receptor refers to a human receptor sequence that can be purified from natural sources and / or prepared using recombinant technology.

[0081] The term "functional portion of a receptor" refers to a full-length or partial fragment of a receptor that retains receptor function; for example, the extracellular portion of a receptor, the extracellular portion of a receptor, and the transmembrane portion of a receptor. It can be purified from natural sources or produced using recombinant technology.

[0082] The term "IL-4" refers to interleukin-4, NCBI Gene ID: 3565, an anti-inflammatory cytokine that induces T cell differentiation into the Th2 type. It is a pleiotropic cytokine produced by activated T cells and is a ligand for the IL4 receptor. GATA3 is a downstream signal after IL-4 activation. Variant IL-4 proteins with high selectivity for IL-13Rα1 (type II receptor) are human IL-4 proteins that contain the following mutations relative to native IL-4 (numbering excludes the methionine at the N terminus): R121K / Y124F / S125R ("KFR" or "KFR4" variants) or R121K / Y124F ("KF" variants).

[0083] A variant IL-4 protein that has higher selectivity for γc (type I receptor) than for IL-13Rαl (type II receptor) is an IL-4 protein that contains the following mutations relative to the sequence of native human IL-4 (numbering excludes the methionine at the N-terminus): R121Q / Y124W / S125F ("RGA" or "super-4" or "S4" variant).

[0084] The term "IL-9 receptor" or "IL-9 receptor" includes native IL-9 receptor protein as well as variant IL-9 receptor protein. As used herein, the sequence of "native" or "wild-type" IL-9 receptor refers to the human IL-9 receptor sequence, which can be purified from natural sources and / or prepared using recombinant technology.

[0085] The term "functional portion of the IL-9 receptor (IL-9 receptor)" refers to a full-length IL-9 receptor or a partial fragment of the IL-9 receptor that retains IL-9 receptor function; for example, the intracellular signaling portion of the IL-9 receptor (SEQ ID NO: 15), the intracellular signaling portion, and the transmembrane portion of the IL-9 receptor. These can be purified from natural sources or produced using recombinant technology.

[0086] The term "IL-2" refers to a type of cell growth factor in the immune system, NCBI Gene ID: 3558, which can regulate the cell activity of white blood cells in the immune system and promote the proliferation of Th0 and CTL. In this application, it can be used to culture T cells and NKT cells.

[0087] It should be understood that the IL-4 receptor proteins according to the present disclosure include fragments that can be shorter than the native IL-4 receptor protein, as long as the IL-4 receptor protein fragments retain the ability to bind IL-4. It should also be understood that the present disclosure encompasses nucleic acid molecules encoding the IL-4 receptor proteins described herein or known in the art, including but not limited to RNA sequences corresponding to the DNA sequences described herein.

[0088] The term "fusion protein" includes immunosuppressive cytokine receptor proteins that are bound to IL-9R using optional additional sequences or moieties (e.g., linkers), as described herein, and nucleic acid molecules encoding these fusion proteins. Also encompassed are recombinant nucleic acid molecules in which the nucleic acid sequence encoding the fusion protein is operably linked to a promoter, vectors containing the molecules, and transgenic cells containing such molecules. Methods for generating these fusion proteins can employ conventional methods in the art, such as using recombinant molecular biology methods.

[0089] The term "binding" refers to the cytokine binding extracellular domain recognizing IL-10, IL-4, IL-13 and / or IL-6 and inducing the signal transduction intracellular domain to transduce the IL-9 receptor intracellular signal.

[0090] The term "signaling intracellular domain" refers to the intracellular portion of a fusion protein. In an embodiment, the signaling intracellular domain transduces IL-9 receptor intracellular signals and directs cells to perform specialized functions.

[0091] The term "first transmembrane domain" may include transmembrane domains of various natural receptor proteins, which play a role in connecting the extracellular region of the immunosuppressive cytokine receptor and the intracellular region of the IL-9 receptor and anchoring them on the cell membrane, including but not limited to the transmembrane regions of the immunosuppressive cytokine receptor and IL-9R.

[0092] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0093] The term "homology" or "identity" refers to the subunit sequence identity between two polymer molecules, such as between two nucleic acid molecules, such as between two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position is occupied by the same monomer subunit in both molecules, such as when two DNA molecules are both occupied by adenosine at a position, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of positions that match or are homologous; for example, when half of the positions in the two sequences (e.g., 5 positions in a polymer that is 10 subunits long) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0094] The term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising at least a targeting extracellular domain (i.e., an extracellular antigen binding domain), a second transmembrane domain (i.e., a transmembrane domain), and a targeting intracellular domain (i.e., a cytoplasmic signaling domain), wherein the cytoplasmic signaling domain comprises a functional signaling domain derived from a stimulatory molecule defined below. On the one hand, the stimulatory molecule is a zeta chain associated with a T cell receptor complex. On the one hand, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined below. On the one hand, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, and / or CD28. On the one hand, CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain contains a functional signaling domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein, and the protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain contains a functional signal transduction domain derived from a costimulatory molecule and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein, and the protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain contains two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein, and the protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain contains at least two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR comprises an optional leader sequence at the amino terminus (N terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0095] The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information into the cell via a defined signaling pathway by producing second messengers to regulate cellular activity, or by functioning as an effector in response to such messengers.

[0096] The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0097] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and may refer to the antigen-binding domain of an intact antibody, e.g., an antigenicity-determining variable region that is sufficient to confer recognition and specific binding to a target, such as an antigen, on the antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (VH or VL), camelid VHH domains, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected by a short, flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated herein, a scFv may have the VL and VH variable regions in either order, e.g., relative to the N- and C-termini of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0098] The term "antigen" refers to a molecule that can provoke an immune response. The immune response can involve the production of antibodies, the activation of specific immunocompetent cells, or both. Those skilled in the art will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will appreciate that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that can elicit an immune response, thus encodes an "antigen" (as the term is used herein). Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, which can be arranged in various combinations to encode polypeptides that can elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene." It will be readily apparent that an antigen can be produced synthetically, can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or fluids, and other biological components.

[0099] The fusion protein, or immune effector cells expressing the fusion protein, can be analyzed using standard methods known in the art or described herein.

[0100] The fusion proteins of the present invention can be used to prepare drugs with various therapeutic uses. In general, the fusion proteins of the present invention can be used to treat or prevent any disease, disorder, or condition involving cells expressing IL-10, IL-4, IL-13, and IL-16 (particularly IL-4) and that would benefit from inhibition of cell proliferation or promotion of cell death. In some embodiments, they can be used to induce apoptosis or cell death, or to treat disorders associated with abnormal apoptosis or cell proliferation, such as cancer.

[0101] As used herein, the terms "cancer," "cancer," or "tumor" refer to cells that have the ability to grow autonomously (e.g., an abnormal state or condition characterized by a surge in the growth of proliferating cells). Hyperproliferative or neoplastic disease states can be classified as pathological types (e.g., because they deviate from a normal state but are not associated with a disease state). Thus, "cancer" or "tumor" refers to any unwanted growth of a cell that has no physiological function. The term cancer includes cell growth that is technically benign but may be at risk of becoming malignant. "Malignant" refers to an abnormal growth of any cell type or tissue. The term malignant includes cell growth that is technically benign but may be at risk of becoming malignant. The term also includes any cancer, carcinoma, neoplasm, neoplasia, or tumor. Thus, these terms are meant to include all types of cancerous growth or tumorigenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or stage of invasion.

[0102] Most cancers fall into three broad histological categories: carcinomas, which are the majority of cancers and cancers of epidermal cells or cells covering the external or internal surfaces of organs, glands, or other body structures (e.g., skin, uterus, lung, breast, prostate, stomach, intestine) and tend to metastasize; sarcomas, which are derived from connective or supporting tissue (e.g., bone, cartilage, tendon, ligament, fat, muscle); and hematologic neoplasms, which are derived from bone marrow and lymphatic tissue. Examples of cancer include, but are not limited to, carcinomas, sarcomas, and hematologic neoplastic disorders such as leukemias.

[0103] The cancer may be an adenocarcinoma (which is typically found in secretory organs or glands such as the breast, lung, colon, prostate, or bladder), or it may be a squamous cell carcinoma (which is derived from squamous epithelium and typically forms in most areas of the body).

[0104] Sarcomas can be osteosarcomas or osteogenic sarcomas (bone), chondrosarcomas (cartilage), leiomyosarcomas (smooth muscle), rhabdomyosarcomas (skeletal muscle), mesotheliomas or mesotheliomas (the membranous lining of body cavities), fibrosarcomas (fibrous tissue), angiosarcomas or hemangioendotheliomas (blood vessels), liposarcoma (fat), gliomas or astrocytomas (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), or mesenchymal or mesodermal mixed tumors (a mixed connective tissue type).

[0105] Hematopoietic neoplastic disorders include those involving hyperplastic / neoplastic cells of hematopoietic origin, such as those derived from the myeloid, lymphoid, or erythroid lineages or their precursors. Preferably, the disease is derived from a poorly differentiated acute leukemia (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Additional exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML); lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-lineage acute lymphoblastic leukemia and T-lineage acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia, and Waldenstrom's macroglobulinemia.

[0106] Other forms of malignant lymphoma include, but are not limited to, non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Strauss disease.

[0107] In embodiments of the present invention, pharmaceutical compositions may comprise the fusion protein of the present invention or the immune effector cell of the present invention and one or more non-toxic pharmaceutically acceptable carriers, diluents, excipients, and adjuvants. These compositions may be suitable for use in the treatment of therapeutic indications described herein.

[0108] In some embodiments, other active ingredients can be added to the composition. The fusion protein or immune effector cell of the present invention can be used to treat one or more cancers in a therapeutically effective amount or in combination with other therapies. The fusion protein or immune effector cell of the present invention can be administered before, during, or after treatment with an anti-tumor or other therapy. The fusion protein or immune effector cell of the present invention can also be used in combination with a sensitizer such as a radiotherapy sensitizer. Generally speaking, a sensitizer is any agent that can increase the activity of the fusion protein. For example, a sensitizer will increase the ability of the fusion protein to inhibit cancer cell growth or kill cancer cells. Exemplary sensitizers include anti-IL-10 antibodies, bone morphogenetic protein, and HDAC inhibitors.

[0109] In embodiments of the invention, the fusion proteins or immune effector cells may be used as part of neoadjuvant therapy (to primary therapy), as part of an adjuvant therapy regimen, where the goal is to cure the cancer in the subject. The fusion proteins of the present invention can be administered at different stages of tumor development and progression, including at various stages of treatment of advanced and / or aggressive neoplasms (e.g., overt disease that is incurable in a subject by local treatment modalities (e.g., surgery or radiation therapy), metastatic disease. Locally advanced disease and / or refractory tumors (e.g., cancer or tumors that do not respond to treatment). "Primary therapy" refers to the first-line treatment following the initial diagnosis of cancer in a subject. Exemplary primary therapies may involve surgery, extensive chemotherapy, and radiation therapy. "Adjuvant therapy" refers to a therapy that is administered to a subject following a primary therapy and when there is a risk of recurrence. Adjuvant systemic therapy is initiated soon after primary therapy, for example, 2, 3, 4, 5, or 6 weeks after the last primary therapy treatment to delay recurrence, prolong survival, or cure the subject. Considering that the fusion protein of the present invention or the immune effector cell can be used alone or in combination with one or more other chemotherapeutic drugs as part of an adjuvant therapy, the combination of the fusion protein of the present invention or the immune effector cell with a standard chemotherapeutic agent can play a role in enhancing the efficacy of chemotherapy and, therefore, can be used to improve standard cancer therapy.

[0110] "Subject" can be a mammal in need of treatment, such as a human or a veterinary patient, and veterinary patients include rodents, such as mice or rats, cats, dogs, cows, horses, sheep, goats, or other livestock. In the embodiments of the present invention, "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like. The subject may be suspected of having a disease characterized by cell proliferation or may be at risk of developing a disease characterized by cell proliferation, diagnosed as having a disease characterized by cell proliferation, or a control subject who is confirmed not to have a disease characterized by cell proliferation. It should be noted that those skilled in the art are aware of the diagnostic methods for diseases characterized by cell proliferation and the clinical divisions of such diagnosis.

[0111] In embodiments of the present invention, the pharmaceutical composition may be in the form of a liquid solution, suspension, emulsion, sustained-release formulation, or powder, and may be formulated with a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated as a suppository using conventional binders and carriers, such as triglycerides. "Pharmaceutically acceptable carrier" refers to a carrier matrix or vehicle that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host or subject.

[0112] The fusion protein or immune effector cell can be delivered together with a pharmaceutically acceptable vehicle. The vehicle can be used to enhance stability and / or as a delivery property. The vehicle can be an artificial membrane vesicle such as a liposome, a nonionic surfactant vesicle (noisome), a nano-microliposome, a microparticle or microcapsule, or a colloidal preparation comprising a pharmaceutically acceptable polymer.

[0113] In an embodiment of the present invention, the pharmaceutical composition of the fusion protein or immune effector cell of the present invention can be prepared into a sterile injectable aqueous or oily suspension using one or more dispersing agents, wetting agents and / or suspending agents by methods known in the art.

[0114] In order to further understand the present invention, the present invention is described in detail below with reference to specific embodiments.

[0115] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available; unless otherwise specified, the methods used in the following examples are all conventional methods in the art.

[0116] Example 1 Construction of immune effector cells expressing chimeric antigen receptors

[0117] Using conventional molecular biological methods in the art, the human IL-4R signal peptide coding sequence (SEQ ID NO: 1), the human IL-4R extracellular region DNA coding sequence (SEQ ID NO: 2), the human IL-4R transmembrane domain coding sequence (SEQ ID NO: 3), and the human IL-9R intracellular domain coding sequence (SEQ ID NO: 4) were sequentially connected, then connected to GPC3-28-BB-z-CAR (SEQ ID NO: 5) with the P2A self-cleavage peptide coding sequence (SEQ ID NO: 6), and inserted into the pPHAGE lentiviral expression vector to obtain the lentiviral plasmid chIL4-9R-GPC3-CAR expressing GPC3-28-BB-z-CAR (amino acid sequence of SEQ ID NO: 7) and IL4-9R fusion protein (amino acid sequence of SEQ ID NO: 8). The nucleotide sequence is shown in SEQ ID NO: 9. The above-mentioned GPC3-28-BB-z-CAR sequence fragment is a chimeric antigen receptor coding sequence, which is composed of a signal peptide encoding nucleotide sequence (SEQ ID NO: 23), a targeting extracellular domain encoding nucleotide sequence (i.e., hYP7 antibody, nucleotide sequence SEQ ID NO: 22, amino acid sequence SEQ ID NO: 32), a Hinge encoding nucleotide sequence (SEQ ID NO: 24), a second transmembrane encoding nucleotide sequence (SEQ ID NO: 25), and a targeting intracellular domain encoding nucleotide sequence, wherein the targeting intracellular domain comprises CD28 (coding sequence SEQ ID NO: 26), CD137 (coding sequence SEQ ID NO: 27) and CD3ζ (coding sequence SEQ ID NO: 28) costimulatory signaling domains connected in sequence, and the above-mentioned Hinge is a hinge (i.e., a linker) connecting the targeting extracellular domain and the second transmembrane encoding nucleotide sequence, so that the targeting extracellular domain can better recognize the antigen.

[0118] The human IL-4R signal peptide coding sequence (SEQ ID NO: 1) and the IL-4R extracellular region DNA coding sequence (SEQ ID NO: 2) were sequentially connected with the human IL-4R transmembrane domain coding sequence (SEQ ID NO: 3) and the human IL-7R intracellular domain coding sequence (SEQ ID NO: 10), and then connected with the GPC3-28-BB-z-CAR (SEQ ID NO: 5) via the P2A self-cleavage peptide coding sequence (SEQ ID NO: 6), and inserted into the pPHAGE lentiviral expression vector to obtain the lentiviral plasmid chIL4-7R-GPC3-CAR expressing GPC3-28-BB-z-CAR (amino acid sequence of SEQ ID NO: 7) and IL4-7R fusion protein (amino acid sequence of SEQ ID NO: 11). The nucleotide sequence is shown in SEQ ID NO: 12.

[0119] The human IL-4R signal peptide coding sequence (SEQ ID NO: 1) and the IL-4R extracellular region DNA coding sequence (SEQ ID NO: 2) were sequentially connected with the human IL-4R transmembrane domain coding sequence (SEQ ID NO: 3) and the human IL-21R intracellular domain coding sequence (SEQ ID NO: 13), and then connected with GPC3-28-BB-z-CAR (SEQ ID NO: 5) via the P2A self-cleavage peptide coding sequence (SEQ ID NO: 6), and inserted into the pPHAGE lentiviral expression vector to obtain the lentiviral plasmid chIL4-21R-GPC3-CAR expressing GPC3-28-BB-z-CAR (amino acid sequence of SEQ ID NO: 7) and IL4-21R fusion protein (amino acid sequence of SEQ ID NO: 14). The nucleotide sequence is shown in SEQ ID NO: 15.

[0120] T cell activation: Human PBMC (purchased from Shanghai Heyou Biotechnology) were cultured in AIM-V medium, supplemented with 2% human AB serum, 500 U / mL recombinant human IL-2, and CD3 / CD28 antibody-conjugated magnetic beads for activation for 48 h before use.

[0121] iNKT cell activation: Isolated single-blood PBMCs were stimulated and cultured with α-GC (α-glucosidase) for 7 days to increase the iNKT ratio. iNKTs were then purified from the PBMCs using Miltenyi magnetic beads. iNKT cells were then activated using Miltenyi CD3 / CD28 transactin and set aside.

[0122] The activated T cells were transduced with the lentiviral plasmids chIL4-9R-GPC3-CAR, chIL4-7R-GPC3-CAR and chIL4-21R-GPC3-CAR, respectively. At the same time, iNKT cells were transduced with the lentiviral plasmid chIL4-9R-GPC3-CAR. The cells were serum-deprived and rested for 24 hours to obtain chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells expressing the fusion proteins IL4-9R (amino acid sequence of SEQ ID NO: 8), IL4-7R (amino acid sequence of SEQ ID NO: 11), IL4-21R (amino acid sequence of SEQ ID NO: 14) and GPC3-28-BB-z-CAR (amino acid sequence of SEQ ID NO: 7) for later use.

[0123] GPC3-28-BB-z-CAR-z (SEQ ID NO: 16) was inserted into the PHAGE lentiviral expression vector. The GPC3-28-BB-z-CAR-z was the GPC3-28-BB-z-CAR sequence shown in SEQ ID NO: 5 with a terminator added. A lentiviral vector expressing GPC3-28-BB-z-CAR was constructed, and then 293T was transfected with the packaged lentivirus to obtain lentivirus. The obtained lentivirus was used to transduce T cells and iNKT cells, respectively, to obtain GPC3-CAR T cells and GPC3-CAR iNKT cells for standby use.

[0124] Please refer to Figure 1, which is a graph of the transduction efficiency of immune effector cells expressing chimeric antigen receptors in Example 1 of the present invention. In the figure, A and G are the GPC3 flow cytometry results of the control group (untreated T cells, no transfection and empty vector), GPC3-CAR T cell group, GPC3-iNKT cell group, chIL4-9R-GPC3-CAR T cell group, chIL4-7R-GPC3-CAR T cell group, chIL4-21R-GPC3-CAR T group, and chIL4-9R-GPC3-CAR iNKT cell group, respectively.

[0125] The results showed that the above lentiviral plasmids had high transduction efficiency in immune effector cells. It should be noted that the number of cells used in subsequent immune effector cell performance testing experiments was converted to the transduction efficiency so that the number of CAR-positive cells in each group of immune effector cells was at the same level.

[0126] Example 2 Detection of the killing efficiency of immune effector cells against IL-4 secreting tumor cells

[0127] A lentiviral expression plasmid encoding Luciferase-GFP-F2A-IL-4 (coding sequence: SEQ ID NO: 17) was constructed, packaged, and used to infect HepG2 cells. Three days later, flow cytometry revealed a positive result for GFP, yielding target IL-4-secreting IL-4-HepG2-GL cells for future use. In this expression plasmid, Luciferase is a luciferase that reacts with a luciferase substrate, indicating viable cells through luminescence; GFP is a green fluorescent protein that serves as a tag; and F2A is a self-cleaving complex that cleaves the translation products of IL4 and Luciferase-GFP.

[0128] The above-mentioned IL-4-HepG2-GL cells were used as tumor cells to compare the killing effect of GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells on IL-4-HepG2-GL cells.

[0129] The UTD T cells prepared in Example 1 (control group, i.e., untreated T cells, no empty transfection), GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT were taken, and IL-4-HepG2-GL was used as the target cell. The effector-target ratio was 1:1, 1:2, and 1:4 for killing. After 24 hours, luciferase substrate was added and the killing efficiency was detected by microplate reader.

[0130] For the results, please refer to Figure 2, which shows the results of the test of the killing efficiency of immune effector cells against IL-4-secreting tumor cells in Example 2 of the present invention. The data in Figure 2 were statistically analyzed using two-way ANOVA with multiple comparisons. Statistical differences are marked as follows: *P<0.05, **P<0.01 vs. UTD T group; #P<0.05, ##P<0.01 vs. GPC3-CAR T group; ΔΔP<0.01 vs. GPC3-CAR iNKT group; @P<0.05, @@P<0.01 vs. chIL4-9R-GPC3-CAR T group. Data are presented as mean ± SEM.

[0131] The killing efficiency is calculated as follows: killing rate = (1-luminescence value of experimental group / tumor background luminescence value) × 100%

[0132] The results showed that when the effector-target ratio was 1:1, UTD T cells, GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells, and chIL4-9R-GPC3-CAR iNKT cells all had a certain killing effect on IL-4-HepG2-GL cells, and the killing effect of GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells, and chIL4-9R-GPC3-CAR iNKT cells was significantly stronger than that of UTD T cells.

[0133] Compared with GPC3-CAR T cells and GPC3-CAR iNKT cells, the killing efficiency of chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells was significantly greater, indicating that the expression of IL4-7R, IL4-21R and IL4-9R can significantly enhance the killing effect of GPC3-CAR T cells and GPC3-CAR iNKT cells on IL-4 secreting tumor cells.

[0134] Moreover, the killing effects of chIL4-9R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells were stronger than those of chIL4-7R- GPC3-CAR T cells and chIL4-21R-GPC3-CAR T cells, indicating that compared with IL4-7R and IL4-21R, the expression of IL4-9R increased the killing effect of GPC3-CAR T cells on IL-4-secreting tumor cells.

[0135] When the effector-target ratio was changed from 1:1 to 1:2 or 1:4, the killing efficiency of GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells, and chIL4-9R-GPC3-CAR iNKT cells against target cells IL-4-HepG2-GL decreased. However, the killing effects of chIL4-9R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells were stronger than those of chIL4-7R-GPC3-CAR T cells and chIL4-21R-GPC3-CAR T cells, further indicating that compared with IL4-7R and IL4-21R, the increased expression of IL4-9R resulted in a more significant killing effect of GPC3-CAR T cells on tumor cells. It should be noted that the expression of IL4-9R significantly improved the tumor-killing effect of GPC3-CAR iNKT cells than the tumor-killing effect of GPC3-CAR T cells, indicating that IL4-9R can significantly improve the effect of iNKT cells in killing tumor cells.

[0136] Example 3 Detection of cytokine secretion levels in immune effector cells co-incubated with IL-4 secreting tumor cells

[0137] The UTD T cells prepared in Example 1 (control group, i.e., untreated T cells, no empty transfection), GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells were co-incubated with the IL-4-HepG2-GL cells prepared in Example 2 at an effector-target ratio of 1:1. On the next day, the co-culture supernatant was taken, and the secretion level of IFN-γ was detected by ELISA.

[0138] For the results, please refer to Figure 3, which shows the cytokine secretion levels detected by co-incubation of immune effector cells with IL-4-secreting tumor cells in Example 3 of the present invention. The data in Figure 3 were statistically analyzed using one-way ANOVA with multiple comparisons. Statistical differences are indicated as follows: **P < 0.01 vs. UTD T group; ##P < 0.01 vs. GPC3-CAR T group; ΔΔP < 0.01 vs. GPC3-CAR iNKT group; @P < 0.05, @@P < 0.01 vs. chIL4-9R-GPC3-CAR T group. Data are presented as mean ± SEM.

[0139] Figure 3 shows that UTD T cells, GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells, and chIL4-9R-GPC3-CAR iNKT cells all secreted IFN-γ, and the level of IFN-γ secreted by GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells, and chIL4-9R-GPC3-CAR iNKT cells was significantly higher than that of UTD T cells.

[0140] Compared with GPC3-CAR T cells and GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells secreted higher levels of IFN-γ, further indicating that the expression of IL4-7R, IL4-21R and IL4-9R can enhance the killing effect of GPC3-CAR T cells and GPC3-CAR iNKT cells on IL-4-secreting tumor cells.

[0141] Moreover, the levels of IFN-γ secreted by chIL4-9R-GPC3-CAR T cells and chIL4-9R-GPC3-CAR iNKT cells were higher than those of chIL4-7R-GPC3-CAR T cells and chIL4-21R-GPC3-CAR T cells, further indicating that compared with IL4-7R and IL4-21R, the expression of IL4-9R makes GPC3-CAR T cells more effective in killing IL-4 secreting tumor cells. It should be noted that the increase in the level of IFN-γ secreted by GPC3-CAR iNKT cells by IL4-9R expression is stronger than that by GPC3-CAR T cells, indicating that IL4-9R can significantly enhance the effect of iNKT cells in killing tumor cells.

[0142] Example 4 Detection of the tumor inhibitory effect of immune effector cells on IL4-HepG2 liver cancer cell-loaded subcutaneous transplanted tumor model mice

[0143] This example uses IL4-HepG2 liver cancer cell-loaded subcutaneous transplanted tumor model mice to detect the tumor suppression effect of immune effector cells, including the following steps:

[0144] 1) Experimental groups: 6-8 week old NCG mice (purchased from Jicui Company) were randomly divided into 6 groups, with 6 mice in each group, namely UTD T cell group (control group, i.e., untreated T cells, not transfected with empty vector), GPC3-CAR T group, GPC3-CAR iNKT cell group, chIL4-9R-CAR T cell group, chIL4-7R-GPC3-CAR T cell group, chIL4-21R-GPC3-CAR T cell group and chIL4-9R-CAR iNKT cell group.

[0145] 2) Establishment of an IL4-HepG2 liver cancer cell-loaded subcutaneous transplant tumor model: A lentiviral expression plasmid encoding GFP-F2A-IL-4 (coding sequence: SEQ ID NO: 18) was constructed. Compared to Luciferase-GFP-F2A-IL-4, the Luciferase portion of this GFP-F2A-IL-4 sequence was omitted. The virus was packaged and used to infect HepG2 cells. Three days later, flow cytometry revealed a GFP-positive result, resulting in IL-4-secreting target cells, IL-4-HepG2. IL4-HepG2 cells in the logarithmic growth phase and in good growth condition were harvested by trypsin digestion, and 5×10 cells were inoculated per mouse. 6 tumor cells were used to obtain IL4-HepG2 liver cancer cell-loaded subcutaneous transplanted tumor model mice.

[0146] 3) CAR-T or CAR-iNKT cell infusion: When the average tumor volume is approximately 80 mm3 -120mm 3 When the disease is severe, T cells or iNKT cells are infused.

[0147] Each group was injected with 2×10 6 / UTD T cells (control group, i.e., untreated T cells, no empty vector transfection), GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells and chIL4-9R-CAR iNKT cells.

[0148] For results, please refer to Figure 4, which shows the changes in tumor volume in mice bearing the IL4-HepG2 liver cancer cell subcutaneous xenograft model described in Example 4. The data in Figure 4 were statistically analyzed using one-way ANOVA with multiple comparisons for the data on day 25. Statistical differences are indicated as follows: **P < 0.01 vs. UTD T group; #P < 0.05, ##P < 0.01 vs. GPC3-CAR T group; ΔΔP < 0.01 vs. GPC3-CAR iNKT group; @@P < 0.01 vs. chIL4-9R-GPC3-CAR T group. Data are presented as mean ± SEM.

[0149] Figure 4 shows that compared with the UTD T cell group, 25 days after the injection of GPC3-CAR T cells, GPC3-CAR iNKT cells, chIL4-9R-GPC3-CAR T cells, chIL4-7R-GPC3-CAR T cells, chIL4-21R-GPC3-CAR T cells, and chIL4-9R-GPC3-CAR NKT cells, all groups showed significant tumor inhibition effects.

[0150] Moreover, as shown in Figure 4, the tumor volume of the UTD-T cell infusion group continued to grow, while the tumor volume growth of the GPC3-CAR T cell group, GPC3-CAR iNKT cell group, chIL4-7R-GPC3-CAR T cell group, and chIL4-21R-GPC3-CAR T cell group slowed down relatively and showed an inhibitory trend, while the tumor growth of the chIL4-9R-CAR T cell group was significantly inhibited, and the tumor volume was significantly smaller than that of the chIL4-7R-GPC3-CAR T cell group, chIL4-21R-GPC3-CAR T cell group, and GPC3-CAR T cell group, indicating that hIL4-9R-GPC3-CAR T has a stronger tumor inhibitory effect, and chIL4-9R significantly improves the tumor inhibitory effect of GPC3-CAR T cells.

[0151] At the same time, compared with the GPC3-CAR iNKT cell group, tumor growth in the chIL4-9R-GPC3-CAR iNKT cell group was also significantly inhibited, indicating that chIL4-9R can also significantly enhance the tumor-suppressing effect of GPC3-CAR iNKT cells. It should be noted that the effect of IL4-9R expression on the tumor-suppressing effect of GPC3-CAR iNKT cells is stronger than that on GPC3-CAR T cells.

[0152] Example 5 Construction of immune effector cells and detection of killing efficiency against IL-4 secreting tumor cells

[0153] In this example, immune effector cells GPC3 (GC33) -CAR T cells, GPC3 (GC33) -CAR iNKT cells, chIL4-9R-GPC3 (GC33) -CAR T cells, chIL4-7R-GPC3 (GC33) -CAR T cells, chIL4-21R-GPC3 (GC33) -CAR T cells and chIL4-9R-GPC3 (GC33) -CAR iNKT cells were constructed. Among them, the difference between GPC3 (GC33) -CAR T cells and the GPC3-CAR T cells in Example 1 is that the targeting extracellular domain coding sequence in CAR, i.e., SEQ ID NO: 22 encoding the hYP7 antibody, is replaced with SEQ ID NO: 29 encoding the GC33 antibody. The encoding nucleotide sequence of GPC3 (GC33) -CAR is SEQ ID NO: 30, and the encoding nucleotide sequence of chIL4-9R-GPC3 (GC33) -CAR is SEQ ID NO: 31. The amino acid sequence of GC33 is SEQ ID NO: 33, and the amino acid sequence of GPC3 (GC33)-CAR is SEQ ID NO: 34.

[0154] Similarly, the difference between GPC3 (GC33) -CAR iNKT cells, chIL4-9R-GPC3 (GC33) -CAR T cells, chIL4-7R-GPC3 (GC33) -CAR T cells, chIL4-21R-GPC3 (GC33) -CAR T cells and chIL4-9R-GPC3 (GC33) -CAR iNKT cells and GPC3-CAR iNKT cells in Example 1, respectively, is that the targeting extracellular domain coding sequence in CAR, i.e., SEQ ID NO: 22 encoding the hYP7 antibody, is replaced with SEQ ID NO: 29 encoding the GC33 antibody.

[0155] Using Example 2IL-4-HepG2-GL cells as tumor cells, GPC3 (GC33) -CAR T cells, GPC3 (GC33) -CAR iNKT cells, chIL4-9R-GPC3 (GC33) -CAR T cells, chIL4-7R-GPC3 (GC33) -CAR T cells, chIL4-21R-GPC3 (GC33) -CAR T cells and chIL4-9R-GPC3 (GC33) -CAR iNKT cells were compared to kill IL-4-HepG2-GL cells. It should be noted that in the test of killing IL-4-HepG2-GL cells by immune effector cells, the number of cells used was converted by transduction efficiency so that the number of CAR-positive cells in each group of immune effector cells was at the same level.

[0156] Take the UTD T cells prepared in Example 1 (control group, i.e., untreated T cells, no empty transfection), GPC3 (GC33)-CAR T cells, GPC3 (GC33)-CAR iNKT cells, chIL4-9R-GPC3 (GC33)-CAR T cells, chIL4-7R-GPC3 (GC33)-CAR T cells, chIL4-21R-GPC3 (GC33)-CAR T cells and chIL4-9R-GPC3 (GC33)-CAR iNKT cells in this example, use IL-4-HepG2-GL as target cells, and kill them with an effector-target ratio of 1:2. After 24 hours, luciferase substrate was added and the killing efficiency was detected by microplate reader. The killing efficiency was calculated as follows: killing rate = (1-luminescence value of experimental group / tumor background luminescence value) × 100%.

[0157] For the results, please refer to Figure 5 , which shows the results of the test of the killing efficiency of immune effector cells against IL-4-secreting tumor cells in Example 5 of the present invention. The data in Figure 5 were statistically analyzed using one-way ANOVA with multiple comparisons. Statistical differences are indicated as follows: **P<0.01 vs. UTD T group; #P<0.05, ##P<0.01 vs. GPC3(GC33)-CAR T group; ΔΔP<0.01 vs. GPC3(GC33)-CAR iNKT group; @@P<0.01 vs. chIL4-9R-GPC3(GC33)-CAR T group. Data are presented as mean ± SEM.

[0158] The results showed that the killing effects of chIL4-9R-GPC3(GC33)-CAR T cells and chIL4-9R-GPC3(GC33)-CAR iNKT cells were stronger than those of chIL4-7R-GPC3(GC33)-CAR T cells and chIL4-21R-GPC3(GC33)-CAR T cells, indicating that compared with IL4-7R and IL4-21R, the expression of IL4-9R increased the killing effect of GPC3(GC33)-CAR T cells on tumor cells more significantly. It should be noted that the expression of IL4-9R significantly enhanced the killing effect of GPC3(GC33)-CAR iNKT cells on tumor cells than the expression of IL4-9R on GPC3(GC33)-CAR T cells, indicating that IL4-9R can significantly enhance the killing effect of iNKT cells on tumor cells.

[0159] In the above embodiments, CAR T cells or CAR iNKT cells targeting GPC3 are exemplarily used. Those skilled in the art can use CAR-T cells or CAR iNKT cells targeting other targets according to the teachings of this application, such as CAR-T cells or CAR iNKT cells targeting EGFR, CAR-T cells or CAR iNKT cells targeting CLD18A2, CART cells or CAR iNKT cells targeting CD19, and CAR-T cells or CAR iNKT cells targeting BCMA.

[0160] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises a cytokine binding extracellular domain and a signal transduction intracellular domain; The cytokine binding extracellular domain is selected from the group consisting of IL-10 receptor extracellular domain, IL-4 receptor extracellular domain, IL-13 receptor extracellular domain, IL-6 receptor extracellular domain, or variants of any of the above receptor extracellular domains; The signal transduction intracellular domain is an IL-9 receptor intracellular domain or a variant thereof.

2. The fusion protein according to claim 1, characterized in that The fusion protein further comprises a first transmembrane domain, and the cytokine binding extracellular domain, the first transmembrane domain and the signal transduction intracellular domain are connected in sequence directly or through a linker; The cytokine binding extracellular domain is used for binding to IL-10, IL-4, IL-13 and / or IL-6, and the signal transduction intracellular domain is used for IL-9 receptor intracellular signal transduction.

3. The fusion protein according to claim 1 or 2, characterized in that The cytokine binding extracellular domain is an IL-4 receptor extracellular domain or a variant thereof.

4. The fusion protein according to claim 3, characterized in that The nucleotide sequence encoding the extracellular domain of the IL-4 receptor is the sequence shown in SEQ ID NO: 2, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO:

2.

5. The fusion protein according to claim 3, characterized in that The nucleotide sequence encoding the intracellular domain of the IL-9 receptor is the sequence shown in SEQ ID NO: 4, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO:

4.

6. The fusion protein according to claim 3, characterized in that The first transmembrane domain is the transmembrane domain of the IL-4 receptor or the transmembrane domain of the IL-9 receptor.

7. The fusion protein according to claim 6, characterized in that The nucleotide sequence encoding the first transmembrane domain is the sequence shown in SEQ ID NO: 3 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO:

3.

8. The fusion protein according to claim 6, characterized in that The amino acid sequence of the fusion protein is the sequence shown in SEQ ID NO: 8, 19, 20 or 21, or the amino acid sequence of the fusion protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 8, 19, 20 or 21.

9. The fusion protein according to claim 1, characterized in that The fusion protein also comprises a signal peptide.

10. The fusion protein according to claim 9, characterized in that The nucleotide sequence encoding the signal peptide is the sequence shown in SEQ ID NO: 1 or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO:

1.

11. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 10.

12. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 11.

13. A host cell, characterized in that The host cell comprises the vector of claim 12.

14. An immune effector cell, characterized in that The immune effector cell expresses the fusion protein of any one of claims 1 to 10, or comprises the nucleic acid molecule of claim 11, or comprises the vector of claim 12.

15. The immune effector cell according to claim 14, characterized in that The immune effector cells are selected from one or more of T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells and / or stem cell-derived immune effector cells.

16. The immune effector cell according to claim 14, characterized in that The immune effector cells are autologous and / or allogeneic cells relative to the individual.

17. The immune effector cell according to claim 14, characterized in that The expression of the fusion protein is constitutive expression or inducible expression.

18. The immune effector cell according to claim 14, characterized in that The immune effector cells also express an exogenous receptor comprising a targeting extracellular domain that specifically binds to a tumor antigen, a second transmembrane domain, and a targeting intracellular domain.

19. The immune effector cell according to claim 18, characterized in that The exogenous receptor is selected from one or more of a chimeric antigen receptor, a modified T cell antigen receptor, a T cell fusion protein and a T cell antigen coupler.

20. The immune effector cell according to claim 19, characterized in that The chimeric antigen receptor is selected from one or more of the following: (i) the targeting extracellular domain is selected from an antibody or a fragment thereof that specifically binds to a tumor antigen, the second transmembrane domain is selected from CD28 or the transmembrane domain of CD8, and the targeting intracellular domain is selected from the costimulatory signaling domain of CD28 and the CD3ζ chain; (ii) the targeting extracellular domain is selected from an antibody or a fragment thereof that specifically binds to a tumor antigen, the second transmembrane domain is selected from the transmembrane domain of CD28 or CD8, and the targeting intracellular domain is selected from the costimulatory signaling domain of CD137 and the CD3ζ chain; (iii) the targeting extracellular domain is selected from an antibody or a fragment thereof that specifically binds to a tumor antigen, the second transmembrane domain is selected from the transmembrane domain of CD28 or CD8, and the targeting intracellular domain is selected from the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137 and the CD3ζ chain.

21. The immune effector cell according to claim 18, characterized in that The tumor antigen is selected from one or more of the following: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostaglandinase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART 1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1.

22. The immune effector cell according to claim 18, characterized in that The tumor antigen is a solid tumor antigen.

23. The immune effector cell according to claim 18, characterized in that The nucleotide sequence encoding the targeted extracellular domain is the sequence shown in SEQ ID NO: 22 or SEQ ID NO: 29, or the nucleotide sequence encoding the targeted extracellular domain is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 22 or SEQ ID NO: 29; The amino acid sequence targeting the extracellular domain is the sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33, or the amino acid sequence targeting the extracellular domain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO: 32 or SEQ ID NO:

33.

24. The immune effector cell according to claim 20, characterized in that The chimeric antigen receptor encoding nucleotide sequence is the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 30, or the chimeric antigen receptor encoding nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 30; The amino acid sequence of the chimeric antigen receptor is the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 34, or the amino acid sequence of the chimeric antigen receptor is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 7 or SEQ ID NO:

34.

25. The immune effector cell according to claim 20, characterized in that The chimeric antigen receptor and the fusion protein are encoded by the nucleotide sequence shown in SEQ ID NO: 9 or SEQ ID NO: 31, or by a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 9 or SEQ ID NO:

31.

26. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises one or more of the fusion protein of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the vector of claim 12, the host cell of claim 13, and the immune effector cell of any one of claims 14 to 25.

27. Use of the fusion protein according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the host cell according to claim 13 and / or the immune effector cell according to any one of claims 14 to 25 in the preparation of an immunotherapy drug.

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