Il15 / il15rα complex and use thereof in field of cell therapy

Abstract

The present invention relates to an IL15 / IL15Rα complex, a nucleic acid molecule, vector, cell, cell product and pharmaceutical composition based on the IL15 / IL15Rα complex, the use thereof for treating diseases such as tumors and autoimmune and infectious diseases, etc.

Description

IL15 / IL15R alpha complex and its use in the field of cell therapy

[0001] CROSS-REFERENCE

[0002] The present invention claims priority to the prior application filed with the China National Intellectual Property Office on August 16, 2024, with the patent application number 2024111308096, and the invention name of "IL15 / IL15R alpha complex and its use in the field of cell therapy". The prior application is incorporated by reference in its entirety into the present invention. TECHNICAL FIELD

[0003] The present invention relates to an IL15 / IL15R alpha complex, cell preparations based on the IL15 / IL15R alpha complex and their use for treating tumors, autoimmune and infectious diseases, etc. BACKGROUND

[0004] Gamma delta T cells are a special type of immune cells with adaptive and innate immune response characteristics. Gamma delta T cells express both the TCR type of gamma chain and delta chain and NKG2D (one of the main functional receptors expressed on NK cells), thus enabling gamma delta T cells to mimic the functions of T cells and NK cells. Compared with traditional alpha beta T cells, which carry TCR of alpha chain and beta chain and recognize antigen-derived peptides presented by MHC molecules (called human leukocyte antigen [HLA] in humans), gamma delta T cells can recognize and kill pathogens independently of MHC (unrestricted). At the same time, gamma delta T cells release various types of cytokines to activate other immune cells such as NKs, macrophages and CD8 + cytotoxic lymphocytes. Gamma delta T cells also exhibit antigen-presenting ability, and in particular, Vγ9Vδ2 T cells (the main gamma delta T cell subpopulation in peripheral blood) can respond to microorganisms, tumors and CD4 + and CD8 + T cell cluster differentiation. Many studies have shown that Vγ9Vδ2 T cells have a wide range of tumor killing effects and are not MHC restricted, and can be used for allogeneic transfer without worrying about GvHD. In the past few decades, many researchers have begun to explore the clinical application of gamma delta T cells in tumor treatment, and the safety and effectiveness of autologous or allogeneic gamma delta T cell therapy have been preliminarily proven. Therefore, gamma delta T is a strong candidate for tumor cell immunotherapy. Although more and more gamma delta T cells are being developed, everyone will face a common problem of the persistence of the cells themselves. Compared with traditional CAR-T, the persistence of adoptive therapy gamma delta T in vivo is poor, which may be a very important factor affecting the treatment effect, therefore, prolonging the persistence of gamma delta T cell products is a key point to improve the clinical effect.

[0005] Interleukin 15 (IL15) is a cytokine discovered in 1994, which is a 14-15KD glycoprotein consisting of 114 amino acids, and is a member of the four-helix bundle cytokine family. IL15 is a cytokine from the interleukin 2 (IL2) family, and the cytokines in the same family also include IL2, IL4, IL7, IL9 and IL21. IL15 binds to the IL15Rα subunit (also known as CD215) to form a dimer with high affinity, and is presented to nearby effector immune cells (such as NK cells or T cells) to function. The IL15Rα subunit is a membrane protein mainly expressed by some activated monocytes, DCs and stromal cells. The IL15Rα subunit structure contains an extracellular region, a transmembrane region and a short intracellular region. The extracellular region from the N-terminal is a signal peptide (amino acids 1-30), a sushi domain (amino acids 31-95, which retains 90% of the binding IL15 activity) and a proline / threonine-rich disordered sequence (amino acids 96-205). When IL15 binds to IL15Rα, it further forms a heterotrimer with IL2 / 15Rβ-γc, which activates NK or CD8 T cells through the following three pathways: 1) JAK1 interacts with IL2 / 15Rβ, JAK3 interacts with γc, STAT proteins (mainly STAT5A and STAT5B) are recruited to IL2 / 15Rβγ, and after phosphorylation, form a heterodimer, and then transported to the nucleus for transcriptional activation; 2) Shc is recruited to the phosphorylation site on the IL2 / 15Rβ chain, and then activates Grb2, which can continue to phosphorylate Akt along the PI3K pathway; 3) binding to guanine nucleotide exchange factor SOS activates RAS-RAF, and finally activates MAPK. These pathways lead to the subsequent expression of Bcl2, Myc, Fos / Jun and NFKB activation. Similar to IL2, IL15 can regulate the adaptive and innate immune systems, unlike IL2, IL15 does not activate Treg cells and does not cause AICD phenomenon, while IL15 can expand and activate natural killer (NK) cells, natural killer T (NKT) cells and memory (m) CD8 T cells. Compared with IL2, almost no capillary leakage is observed. In view of the unique advantages of IL15, it has been widely used in cancer immunotherapy in recent years.

[0006] However, due to the short half-life of IL15 in vivo and the low monomer bioactivity, its application is greatly limited. Several different types of IL15 agonists have been developed to increase the half-life of IL15 in vivo. The first is IL15 N72D:IL15Rαsu / Fc, also known as N-803 (formerly ALT-803), which is a mutated IL15 (N72D) protein non-covalently linked to the sushi domain of IL15Rα, which is in turn fused to human immunoglobulin IgG1 Fc. The half-life of N-803 in serum is significantly longer than that of IL15 monomer (25h vs 0.64h), and in addition, at equimolar concentrations, N-803 is more potent than IL15 monomer in inducing NK cell proliferation in vivo. Increasing studies have shown that fusion proteins of IL15 and partial or complete soluble IL15Rα (IL15 super agonists, IL15SA) can enhance the half-life of IL15 in vivo, thereby improving the cytotoxicity of effector cells.

[0007] IL15 can improve the function of cell drugs in a co-secreted form. Generally, IL15 monomers have a short half-life and low bioactivity, which limits their application. Although the complex of traditional secreted IL15 and IL15Rα can prolong the half-life to some extent, it can cause systemic toxicity, thus limiting the application dose and efficacy to some extent. In order to reduce systemic toxicity, relatively increase local concentration, and specifically enhance effector cell expansion and toxicity, and improve efficacy, some studies rivet exogenous expressed IL15 or IL15 / IL15Rα complex on the cell membrane to make it function as a membrane rivet, for example, the existing document 1 mentions that the IL15 / IL15Rα complex rivet is connected to the back of the CD19 CAR structure, the IL15 / IL15Rα complex structure is that the full-length wild-type IL15 is covalently connected to the full-length IL15Rα through a connecting peptide, and the IL15 / IL15Rα complex rivet enhances the anti-tumor effect of CD19 CAR-T cells. Furthermore, the existing document 2 discloses a chimeric antigen receptor Mesothelin scFv-CD8H&TM-CD28-41BB-CD3ζ-mbIL15 and its use, the mbIL15 structure is also that the full-length wild-type IL15 is covalently connected to the full-length IL15Rα through a connecting peptide, and this complex is connected to the back of the Meso CAR structure, the Meso-mbIL15 CAR-T cells prepared by the invention have strong killing function to specific tumor cells and have good proliferation capacity. Current studies show that the IL15 / IL15Rα complex rivet is mostly the C-terminal of IL15 covalently connected to the N-terminal of IL15Rα through a connecting peptide, and riveted on the cell membrane through the transmembrane region of IL15Rα itself.

[0008] In addition, free IL-15Rα (sIL-15Rα) is detected in human serum, which is attributed to its proteolysis. Studies have shown [Prior Art 3] that the key cleavage site of this hydrolysis process is serine at position 181 (S181), and the deletion of this site can effectively inhibit the hydrolysis of IL-15Rα.

[0009] Prior Art 1: PNAS article in 2016 (https: / / doi.org / 10.1073 / pnas.1610544113)

[0010] Prior Art 2: Patent Document CN 108728458 B

[0011] Prior Art 3: doi:10.1073 / pnas.1405514111 SUMMARY

[0012] In order to further solve the various problems existing in the prior art described above, especially to solve the technical problem of how to prolong the persistence of γδ T cell products, the inventors of the present application have conducted in-depth research and found a new IL15 / IL15Rα complex. It is confirmed that the IL15 / IL15Rα complex significantly promotes the proliferation of γδ T cells in the absence of cytokines and the killing of target cells, and significantly prolongs the persistence of γδ T cells in mice and enhances their anti-tumor effect.

[0013] In addition, the inventors of the present application have creatively constructed an IL-15 / IL-15Rα complex with anti-hydrolysis properties by deleting the hydrolysis site of IL-15Rα. This design significantly reduces the shedding of the complex due to IL-15Rα hydrolysis, increases its expression level on the cell membrane, and further enhances downstream signal transduction and cell proliferation. Functional verification shows that this anti-hydrolysis complex can strongly drive γδ T cell proliferation and target cell killing in the absence of cytokines; in mice, it can effectively prolong the survival time of γδ T cells and enhance the anti-tumor effect.

[0014] The present application is based on the above-mentioned IL15 / IL15Rα complex, and takes the chimeric antigen receptor targeting B7H3 as an example to prepare B7H3 CAR-IL15 / IL15Rα-γδT cells. The structure is composed of the heavy chain and light chain variable regions of the anti-human B7H3 monoclonal antibody (Anti-B7H3 scFv), the human CD8α hinge region, the human CD8 transmembrane region, and the human CD28 intracellular region, and the human CD3ζ intracellular region and IL15 / IL15Rα or IL15(N72D) / IL15Rα structure in series. The B7H3 CAR-IL15 / IL15Rα-γδT cells prepared by the present application have better proliferation capacity under factor-free conditions and stronger killing function on antigen-specific tumor cells under factor-free conditions.

[0015] The first aspect of the present application provides an IL15 / IL15Rα complex in a membrane-bound form, which structure comprises IL15 and IL15Rα, wherein the IL15 comprises IL15 wild type and IL15 mutant, and the IL15Rα comprises, from N-terminal to C-terminal, (1) IL15Rα sushi domain (65 aa), (2) the region between IL15Rα sushi domain and IL15Rα transmembrane region, (3) IL15Rα transmembrane region, and (4) IL15Rα intracellular region, wherein (1) IL15Rα sushi domain (65 aa) and (2) the region between IL15Rα sushi domain and IL15Rα transmembrane region are extracellular regions, and the region between IL15Rα sushi domain and IL15Rα transmembrane region is the amino acid sequence naturally existing between IL15Rα sushi domain and IL15Rα transmembrane region, or a combined fragment of the amino acid sequence naturally existing between IL15Rα sushi domain and IL15Rα transmembrane region and other natural or engineered amino acid fragments.

[0016] The second aspect of the present application provides the IL15 / IL15Rα complex as described in the above-mentioned first aspect, wherein the region between IL15Rα sushi domain and IL15Rα transmembrane region is the full-length amino acid sequence of the amino acid sequence naturally existing between IL15Rα sushi domain and IL15Rα transmembrane region, or a truncated fragment or a combination of truncated fragments of the full-length amino acid sequence.

[0017] The third aspect of the present application provides the IL15 / IL15Rα complex as described in the above-mentioned first or second aspect, wherein the IL15 / IL15Rα complex is IL15 / IL15Rα (truncated type).

[0018] The fourth aspect of the present application provides the IL15 / IL15Ra complex as described in any one of the first to third aspects, wherein the amino acid sequence of the extracellular region of IL15Ra is selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 27, the amino acid sequence represented by SEQ ID NO: 28, the amino acid sequence represented by SEQ ID NO: 29, the amino acid sequence represented by SEQ ID NO: 30, the amino acid sequence represented by SEQ ID NO: 31, the amino acid sequence represented by SEQ ID NO: 25, the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence represented by SEQ ID NO: 33, the amino acid sequence represented by SEQ ID NO: 34, and the amino acid sequence represented by SEQ ID NO: 35, and combinations thereof.

[0019] The fifth aspect of the present application provides the IL15 / IL15Ra complex as described in any one of the first to fourth aspects, wherein the amino acid sequence of the extracellular region of IL15Ra is selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 28, the amino acid sequence represented by SEQ ID NO: 31, the amino acid sequence represented by SEQ ID NO: 32, and the amino acid sequence represented by SEQ ID NO: 34.

[0020] The sixth aspect of the present application provides the IL15 / IL15Ra complex as described in any one of the first to fifth aspects, wherein the DNA sequence of the wild-type IL15 is represented by SEQ ID NO: 5, the amino acid sequence of the wild-type IL15 is represented by SEQ ID NO: 23, the DNA sequence of the mutant IL15 is represented by SEQ ID NO: 8, and the amino acid sequence of the mutant IL15 is represented by SEQ ID NO: 24.

[0021] The seventh aspect of the present application provides the IL15 / IL15Ra complex as described in any one of the first to sixth aspects, wherein the amino acid sequence of the IL15 / IL15Ra complex is represented by SEQ ID NO: 36 or SEQ ID NO: 37.

[0022] The eighth aspect of the present application provides the IL15 / IL15Ra complex as described in any one of the first to eleventh aspects, wherein the other natural or engineered amino acid fragment is an amino acid fragment for linking two polypeptide fragments, the other natural or engineered amino acid fragment is a GGGGS flexible linker, or the other natural or engineered amino acid fragment is a hinge region of IgG.

[0023] The ninth aspect of the present application provides the IL15 / IL15Ra complex as described in the first or second aspect, wherein the hydrolysis site of IL15Ra is deleted in the amino acid sequence of IL15Ra.

[0024] The tenth aspect of the present application provides the IL15 / IL15Ra complex according to the ninth aspect described above, wherein the serine at position 181 in the amino acid sequence of IL15Ra is deleted.

[0025] The eleventh aspect of the present application provides the IL15 / IL15Ra complex according to the tenth aspect described above, wherein the amino acid sequence is shown in SEQ ID NO: 49.

[0026] The twelfth aspect of the present application provides a nucleic acid molecule encoding the IL15 / IL15Ra complex according to the first to eleventh aspects described above.

[0027] The thirteenth aspect of the present application provides a vector comprising the nucleic acid molecule according to the twelfth aspect described above.

[0028] The fourteenth aspect of the present application provides the vector according to the thirteenth aspect described above, which is a lentivirus vector, a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector.

[0029] The fifteenth aspect of the present application provides a cell comprising the IL15 / IL15Ra complex according to any one of the first to eleventh aspects described above, the nucleic acid molecule according to the twelfth aspect, or the vector according to the thirteenth or fourteenth aspect.

[0030] The sixteenth aspect of the present application provides the cell according to the eleventh aspect described above, wherein the cell comprises autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, red blood cells, and the T cells comprise αβ T cells, γδ T cells, regulatory T cells.

[0031] The seventeenth aspect of the present application provides the cell according to the sixteenth aspect described above, wherein the γδ T cells comprise δ1, δ2, δ3, δ4, and δ5 T cells.

[0032] The eighteenth aspect of the present application provides a cell product comprising the IL15 / IL15Ra complex according to any one of the first to eleventh aspects described above, the nucleic acid molecule according to the twelfth aspect, the vector according to the thirteenth or fourteenth aspect, or the cell according to any one of the fifteenth to seventeenth aspects.

[0033] The nineteenth aspect of the present application provides the cell product according to the eighteenth aspect described above, wherein the cell product is a CAR-γδ T cell product.

[0034] The twentieth aspect of the present application provides the cell product of the eighteenth or nineteenth aspect described above, wherein the cell product expresses a CAR comprising an antigen binding domain targeting B7H3, CD19.

[0035] The twenty-first aspect of the present application provides the cell product of the eighteenth or nineteenth aspect described above, wherein the CAR structure comprises an optional signal peptide, a polypeptide binding to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an immune active cell activation signal transduction region, wherein the immune active cell activation signal transduction region further comprises a costimulatory domain and a signal transduction domain.

[0036] The twenty-second aspect of the present application provides a pharmaceutical composition comprising: the IL15 / IL15Rα complex of any one of the first to eleventh aspects described above, the nucleic acid molecule of the twelfth aspect described above, the vector of the thirteenth or fourteenth aspect described above, the cell of any one of the fifteenth to seventeenth aspects described above, or the cell product of any one of the eighteenth to twenty-first aspects described above, and a pharmaceutically acceptable excipient as needed.

[0037] The twenty-third aspect of the present application provides the use of the IL15 / IL15Rα complex of any one of the first to eleventh aspects described above, the nucleic acid molecule of the twelfth aspect described above, the vector of the thirteenth or fourteenth aspect described above, the cell of any one of the fifteenth to seventeenth aspects described above, the cell product of any one of the eighteenth to twenty-first aspects described above, or the pharmaceutical composition of the twenty-second aspect described above, in the preparation of a medicament for preventing or treating a disease, including a malignant / benign hematological tumor, a malignant / benign solid tumor, an autoimmune disease, a bacterial infection, a fungal infection, a viral infection, a parasitic infection, a bone growth abnormality, an allogeneic transplantation, a transplant rejection, aging.

[0038] The twenty-fourth aspect of the present application provides the use according to the twenty-third aspect above, wherein the disease comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, renal tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome; systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, multiple sclerosis, Sjogren's syndrome, ankylosing spondylitis, autoimmune glomerulonephritis, autoimmune hepatitis, idiopathic leukopenia, psoriasis, vitiligo, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmunity, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis; epidemic cerebrospinal meningitis, bacterial dysentery, whooping cough, scarlet fever, acute bacterial conjunctivitis, typhoid fever, diphtheria, tuberculosis, plague, cholera; candidiasis, aspergillosis, cryptococcosis, histoplasmosis, mucormycosis, paracoccidioidomycosis, sporotrichosis, dermatophytosis; viral hepatitis, poliomyelitis, hand-foot-and-mouth disease, influenza, measles, rubella, infantile acute exanthema, varicella, smallpox, herpes simplex virus infection, foot-and-mouth disease, mumps, hemolytic-uremic syndrome, human papillomavirus infection, rabies, cytomegalovirus infection, acquired immunodeficiency syndrome, epidemic hemorrhagic fever, yellow fever, Rift Valley fever, Argentine hemorrhagic fever, Lassa fever, Marburg hemorrhagic fever, Ebola hemorrhagic fever, dengue fever and dengue hemorrhagic fever; amebiasis, helminthiasis, nematodiasis, malaria, leishmaniasis, ascariasis, enterobiasis, ancylostomiasis, trichuriasis, fascioliasis, paragonimiasis, schistosomiasis, filariasis; Alzheimer's disease.

[0039] The twenty-fifth aspect of the present application provides a method for treating or preventing a disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the cell preparation according to the eighteenth to twenty-first aspects above, or the pharmaceutical composition according to the twenty-second aspect above.

[0040] The twenty-sixth aspect of the present application provides the method of the twenty-fifth aspect above, wherein the disease comprises malignant / benign hematological tumors, malignant / benign solid tumors, autoimmune diseases, bacterial infections, fungal infections, viral infections, parasitic infections, bone growth abnormalities, allogeneic transplantation, transplant rejection, aging.

[0041] The twenty-seventh aspect of the present application provides the method of the twenty-sixth aspect, wherein the disease comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome; systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, multiple sclerosis, Sjogren's syndrome, ankylosing spondylitis, autoimmune glomerulonephritis, autoimmune hepatitis, idiopathic leukopenia, psoriasis, vitiligo, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmunity, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis; epidemic cerebrospinal meningitis, bacterial dysentery, whooping cough, scarlet fever, acute bacterial conjunctivitis, typhoid fever, diphtheria, tuberculosis, plague, cholera; candidiasis, aspergillosis, cryptococcosis, histoplasmosis, mucormycosis, paracoccidioidomycosis, sporotrichosis, dermatophytosis; viral hepatitis, poliomyelitis, hand-foot-mouth disease, influenza, measles, rubella, infantile acute exanthema, varicella, smallpox, herpes simplex virus infection, foot-and-mouth disease, mumps, hemolytic-uremic syndrome, human papillomavirus infection, rabies, cytomegalovirus infection, acquired immunodeficiency syndrome, epidemic hemorrhagic fever, yellow fever, Rift Valley fever, Argentine hemorrhagic fever, Lassa fever, Marburg hemorrhagic fever, Ebola hemorrhagic fever, dengue fever and dengue hemorrhagic fever; amebiasis, helminthiasis, nematodiasis, malaria, leishmaniasis, ascariasis, enterobiasis, ancylostomiasis, trichuriasis, liver fluke disease, lung fluke disease, blood fluke disease, filariasis; Alzheimer's disease. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1: Figure 1A is a structural schematic diagram of IL15 / IL15Ra complex; Figure IB is a structural schematic diagram of CAR expressing different forms of IL15 / IL15Ra complex; Figure 1C is a structural schematic diagram of B7H3 CAR.

[0043] Figure 2: shows the expansion and viability changes of B7H3 CAR-V62 T cells expressing different forms of IL15 / IL15Ra complex, wherein Figure 2A shows the number of cell expansion changes, Figure 2B shows the proliferation fold changes, and Figure 2C shows the viability changes.

[0044] Figure 3: shows the V62 T cell ratio and CAR expression positive rate after 7 days of culture, wherein Figure 3A is a flow chart, and Figure 3B is a columnar statistical chart.

[0045] Figure 4: shows the proliferation changes of B7H3 CAR-V62 T cells in a factor-free system after 9 days of culture, wherein Figure 4A shows the CAR + cell expansion fold changes, and Figure 4B shows the V62 T cell expansion fold changes.

[0046] Figure 5: shows the proliferation and viability changes of B7H3 CAR-V62 T cells in a factor-free system after 9 days of culture, wherein Figure 5A shows the number of V62 T cells, Figure 5B shows the V62 T cell proliferation fold changes, and Figure 5C shows the changes of V62 T cell viability.

[0047] Figure 6: shows the effects of CAR expressing different forms of IL15 / IL15Ra complex on the pSTAT5 level of V62 T cells, wherein Figure 6A and Figure 6B respectively show the pSTAT5 mean fluorescence intensity of V62 T cells and CAR + cells.

[0048] Figure 7: shows the results of flow cytometry detection of the B7H3 expression abundance of target cells THP-1 cells (Figure 7A) and SKOV3 cells (Figure 7B).

[0049] Figure 8: shows the effect of B7H3 CAR-V62 T cells on continuous killing of THP-1 in a factor-free system after 9 days of culture.

[0050] Figure 9: shows the IFN-γ secretion level of B7H3 CAR-V62 T cells when co-incubated with target cells for 18h.

[0051] Figure 10: shows the tumor killing effect of CAR-V62 T cells expressing different forms of IL15 / IL15Ra complex CAR structure in a THP-1 mouse model, wherein Figure 10A and 10B are tumor fluorescence charts, Figure 10C is a survival curve, and Figure 10D is a B7H3 CAR-V62 T content change chart.

[0052] Figure 11: shows the tumor killing effect of CAR-Vδ2T expressing different forms of IL15 / IL15Rα complex in SKOV3 mouse model, wherein Figure 11A and 11B are tumor fluorescence images, and Figure 11C is a graph showing the change of B7H3 CAR-Vδ2T content.

[0053] Figure 12: Figure 12A is a structural schematic diagram of IL15 / IL15Rα complex; and Figure 12B is a structural schematic diagram of CAR expressing different forms of IL15 / IL15Rα complex.

[0054] Figure 13: shows the expansion and viability change of B7H3 CAR-Vδ2T cells expressing different forms of IL15 / IL15Rα complex, wherein Figure 13A shows the change of cell expansion number, Figure 13B shows the change of proliferation fold, and Figure 13C shows the change of viability.

[0055] Figure 14: shows the proliferation change of B7H3 CAR-Vδ2T cells cultured for 9 days in factor-free condition, wherein Figure 14A shows the change of Vδ2T cell expansion fold, and Figure 14B shows the change of Vδ2T cell viability.

[0056] Figure 15: shows the effect of CAR expressing different forms of IL15 / IL15Rα complex on the pSTAT5 level of Vδ2T cells, wherein Figure 15A and Figure 15B respectively show the pSTAT5 mean fluorescence intensity of Vδ2T cells and CAR + cells.

[0057] Figure 16: shows the structural schematic diagram of B7H3 CAR expressing different forms of IL15 / IL15Rα complex.

[0058] Figure 17: shows the effect of B7H3 CAR expressing different forms of IL15 / IL15Rα complex on the pSTAT5 level of Vδ2T cells, wherein Figure 17A and 17B respectively show the pSTAT5 mean fluorescence intensity of CAR + cells and CAR - cells of donor 3 and donor 4.

[0059] Figure 18: shows the proliferation change of B7H3 CAR-Vδ2T cells cultured for 9 days in factor-free condition, wherein Figure 18A and Figure 18C are the proliferation fold of CAR + cells, and Figure 18B and Figure 18D are the viability change of Vδ2T cells.

[0060] Figure 19: shows the effect of B7H3 CAR-Vδ2 T cells cultured for 9 days in killing THP-1 continuously in factor-free condition, wherein Figure 19A and 19B are the statistical graphs of B7H3 CAR-Vδ2 T killing THP-1 tumor fluorescence residue of donor 3 and donor 4, respectively.

[0061] Figure 20: shows the expansion of Vδ2 T cells during the process of B7H3 CAR-Vδ2 T cells killing THP-1 continuously, wherein Figure 20A and 20B show the expansion of Vδ2 T cells during the process of B7H3 CAR-Vδ2 T cells killing THP-1 of donor 3 and donor 4, respectively.

[0062] Figure 21: shows the level of IFN-γ secretion of B7H3 CAR-Vδ2 T cells when co-incubated with target cells for 18h.

[0063] Figure 22: shows the level of IL15 secretion of B7H3 CAR-Vδ2 T cells supernatant.

[0064] Figure 23: shows the tumor killing effect of CAR-Vδ2 T expressing different forms of IL15 / IL15Rα complex B7H3 CAR structure in THP-1 mouse model, wherein Figure 23A is the fluorescence imaging of mouse tumor, and Figure 23B, Figure 23C, Figure 23D, Figure 23E are the statistical graphs of tumor fluorescence of Day 14, Day 23, Day 30, Day 36, respectively.

[0065] Figure 24: shows the sequence comparison between IL15Rα (73aa) with S181 amino acid deleted and wild type IL15Rα, wherein “*” represents the same amino acid compared with the sequence of wild type IL15Rα, and “-” represents the deleted amino acid compared with the sequence of wild type IL15Rα. DETAILED DESCRIPTION

[0066] The present application is further explained by the following description of specific embodiments with reference to the figures, which are by way of illustration, and without limitation, of the principles of the application. Various modifications and improvements can be made by those skilled in the art to the present application, without departing from the spirit and scope of the application, which are limited only by the scope of the claims.

[0067] It should be noted that the technical terms or scientific terms in the present specification should be understood as the general meanings understood by the skilled person in the art, unless otherwise defined. In addition, it should also be understood that the terms used in the present application are only for the purpose of describing the specific embodiments, and are not intended to be limiting.

[0068] The “γδ T cell” in the present specification refers to an immune cell expressing CD3 with TCR chain being γ chain and δ chain.

[0069] The "Vδ2 T cell" in the present specification refers to a γδ T cell with a δ chain of type 2.

[0070] The "membrane-bound form" in the present specification refers to a protein or protein complex riveted in the cell membrane by a transmembrane protein structure; preferably, the "IL15 / IL15Rα complex" in the present specification is riveted in the cell membrane by the transmembrane domain of IL15Rα.

[0071] The "IL15 / IL15Rα complex" in the present specification refers to a protein combination of IL15 protein and IL15Rα protein connected by an amino acid fragment, and the amino acid fragment used for connecting the protein combination is well known in the art, such as a flexible connecting fragment, a rigid connecting fragment or a cleavable connecting fragment; the present specification preferably uses a flexible connecting fragment, such as a connecting fragment comprising a glycine polymer (G)n or a glycine-serine polymer (GmS)n (wherein m and n are integers greater than 1, such as 2, 3, 4, 5, 6, 7 or 8), which can be exemplified as SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 53). In the detailed embodiments of the present specification, a natural amino acid fragment such as the hinge region of IgG can also be used. Among them, the IL15 protein includes its wild type and mutants, and the IL15Rα protein includes its full-length type and truncated type.

[0072] The "IL15" in the present specification refers to interleukin 15, which is a 114-amino-acid 14-15KD glycoprotein, including its wild type and mutants; the term "IL15 mutant" refers to a mutant molecule obtained by one or more amino acid substitutions, additions or deletions, which improves or reduces the affinity between IL15 and its receptor, or increases or reduces the activity of stimulating the proliferation of specific cell lines, T cells or NK cells, or the activity of releasing cytokines.

[0073] The "IL15(N72D)" in the present specification refers to IL15 with the 72nd amino acid mutated from Asn to Asp, and its amino acid sequence is shown in SEQ ID NO: 24.

[0074] IL15Rα" in the present application refers to a membrane protein, which is mainly expressed by some activated monocytes, DCs and stromal cells, and its structure comprises an extracellular region, a transmembrane region and a short intracellular region. The extracellular region from the N-terminal is a sushi domain (amino acids 1-65, which retains 90% of the binding activity of IL15) and a proline / threonine-rich disordered sequence (amino acids 66-175), respectively. The full-length amino acid sequence of the proline / threonine-rich disordered sequence is the "full-length amino acid sequence of the amino acid sequence naturally existing between the sushi domain of IL15Rα and the transmembrane region of IL15Rα" in the present specification.

[0075] "IL15Rα (truncated)" in the present specification refers to a combination fragment of the sushi domain and a truncated proline / threonine-rich disordered sequence fragment, or a combination fragment of the sushi domain and other natural or engineered amino acid fragments in the extracellular region, with the transmembrane region and intracellular region unchanged. The "truncated proline / threonine-rich disordered sequence fragment" refers to a truncated fragment or a combination of truncated fragments of the full-length amino acid sequence of the proline / threonine-rich disordered sequence (amino acids 66-175); the above-mentioned "other natural or engineered amino acid fragments" and "amino acid fragments for protein combination connection" have the same meaning, including naturally occurring amino acid fragments of proteins or artificially designed and synthesized amino acid fragments, which can connect two polypeptide fragments together, such as the hinge region of IgG, GGGGSGGGGSGGGGS (i.e. (G4S)3, SEQ ID NO: 55).

[0076] As an example of "IL15Rα (truncated)", IL15Rα (73aa) means a truncated IL15Rα whose extracellular region is truncated at the sushi domain (amino acids 1-65, 65 amino acids in total) and the first 8 amino acids (amino acids 66-73, 8 amino acids in total) of the proline / threonine-rich disordered sequence of IL15Rα, and whose transmembrane region and intracellular region are unchanged; IL15Rα (66aa) means a truncated IL15Rα whose extracellular region is truncated at the sushi domain (amino acids 1-65, 65 amino acids in total) and the first 1 amino acid (amino acid 66) of the proline / threonine-rich disordered sequence of IL15Rα, and whose transmembrane region and intracellular region are unchanged; IL15Rα (DeEXON3), IL15Rα (DeEXON4), and IL15Rα (DeEXON5) mean IL15Rα whose extracellular region is deleted at the third or fourth or fifth exon sequence (the three exons encode positions in the proline / threonine-rich disordered sequence) of the IL15Rα gene, and whose transmembrane region and intracellular region are unchanged; IL15Rα (sushi + (G4S)3) means IL15Rα whose sushi domain is connected to the transmembrane region via a (G4S)3 linker, and whose transmembrane region and intracellular region are unchanged. IL15Rα (73aa) is preferred. Note that IL15Rα (65aa) is not "IL15Rα (truncated)" in the present application, and in "IL15Rα (truncated)" in the present specification, the truncated proline / threonine-rich disordered sequence contains 1-109 amino acid residues, and preferably, for example, 5-40 amino acid residues.

[0077] "IL15 / IL15Rα (truncated)" in the present specification means a protein complex of IL15 and IL15Rα (truncated), and "IL15(N72D) / IL15Rα (truncated)" means a protein complex of IL15(N72D) and IL15Rα (truncated).

[0078] "IL15 / IL15Rα (full-length)" in the present specification means a protein complex of IL15 and IL15Rα (full-length), and "IL15(N72D) / IL15Rα (full-length)" means a protein complex of IL15(N72D) and IL15Rα (full-length).

[0079] The "other natural or engineered amino acid fragment" in the present specification has the same meaning as the "amino acid fragment for protein combination connection" described above, and refers to an amino acid fragment of a naturally occurring protein or an artificially designed and synthesized amino acid fragment, which is an amino acid fragment capable of connecting two polypeptide fragments, such as polypeptide fragment A and polypeptide fragment B, together, and can be exemplified by a GGGGS flexible linker, a hinge region of IgG, and more specifically can be exemplified by (G4S)3, (G4S)4, (G4S)5.

[0080] The "EXON3" in the present specification refers to a third exon sequence encoding IL15Rα, and the corresponding encoded amino acid sequence is: DPALVHQRPAPPSTVTTAGVTPQPESLSPSGK (SEQ ID NO: 56), the "EXON4" refers to a fourth exon sequence encoding IL15Rα, and the corresponding encoded amino acid sequence is: PAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPP (SEQ ID NO: 57), and the "EXON5" refers to a fifth exon sequence encoding IL15Rα, and the corresponding encoded amino acid sequence is: GVYPQGHSDTT (SEQ ID NO: 58).

[0081] The "hinge region of IgG" in the present specification refers to the hinge region of IgG protein, and IgG includes IgG1, IgG2, IgG3, and IgG4.

[0082] The "wild type" in the present specification refers to an individual or a gene directly obtained from nature, which is not artificially mutagenized, and which carries the most common genome in nature.

[0083] The "mutant" in the present specification refers to an individual or a gene in which a base pair is added, deleted or changed, thereby causing a change in the structure of the gene.

[0084] The "nucleic acid molecule" in the present specification refers to a biomolecular compound polymerized from deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Its constituent unit is a nucleotide, and the nucleotide monomer is composed of a five-carbon sugar, a phosphate group and a nitrogen-containing base.

[0085] The "coding" in the present specification, when applied to a nucleic acid sequence, refers to a polynucleotide "coding" a polypeptide, which, in its natural state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or its fragments.

[0086] The "vector" in the present specification refers to a recombinant vector which retains the ability to infect non-dividing and / or slowly dividing cells and to integrate into the genome of target cells. In some preferred embodiments, the vector is derived from or based on a wild-type virus. In some further preferred embodiments, the vector is derived from or based on a wild-type lentivirus. As examples thereof, one can cite, for example, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, and the like. More particularly, in a preferred embodiment of the present application, the vector is a lentiviral vector.

[0087] The cells in the present application can be exemplified by T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NKT cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells and cells derived therefrom, red blood cells, among which the T cells can be exemplified by αβ T cells, γδ T cells, regulatory T cells. In a preferred embodiment of the present application, γδ T cells are used.

[0088] The "cell preparation" in the present specification refers to a preparation made from living cells as starting raw materials, using biological techniques, for use in the treatment of diseases.

[0089] The "CAR" in the present specification refers to "chimeric antigen receptor", which is an artificially modified receptor that can anchor a specific molecule (such as an antibody) that recognizes a tumor cell surface antigen on an immune cell (such as a T cell), allowing the immune cell to recognize a tumor antigen or a viral antigen and kill tumor cells or virus-infected cells. The CAR usually comprises, in order, an optional signal peptide, a polypeptide (such as a single-chain antibody) that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an immune-active cell activation signal transduction region, wherein the immune-active cell activation signal transduction region further comprises a costimulatory domain and a signal transduction domain.

[0090] Among the above "CAR" structure, the "hinge region" refers to a hydrophilic region between the antigen recognition domain and the transmembrane domain. The hinge region can use the hinge region of various different antibodies or antigen receptors, in particular the hinge region of CD molecules. In a specific embodiment, the hinge region can be selected from, for example, CD4, CD8α, CD28, IgG1, IgG4. In a preferred embodiment of the present application, the CD8α hinge region is used.

[0091] The "transmembrane region" in the above "CAR" structure can be any peptide that can cross the cell membrane. The transmembrane region used is preferably that of a CD molecule, and can be selected from, for example, the transmembrane region of CD4, CD8, CD28, CD3 zeta, the alpha, beta chain of T cell receptor, CD3 zeta, CD3 epsilon, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor), NKG2D, or GITR. In a preferred embodiment of the present application, the CD8 alpha transmembrane region is used.

[0092] The "co-stimulatory domain" refers to a portion of the CAR that enhances the proliferation, survival, and / or development of memory cells. The CAR of the present application can comprise one or more co-stimulatory domains. Each co-stimulatory domain comprises, for example, the co-stimulatory domain of any one or more of CD28, 4-1BB, ICOS, OX40, CD27, CD40, Myd88, HVEM, GITR, Fc Receptor-associated gamma chain, and the like. In a preferred embodiment of the present application, the CD28 co-stimulatory domain is used.

[0093] The "signaling domain" refers to a portion of the CAR that transduces an effector function signal and directs the cell to perform its specialized function. Examples of domains that transduce an effector function signal include, but are not limited to, CD3 zeta, Fc epsilon RI gamma. In a preferred embodiment of the present application, the CD3 zeta domain is used.

[0094] The "pharmaceutically acceptable excipient" in the pharmaceutical composition can be any of the conventionally used pharmaceutically acceptable excipients, and the person skilled in the art can select a suitable pharmaceutically acceptable excipient according to the chemical-physical conditions and conditions such as the administration route. Examples include carriers, adjuvants, excipients, and diluents. The pharmaceutically acceptable excipient is preferably a carrier that is chemically inert to the active agent and an excipient that has no harmful side effects or toxicity under the conditions of use.

[0095] "Treat" or "treatment" in the present specification means a manner by which a disease is removed after the disease occurs by some means or a manner by which the disease is avoided from occurring by some means. "Treat" and "treatment" in the present specification do not necessarily mean 100% or complete treatment or prevention. Rather, there are different degrees of treatment or prevention, which a person skilled in the art acknowledges as having potential beneficial or therapeutic effects. In addition, the treatment or prevention provided by the method of the present application can include treatment or prevention of one or more conditions or symptoms of the disease (e.g., cancer) being treated or prevented. Also, "prevention" can include delaying the onset of the disease or its symptoms or conditions when used for the purposes of the present application.

[0096] In the present specification, any domain / region of the CAR of the present application can be connected together using a "flexible linker peptide chain". The "flexible linker peptide chain" refers to an oligo- or polypeptide region of about 1 to 100 amino acids in length, which can be composed of flexible residues such as glycine and serine, so as to allow adjacent protein domains to move freely relative to each other, and a longer linker can be used when it is desired to ensure that two adjacent domains do not interfere with each other in space. In a preferred embodiment of the present application, (GGGGS)3, i.e., (G4S)3 is used.

[0097] "Expression positive rate" in the present specification refers to the proportion of detection of expression of an exogenous gene on T cells, for example, the expression positive rate of a CAR on T cells, which reflects the proportion of CAR-positive T cells to total T cells.

[0098] "Factor-free condition", "factor-free system", and "cell factor-free system" in the present specification mean that there is no cell factor in the experimental condition or experimental system.

[0099] "Hydrolysis site" or "proteolysis site" in the present specification both refer to a specific chemical bond / peptide bond on a protein chain that is easily recognized and cleaved by a protease, and the cleavage can cause a change in the structure of the protein (e.g., release of fragments). In a preferred embodiment, the hydrolysis site of IL-15Rα is the peptide bond region in which the 181st serine residue (S181) is located. The hydrolysis site of IL-15Rα in the present specification includes, but is not limited to, the peptide bond region in which the 181st serine residue (S181) is located.

[0100] Experimental materials and methods

[0101] Cell lines

[0102] The 293T cell line was cultured in Dulbecco's modified medium (DMEM) (Gibco), and the THP-1, SKOV3 cell lines were cultured in 1640 medium (Gibco), and 10% fetal bovine serum (FBS) (Gibco) was added to the above cells.

[0103] Plasmid construction

[0104] The IL15 and IL15Ra mature peptide sequence information of human was searched from the database of NCBI website, and a linker (SGGGSGGGGSGGGGSGGGGSGGGSLQ, SEQ ID NO: 53) was added in the middle of IL15 and IL15Ra. The signal peptide nucleotide sequence of the complex was ATGTATAGAATGCAGCTGCTGTCATGTATTGCCCTGTCTCTGGCCCTGGTGACTAATTCT (SEQ ID NO: 54). The IL15 / IL15Ra complex gene sequence was codon-optimized to ensure that the coding amino acid sequence was unchanged and more suitable for human T cell expression. Finally, the synthetic gene sequence was obtained.

[0105] The gene sequence information is as follows:

[0106] The synthetic gene sequence was constructed to the rear of the prepared B7H3 CAR molecule by homologous recombination, and the B7H3 CAR molecule and the IL15 / IL15Ra complex were connected by the P2A element. The vector used was pHR lentivirus vector. The recombinant product was transformed into competent E. coli (DH5α), and after overnight culture, single colonies were picked and subjected to colony PCR. The positive clones were preliminarily confirmed, and the plasmid was sequenced. The sequencing results were compared with the synthetic IL15 / IL15Ra complex sequence to verify whether the sequence was correct.

[0107] The sequencing primer was:

[0108] Sense sequence: ACGATGGCCTTTACCAGGGTCT (SEQ ID NO: 39)

[0109] Antisense sequence: CCAGAGGTTGATTATCGATAAGC (SEQ ID NO: 40)

[0110] After sequencing, the plasmid was extracted and purified using the Qiagen plasmid purification kit.

[0111] IL15 (N72D) / IL15Ra (truncated) construction

[0112] The above constructed CAR-IL15 / IL15Ra plasmid was used as a template, and the mutation site and truncated primer were designed to construct the target plasmid by homologous recombination.

[0113] Lentivirus preparation

[0114] This method uses VSV-G lentivirus vector. 1×10 7293T cells were plated in poly-d-lysine coated 100 mm dishes. The next day, 6 pg of pHR-EFl-B7H3 CAR plasmid (original backbone plasmid from Addgene, plasmid #72263), 4 pg of pspAx2 (Addgene, plasmid #12260), 2 pg of pCMV-VSV-G (Addgene, plasmid #8454) were transfected using 30 pg of PEI transfection reagent, respectively. Eight hours post transfection, the cell culture medium was changed. Supernatants were collected at 48 and 72 hours and used for lentix TM Concentrator (Takara) to concentrate the virus, the virus titer was monitored by transducing Jurkat cells, and the concentrated virus was stored at -80 °C for later use.

[0115] Primary cell culture

[0116] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation and washed twice with phosphate-buffered saline (PBS). Cell count and viability were determined by AO / PI staining. V52 T cell expansion: PBMCs were cultured in serum-free medium at a starting concentration of 2 x 10 6 cells / mL with the addition of 1000 U / mL of rhIL2 and 5 mM ZOL.

[0117] Lentivirus transduction of gd T

[0118] After 24 hours of in vitro culture in the above-mentioned gd T cell medium (serum-free medium with rhIL2 and ZOL), lentivirus was directly added to the cells to be transfected at the appropriate MOI, with the addition of BX795, mixed well, and incubated at 37 °C, 5% CO2.

[0119] Flow cytometry

[0120] The cells to be tested were washed once with FACS buffer, then stained with antibodies diluted in FACS buffer (PBS + 0.5% FBS + 2.5 mM EDTA) for 30 minutes at 4 °C. 2 x 10 5 The incubation buffer for 2 x 10

[0121] Flow detection of pSTAT5: Take 1 x 10 6 The cells to be tested were replaced with factor-free medium and starved for 24 hours for pSTAT5 staining. The positive control was stimulated with 50 ng / mL IL15 for 30 minutes. The staining steps were as follows: extracellular huFc-labeled B7H3 protein and TCR Vδ2 (Biolegend, 331428) TCR Vδ2 antibody were diluted according to the instructions, and the use concentration of B7H3 protein was 1 μg / mL. Staining was performed at 4°C for 30 minutes, and the cells were washed twice with FACS buffer, and then stained with AF647 anti-huFc secondary antibody. After the membrane surface antibody labeling was completed, 1 mL of Stain Buffer was added for washing once, and the supernatant was discarded; then cell fixation was performed, 250 μL of Fixation Buffer (BD, 554655) was added to resuspend the cells, and the cells were mixed gently before resuspension, and incubated at 4°C in the dark for 30 minutes; then cell membrane rupture was performed, 500 μL of Perm Buffer III (BD, 558050) was added to each tube, and incubated at ice for 30 minutes in the dark; then p-STAT5 staining was performed, the antibody was diluted according to the instructions, and a FMO control was set at the same time, and incubated at room temperature in the dark for 1 hour; finally, 200 μL of Stain Buffer (BD, 554656) was used to resuspend the stained cells, and the samples were collected on a flow cytometer, and the data were analyzed by FlowJo software.

[0122] Continuous killing experiment in vitro

[0123] THP-1 target cells were replaced with fresh 1640 complete medium, and the cell density was adjusted to 1 x 10 6 / mL. The B7H3 CAR-Vδ2 T cells cultured for 9 days were collected, the factor-free medium was replaced, and the cell density was adjusted to 1 x 10 6 / mL. According to the ratio of effector cells to target cells = 1:1, the culture was performed in a 24-well plate (NEST, 702001), and in each co-culture group, the effector cells were 500,000 (500 μL), and the target cells were 500,000 (500 μL). At the same time, an experimental group containing only target cells was set up. Then 50 μL of sample was taken every 24 hours to detect the Luciferase value, and the next round of killing experiment was set every 48 / 72 hours. For each group of co-cultured Vδ2 T cells and target cells, the cytotoxicity killing cells % = 100% - calibrated target cell survival %, that is

[0124] IFN-γ secretion detection

[0125] Take the continuous killing experiment effector cells and target cells co-incubation 18 hours of cell supernatant, carry out Elisa detection IFN-γ.

[0126] The specific experimental steps are as follows:

[0127] Coating antibody: Coating Buffer coating buffer: 10 times dilution with pure water, get 1x coating buffer dilution. Prepare 96 well plates according to the reaction number, add 100 μL of diluted coating antibody to each well, cover tightly with sealing film, 4℃ overnight, standby.

[0128] Washing: PBST: 10 times dilution with pure water, get 1x PBST dilution. Remove the coating antibody, add 250 μL of PBST to each well, directly remove, repeat the process, a total of 3 times.

[0129] Blocking: 1x Elisa: 5 times dilution of 5x Elisa / ELISPOT Diluent Elisa with pure water, get 1x Elisa dilution. Add 200 μL of 1x Elisa dilution to each well, incubate at room temperature for 1 hour.

[0130] Prepare standard: a total of 8 concentration gradients, from high concentration to dilution. Washing: remove the blocking liquid, add 250 μL of PBST to each well, directly remove, wash once.

[0131] Add sample: add standard, 100 μL per well, add sample, 100 μL per well, incubate at room temperature for 2 hours.

[0132] Washing: remove the standard and sample, add 250 μL of PBST to each well, directly remove, repeat the process, a total of 3 times.

[0133] Add antibody: Detection Antibody: 250 times dilution with 1x Elisa dilution, get 1x antibody dilution, add 100 μL of antibody to each well, incubate at room temperature for 1 hour.

[0134] Washing: remove the antibody, add 250 μL of PBST to each well, directly remove, repeat the process, a total of 3 times.

[0135] Add secondary antibody: Streptavidin-HRP: 100 times dilution with 1x Elisa dilution, get 1x enzyme substrate dilution, add 100 μL to each well, incubate at room temperature for 30 minutes.

[0136] Wash: Remove enzyme substrate, add 250 μL of PBST to each well, remove directly, repeat this process for a total of 5 times.

[0137] Add Substrate: Add 100 μL of TMB Reagent to each well, incubate at room temperature for 15 minutes in the dark.

[0138] Add Stop Solution: Prepare Stop Solution: 85% Phosphoric Acid (15 mol / L), dilute with pure water to 15 times to obtain 1 mol / L dilution, add 100 μL of Stop Solution Reagent to each well, immediately detect on the machine.

[0139] IL15 secretion detection

[0140] Take CAR-Vδ2 T cells cultured for 9 days, replace with factor-free culture medium, adjust the cell density to 1×10 6 / mL, take 1×10 6 Cell suspension was placed in a 24-well plate, cultured at 37°C, 5% CO2 for 48 hours, and the supernatant was collected for IL15 Elisa detection.

[0141] IL15 Elisa kit was purchased from Yikosai, item number EH057-48.

[0142] The specific experimental steps are as follows:

[0143] Reagent preparation:

[0144] Take the kit out of the refrigerator 20 minutes in advance to balance to room temperature;

[0145] Dilute 20x Wash Buffer Concentrate with deionized water to 1x wash working solution, and return the unused to the refrigerator;

[0146] Biotinylated antibody working solution: according to the amount required for the experiment, dilute Human IL-15100x Biotin-Antibody 100 times with Assay Diluent to prepare the biotinylated antibody working solution, prepare 30 minutes before use, and only for use on the same day;

[0147] Enzyme conjugate working solution: according to the amount required for the experiment, dilute 100x HRP-Streptavidin 100 times with Assay Diluent to prepare the enzyme conjugate working solution. Prepare 30 minutes before use, and only for use on the same day.

[0148] Operation steps

[0149] Take the required strips from the sealed bag that has been balanced to room temperature, and return the unused strips and desiccant to the aluminum foil bag and store in the 2-8°C refrigerator;

[0150] Blank hole;

[0151] Prepare sample, standard and biotinylated antibody working solution in advance;

[0152] Add sample to corresponding hole respectively, 100 μL / hole, then add 50 μL biotinylated antibody working solution to sample and standard hole, seal the reaction hole with sealing tape, incubate at room temperature (20-25℃) for 120 minutes, use micro-vibration device (frequency 300 rpm);

[0153] Prepare enzyme conjugate working solution in advance;

[0154] Spin off the liquid in the hole, wash the plate with 250 μL 1x washing working solution for 5 times;

[0155] Add enzyme conjugate working solution to the hole (except blank hole), 100 μL / hole, seal the reaction hole with sealing tape, incubate at room temperature (20-25℃) for 60 minutes in dark, use micro-vibration device (frequency 300 rpm);

[0156] Spin off the liquid in the hole, wash the plate with 250 μL 1x washing working solution for 5 times;

[0157] Add Substrate Solution (including blank hole) to the hole, 100 μL / hole, incubate at room temperature (20-25℃) for 15 minutes in dark;

[0158] Add Stop Solution (including blank hole) to the hole, 100 μL / hole, mix immediately and then measure OD450 value with enzyme label instrument, the final OD value is obtained by subtracting OD450 value of blank hole from OD450 value of each standard and sample.

[0159] CAR-γδT proliferation experiment

[0160] Collect CAR-Vδ2T cultured for 9 days, replace the medium with factor-free condition, and adjust the cell density to 1x10 6 / mL, take 1x10 6 Cell suspension is placed in 24-well plate and cultured at 37℃, 5% CO2. Subsequently, count and pass every 48 hours, i.e. continue to take 1x10 6 cells and supplement fresh factor-free culture medium.

[0161] Mouse model

[0162] THP-1 model: (Day 0) i.v. return THP-1 cells (resuspended in 200 μL PBS) through tail vein, 2x10 6Day 3, weigh the body weight, and randomly divide into 5 groups according to the data of live imaging and body weight, 5 mice in each group, and then CAR-Vδ2 T cells are transfused on the same day, 2×10 6 CAR + cells, and the control group is transfused with the same volume of PBS, and the tumor fluorescence is detected the next day, and then live imaging and body weight measurement are performed every 7-10 days until the mice die, and the tumor fluorescence and survival curve are calculated. The peripheral blood of the mice is collected on the 7th day and the 14th day after the transfusion of CAR-Vδ2 T cells, and the CAR-Vδ2 T content is detected.

[0163] SKOV3 model: (Day 0) SKOV3-Luciferase cells (resuspended in 200 μL PBS) are transfused intraperitoneally, 1.5×10 6 , Day 12, weigh the body weight, and randomly divide into 4 groups according to the data of live imaging and body weight, 4 mice in each group, and then CAR-Vδ2 T cells are transfused on the next day, 5×10 5 CAR + cells, and the control group is transfused with the same volume of PBS, and then live imaging and body weight measurement are performed every 7-10 days until the mice die, and the tumor fluorescence is calculated. The peripheral blood of the mice is collected on the 7th day, the 14th day, and the 19th day after the transfusion of CAR-Vδ2 T cells, and the CAR-Vδ2 T content is detected.

[0164] Example

[0165] The following specific examples further describe the technical solutions provided by the present application, which are intended to merely illustrate the present application and not limit the scope of the present application

[0166] In addition, various experimental methods in the examples, such as no special instructions, are conventional methods. Various reagents and materials used in the examples, such as no special instructions, are commercially available products.

[0167] Example 1

[0168] The inventors constructed a CAR expressing an IL15 and a covalently bound complex of IL15Rα of different structures, wherein the structure of the IL15 / IL15Rα complex is shown in FIG. 1A, and the structure of the CAR expressing an IL15 and a covalently bound complex of IL15Rα of different structures is shown in FIG. 1B, wherein the B7H3 CAR chimeric antigen receptor is composed of the heavy chain and light chain variable region of an anti-human B7H3 monoclonal antibody (Anti-B7H3 scFv), a human CD8α hinge region and a human CD8 transmembrane region (CD8 hinge and tm), a human CD28 intracellular region (CD28 cyto), and a human CD3ζ intracellular region (CD3ζ), and the structure of the B7H3 CAR is shown in FIG. 1C.

[0169] Example 2 CAR-Vδ2 T cells expressing different CAR structures were expanded, viability and expression positive rate

[0170] Lentivirus with different CAR structures in Example 1 were prepared respectively, and the corresponding B7H3 CAR-Vδ2 T cells were prepared. The γδ T cells were activated in vitro on Day 0, and lentivirus with different CAR structures was transduced on Day 1, 2 x 10 6 cells were transduced by each virus, NTD was the control group, no virus transduction, the initial number was 4 x 10 6 , Day 3 was supplemented with an equal volume of culture medium, and the subsequent AOPI counting was performed every 2 days, and the cell density was adjusted to 1 x 10 6 / mL, until Day 15, the number of cell expansion (Figure 2A), proliferation fold (Figure 2B) and viability (Figure 2C) were counted. The results showed that during the expansion period, Vδ2 T cells expressing different CAR structures could maintain a high cell viability, and there was no significant difference in cell viability between different CAR structures. From the proliferation fold, B7H3 CAR1, B7H3 CAR2 and B7H3 CAR7 could better expand than other groups.

[0171] 2 x 10 5 cells were taken after 7 days of culture, and the proportion and expression positive rate of Vδ2 T were detected by flow cytometry (Figures 3A and 3B: 3A is a flow cytometry graph, and 3B is a columnar statistical graph), the results showed that the proportion of Vδ2 T cells expressing different CAR structures was similar, which indicated that different CAR structures did not affect the harvest of Vδ2 T cells, and from the expression positive rate, B7H3 CAR1, B7H3 CAR5 and B7H3 CAR7 had higher expression positive rate than other groups.

[0172] Example 3 Changes in CAR-Vδ2 T cells expressing different CAR structures under factor-free conditions

[0173] B7H3 CAR-Vδ2 T cells cultured for 9 days were taken, and the medium was replaced with factor-free medium for starvation culture, and the cell density was adjusted to 1 x 10 6 / mL, and an appropriate amount of cells was taken after 3 days for flow cytometry counting, and the CAR +Cell (Figure 4A) and Vδ2 T cell expansion fold (Figure 4B). The results show that under the factor-free condition, B7H3 CAR2 expressing IL15 / IL15Rα (full length) and B7H3 CAR3 expressing IL15(N72D) / IL15Rα (full length) proliferate better than B7H3 CAR1 not expressing IL15 / IL15Rα, indicating that the IL15 / IL15Rα (full length) structure has the effect of improving Vδ2 T cell expansion.

[0174] Among all the structures, B7H3 CAR5 expressing IL15 / IL15Rα (73aa) and B7H3 CAR7 structure expressing IL15(N72D) / IL15Rα (73aa) have the best CAR + The number of cells and Vδ2 T cells is significantly higher than that of other groups, and the Vδ2 T cells expressing the B7H3 CAR7 structure proliferate best.

[0175] For the IL15 / IL15Rα complex structure, the truncated IL15Rα (73aa) significantly promotes the persistence of Vδ2 T cells under the factor-free condition compared with the full-length IL15Rα, while the IL15Rα (65aa) with only the sushi domain in the extracellular region cannot promote the proliferation of Vδ2 T cells compared with the full-length IL15Rα, and is close to the effect of B7H3 CAR1 not expressing IL15 / IL15Rα, so IL15 / IL15Rα (65aa) has no biological activity and cannot promote the persistence of γδ T cells under the factor-free condition.

[0176] To further verify the experimental results, the inventors activated Vδ2 T cells in vitro again and expressed B7H3 CAR2-7, successfully prepared CAR-Vδ2 T cells, took the CAR-Vδ2 T cells cultured for 9 days, replaced the culture medium under the factor-free condition for starvation culture, counted by AOPI and adjusted the cell density to 1×10 6 / mL and adjusted the CAR positive rate to be consistent, to exclude the difference in cell proliferation due to different CAR positive rates, and then counted the AOP1 every 2 / 3 days, and counted the number of cell expansion (Figure 5A), the proliferation fold (Figure 5B) and the viability (Figure 5C). The results were consistent with the previous results. The number of Vδ2 T cells expressing IL15 / IL15Rα (73aa) of B7H3 CAR5 and IL15(N72D) / IL15Rα (73aa) of B7H3 CAR7 structure was significantly higher than that of other groups, and in the absence of factors, it could maintain a high cell viability. The extracellular region only expressed IL15Rα (65aa) of sushi domain (B7H3 CAR4 expressing IL15 / IL15Rα (65aa) and B7H3 CAR6 expressing IL15(N72D) / IL15Rα (65aa)) compared with full-length IL15Rα (B7H3 CAR2 expressing IL15 / IL15Rα (full-length) and B7H3 CAR3 expressing IL15(N72D) / IL15Rα (full-length)), could not promote the proliferation of Vδ2 T cells. The above experimental results showed that the IL15 / IL15Rα complex of B7H3 CAR5 and B7H3 CAR7 structure could effectively help γδ T cells to resist factor deficiency and improve the persistence of γδ T cells.

[0177] Example 4 Effect of different CAR structures on pSTAT5 level of CAR-Vδ2 T cells

[0178] The above-mentioned CAR-Vδ2 T cells cultured for 9 days were replaced with factor-free medium for starvation culture, and pSTAT5 detection was performed after 24 h, with 50 ng / mL IL15 stimulation for 30 min as a positive control. The pSTAT5 mean fluorescence intensity (MFI) of Vδ2 T cells (Figure 6A) and CAR + cells (Figure 6B) was counted. Whether it was Vδ2 T cell population or CAR +The cell population expressing B7H3 CAR2 with IL15 / IL15Rα (full length) and B7H3 CAR3 with IL15(N72D) / IL15Rα (full length) has a higher pSTAT5 level than B7H3 CAR1 without IL15 / IL15Rα. The Vδ2 T cells expressing B7H3 CAR5 with IL15 / IL15Rα (73aa) structure and B7H3 CAR7 with IL15(N72D) / IL15Rα (73aa) structure have the highest pSTAT5 level, indicating that the IL15 / IL15Rα complex of B7H3 CAR5 and B7H3 CAR7 structure has the strongest activation effect on the downstream signaling pathway. It has been confirmed in many studies that the pSTAT5 level has a strong positive correlation with the viability and proliferation of various immune cells, which provides a molecular mechanism for the previously observed stronger survival ability of Vδ2 T cells expressing B7H3 CAR5 and B7H3 CAR7 structure under factor-deficient conditions and the realization of sustained expansion within a certain period of time.

[0179] CAR-Vδ2 T cells expressing membrane-bound IL15 / IL15Rα complex are both presenting cells and responding cells: as presenting IL15 / IL15Rα cells, they can activate peripheral cells in trans; as responding cells, they can continuously synthesize IL15 / IL15Rα complex themselves, and the intracellular stored complex can act as a circulating pool to act on themselves, promoting resistance to factor-deficient environment and enhancing sustainability within a certain period of time.

[0180] Example 5 In vitro tumor killing effect of CAR-Vδ2 T cells expressing different CAR structures

[0181] To further study whether CAR-Vδ2 T cells expressing different CAR structures have the function of enhancing in vitro tumor killing, the inventors performed the following experiments.

[0182] (1) Antigen expression level of target cells

[0183] The inventors performed flow cytometry on the B7H3 antigen expression abundance of target cells THP-1 cells (Figure 7A) and SKOV3 cells (Figure 7B), and the results showed that THP-1 cells and SKOV3 cells were both B7H3 positive, so these two tumor cells can be used as antigen-positive target cells for in vitro killing experiments and animal models.

[0184] (2) Effect of killing THP-1

[0185] To take the B7H3 CAR-Vδ2T cells cultured for 9 days to continuously kill THP-1 (Figure 8), there is no factor in the killing system, the inventors found that after three rounds of killing, the CAR-Vδ2T cells with the structure of B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) have the best effect on killing the target cells THP-1, the CAR-Vδ2T cells with the structure of B7H3 CAR2 (expressing IL15 / IL15Rα (full length)) are the second, the CAR-Vδ2T cells with the structure of B7H3 CAR3 (expressing IL15(N72D) / IL15Rα (full length)) are weaker than the CAR-Vδ2T cells with the structure of B7H3 CAR2 (expressing IL15 / IL15Rα (full length)) and stronger than the CAR-Vδ2T cells with the structure of B7H3 CAR4 (expressing IL15 / IL15Rα (65aa)), and the CAR-Vδ2T cells with the structure of B7H3 CAR6 (expressing IL15(N72D) / IL15Rα (65aa)) have almost no killing effect.

[0186] (3) IFN-γ secretion amount of B7H3 CAR-Vδ2T cells

[0187] The culture supernatant of the effector target cells co-incubated for 18 h in the first round of killing experiment in the above (2) was collected, and the content of IFNγ was detected by Elisa (Figure 9). Consistent with expectations, the Vδ2T cells with the structure of B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) secrete the most IFN-γ, the Vδ2T cells with the structure of B7H3 CAR6 (expressing IL15(N72D) / IL15Rα (65aa)) secrete the least, and the other three CAR structures have no obvious difference.

[0188] Example 6 In vivo tumor killing effect of CAR-Vδ2T cells expressing different CAR structures

[0189] The inventors further constructed two CDX mouse models of target cells, and used the two models to investigate the in vivo tumor killing function of B7H3 CAR-Vδ2T cells expressing different CAR structures. The two CDX mouse models of target cells are THP-1 and SKOV3 models.

[0190] (1) THP-1 model

[0191] The THP-1 mouse model was constructed according to the steps described in the experimental materials and methods, and the tumor fluorescence (Figure 10A, B) and survival curve (Figure 10C) were counted.

[0192] Results showed that the tumor inhibition ability of B7H3 CAR2 (expressing IL15 / IL15Rα (full length)) was higher than that of B7H3 CAR1 (not expressing IL15 / IL15Rα). The tumor inhibition effects of B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) were the most significant, which were significantly higher than those of B7H3 CAR2 (expressing IL15 / IL15Rα (full length)) and B7H3 CAR1 (not expressing IL15 / IL15Rα), and there was no obvious difference between B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)).

[0193] In addition, the peripheral blood of mice was collected on the 7th day and the 14th day after the B7H3 CAR-Vδ2T cells were reinfused, and the content of B7H3 CAR-Vδ2T was detected and counted (Figure 10D). The results showed that the persistence of Vδ2T cells expressing B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) was significantly higher than that of B7H3 CAR1 (not expressing IL15 / IL15Rα) and B7H3 CAR2 (expressing IL15 / IL15Rα (full length)).

[0194] (2) SKOV3 model

[0195] The SKOV3 mouse model was constructed according to the steps described in the experimental materials and methods, and the tumor fluorescence was counted (Figures 11A and 11B).

[0196] Results showed that the tumor inhibition effects of B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) were the most significant compared with B7H3 CAR1 (not expressing IL15 / IL15Rα), and there was no obvious difference between B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)).

[0197] In addition, the peripheral blood of the mice was collected on day 7, 14 and 19 after the B7H3 CAR-Vδ2 T cells were returned, and the content of CAR-Vδ2 T was detected and counted (Figure 11C). The Vδ2 T cells with B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) structures were significantly higher than B7H3 CAR1 (not expressing IL15 / IL15Rα), and the Vδ2 T cells with B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) structure were significantly higher than B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) in vivo.

[0198] The experimental results of the above two CDX mouse models showed that the CAR-Vδ2 T cells expressing B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR7 (expressing IL15(N72D) / IL15Rα (73aa)) structures had enhanced tumor inhibition effect, and their in vivo persistence was significantly enhanced.

[0199] Example 7

[0200] Based on the results of the above examples, the inventors proposed a hypothesis that in the structure of the IL 15 / IL15Rα complex expressed in the γδT, a specific structure of the flexible connecting peptide between the sushi domain and the transmembrane region of IL15Rα is required, and there may be a certain range for the length of the connecting peptide, and the length of the connecting peptide may affect the biological activity of the IL15 / IL15Rα complex.

[0201] To verify the above hypothesis, the inventors newly constructed IL15 / IL15Rα complexes covalently combined with IL15 and IL15Rα of different structures according to the structure shown in Figure 12A, and in turn constructed CARs expressing the above IL15 / IL15Rα complexes, which were sequentially named B7H3 CAR8 / 9 / 10 / 11 / 12 / 13 / 14 / 15. The structure of the IL15 / IL15Rα complex is shown in Figure 12A. The structure of the CAR is shown in Figure 12B. Among them, the structure of IL15Rα in the IL15 / IL15Rα complex of B7H3 CAR15 is that the sushi domain is connected to the transmembrane region through a (G4S)3 connecting peptide.

[0202] Example 8 CAR-Vδ2 T cells expressing different CAR structures are expanded and have a survival rate

[0203] The lentivirus of B7H3 CAR1 / 2 / 5 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 was prepared respectively, and the corresponding B7H3 CAR-Vδ2 T cells were prepared. The operation steps were the same as Example 2. The number of cell expansion (Figure 13A), the proliferation fold (Figure 13B) and the viability (Figure 13C) were counted. The results showed that during the expansion cycle, Vδ2 T cells expressing different CAR structures could maintain a high cell viability, and there was no significant difference in cell viability between different CAR structures. In terms of proliferation fold, B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) and B7H3 CAR8 (expressing IL15 / IL15Rα (DeEXON3)) could better expand than other groups.

[0204] Example 9 Expansion changes of CAR-Vδ2 T cells expressing different CAR structures under factor-free conditions

[0205] The B7H3 CAR-Vδ2 T cells cultured for 9 days in Example 8 above were replaced with factor-free medium for starvation culture, and the cell density was adjusted to 1×10 6 / mL. After 11 days and 15 days of culture, an appropriate amount of cells was taken for counting, and the Vδ2 T cell expansion fold (Figure 14A) and Vδ2 T cell viability (Figure 14B) were counted. The results showed that under factor-free conditions, B7H3 CAR1 (not expressing IL15 / IL15Rα) structure Vδ2 T cells expanded the worst, the viability continued to decrease and was significantly lower than other CARs at Day 15, indicating that the newly constructed different forms of IL15 / IL15Rα all had biological activity, and B7H3 CAR5 (expressing IL15 / IL15Rα (73aa)) structure Vδ2 T cells proliferated best.

[0206] As for the structures of these newly constructed different forms of IL15 / IL15Rα complexes, different structural fragments are connected between the sushi domain and the transmembrane region of the IL15Rα of these complexes. Combined with the results of the previous examples (the IL15 / IL15Rα complex of IL15Rα (65aa) expressing only the sushi domain in the extracellular region has no biological activity and cannot promote the proliferation of Vδ2 T cells, and has no difference with B7H3 CAR1 not expressing IL15 / IL15Rα), it can be known that only the IL15 / IL15Rα complex with a specific structural fragment connected between the sushi domain and the transmembrane region of IL15Rα can promote the proliferation of Vδ2 T cells and maintain a certain Vδ2 T cell viability under factor-free conditions. Among them, the IL15 / IL15Rα complex structure of IL15Rα (73aa) (B7H3 CAR5) is the best.

[0207] Example 10 Effect of different CAR structures on pSTAT5 level of CAR-Vδ2 T cells

[0208] The CAR-Vδ2 T cells cultured for 9 days in Example 8 above were replaced with factor-free conditioned medium for starvation culture, and pSTAT5 detection was performed 24 h later. The pSTAT5 mean fluorescence intensity (MFI) of Vδ2 T cells (Fig. 15A) and CAR + cells (Fig. 15B) was counted. From the Vδ2 T cell population, the pSTAT5 levels of Vδ2 T cells expressing IL15 / IL15Rα complex were increased to varying degrees. The same phenomenon was observed in the CAR + cell population, indicating that IL15 / IL15Rα complexes of different structures have biological activity and can activate downstream signaling pathways. Among them, the IL15 / IL15Rα complex structure of IL15Rα (73 aa) (B7H3 CAR5) has the strongest stimulating effect.

[0209] Example 11

[0210] The inventors constructed a CAR expressing a hydrolysis-resistant form of IL15 / IL15Rα complex, the structure of which is shown in FIG. 16.

[0211] Using the constructed B7H3 CAR7 plasmid as a template, PCR was performed using a primer lacking S181. The PCR product was transformed into competent E. coli (DH5α), and after overnight culture, a single colony was picked and sent for sequencing. The sequencing results were compared with the B7H3 CAR7 sequence to verify whether the newly constructed clone sequence lacked S181. The clone lacking S181 was named B7H3 CAR16.

[0212] The construction primer is:

[0213] Sense sequence: TGGCCATCAGCACCACCGTGCTGCTGTGCGGCCTGA

[0214] (SEQ ID NO: 50)

[0215] Antisense sequence: GCACAGCAGCACGGTGGTGCTGATGGCCACCCTCTGGTGCA

[0216] (SEQ ID NO: 51)

[0217] After sequencing, the plasmid was extracted and purified using the Qiagen plasmid purification kit.

[0218] Example 12 Effect of different CAR structures on pSTAT5 level of CAR-Vδ2 T cells

[0219] B7H3 CAR-1 / 7 / 16 Vδ2T cells were prepared, and pSTAT5 was detected to statistically analyze CAR levels. + Cells and CAR - Mean fluorescence intensity (MFI) of pSTAT5 in cells (Figure 17). Both donors showed expression of B7H3 CAR7 and B7H3 CAR16 CARs. + Cellular pSTAT5 levels are higher than those of B7H3 CAR1 CAR + Cellular pSTAT5 levels, in which donor 3 expressed B7H3 CAR16 CAR + The pSTAT5 level in cells was the highest, significantly higher than that in B7H3 CAR7. + Cells, donor 4, expressed B7H3 CAR16 and B7H3 CAR7 CARs + The pSTAT5 levels in the cells were similar.

[0220] Example 13: Changes in the expansion of CAR-Vδ2T cells expressing different CAR structures under factor-free conditions.

[0221] B7H3 CAR-Vδ2T cells cultured for 9 days in Example 12 were subjected to starvation culture in a factor-free medium, and AOPI counts were performed to adjust the cell density to 1×10⁻⁶. 6 / mL, cell counts and flow cytometry were performed every 2 days to detect CAR positivity rate, and CAR counts were statistically analyzed. + Cell expansion fold (Fig. 18A, C) and cell viability (Fig. 18B, D). The results showed that, under factor-free conditions, the expression of B7H3 CAR16 (a CAR expressing the IL15(N72D) / IL15Rα(73aa) with S181 deleted) structure was significantly reduced. + The cells exhibited the best proliferation, expressing CAR7H3 CAR7 (expressing the IL15(N72D) / IL15Rα(73aa)) structure. + Cell proliferation is secondary, followed by B7H3 CAR1 (which does not express IL15 / IL15Rα) CAR. + Cell proliferation was poor. In terms of viability, Vδ2T cells expressing B7H3 CAR7 and B7H3 CAR16 maintained a high viability, while the viability of cells expressing B7H3 CAR1 continued to decline and was significantly lower than that of other CAR-Vδ2T cells on Day 13.

[0222] Example 14: In vitro tumor-killing effect of CAR-Vδ2T cells expressing different CAR structures

[0223] (1) Effects of killing THP-1

[0224] B7H3 CAR-Vδ2 T cells were taken after 9 days of culture to continuously kill THP-1, and the residual tumor fluorescence was counted (Figure 19). No factors were added in the killing system, and THP-1 tumor cells were added every 48 h. After three rounds of killing, the killing effect of CAR-Vδ2 T cells with the structure of B7H3 CAR7 (expressing IL15(N72D) / IL15Rα(73aa)) and B7H3 CAR16 (expressing IL15(N72D) / IL15Rα(73aa) with S181 deleted) was better than that of CAR-Vδ2 T cells with the structure of B7H3 CAR1 (not expressing IL15 / IL15Rα), and the killing efficiency of B7H3 CAR16 tended to be higher than that of B7H3 CAR7 in donor 4.

[0225] (2) Expansion of Vδ2 T cells during killing

[0226] Cell samples were taken after 168 h of killing for absolute counting by flow cytometry. Vδ2 T cells in the killing system were labeled by flow cytometry antibody BV421 anti-human TCR Vδ2 to compare the expansion of Vδ2 T cells in different experimental groups (Figure 20). The expansion of CAR-Vδ2 T cells with the structure of B7H3 CAR7 (expressing IL15(N72D) / IL15Rα(73aa)) and B7H3 CAR16 (expressing IL15(N72D) / IL15Rα(73aa) with S181 deleted) was better than that of CAR-Vδ2 T cells with the structure of B7H3 CAR1 (not expressing IL15 / IL15Rα), and the expansion of B7H3 CAR16 was higher than that of B7H3 CAR7 in both donors.

[0227] (3) IFN-γ secretion of B7H3 CAR-Vδ2 T cells

[0228] Culture supernatant of effector target cells co-incubated for 18 h in the first round of killing experiment in (1) above was collected, and the content of IFN-γ was detected by Elisa (Figure 21). Consistent with expectations, the IFN-γ secretion of CAR-Vδ2 T cells with the structure of B7H3 CAR7 and B7H3 CAR16 was higher than that of CAR-Vδ2 T cells with the structure of B7H3 CAR1 in both donors, and the IFN-γ secretion of B7H3 CAR16 was higher than that of B7H3 CAR7 in donor 4.

[0229] Example 15 Detection of IL15 level in supernatant of CAR-Vδ2 T cells expressing different CAR structures

[0230] CAR-Vδ2 T cells cultured for 9 days in Example 12 above were collected, and the culture medium was replaced with factor-free medium, and the cell density was adjusted to 1×106 1 x 10 6 The cell suspension was placed in a 24-well plate, and after 48 hours of culture at 37°C in 5% CO2, the supernatant was collected and subjected to Elisa detection of IL15. The IL15 content was detected by Elisa (Figure 22), and 500 pg / mL of IL15 was used as a positive control (PC) of the experimental system. The IL15 in the supernatant of the CAR-Vδ2T expressing B7H3 CAR16 (CAR expressing IL15(N72D) / IL15Rα(73aa) deletion S181) structure in two donors was lower than that of B7H3 CAR7 (CAR expressing IL15(N72D) / IL15Rα(73aa)), which showed that the IL15 / IL15Rα complex in the hydrolysis-resistant form to some extent reduced the free IL15 generated by the hydrolysis of IL15Rα.

[0231] Example 16 In vivo tumor killing effect of CAR-Vδ2T cells expressing different CAR structures

[0232] The inventors investigated the efficacy of CAR-Vδ2T cells expressing different CAR structures in a THP-1 mouse model, and the tumor fluorescence was counted (Figure 23). Figure 23A is a fluorescence imaging image of the mouse tumor, and Figures 23B, 23C, 23D, and 23E are tumor fluorescence statistical graphs of Day 14, Day 23, Day 30, and Day 36, respectively.

[0233] The results showed that compared with the G1 control group, the experimental groups G2 / 3 / 4 that were reinfused with B7H3 CAR-Vδ2T cells all showed different degrees of tumor inhibition effect, among which the tumor inhibition ability of the G2 group (B7H3 CAR1 (not expressing IL15 / IL15Rα)) was weak, while the tumor inhibition effect of the G3 group (B7H3 CAR7) and the G4 group (B7H3 CAR16) were both good, and the tumor inhibition effect of the G4 group (B7H3 CAR16) was more persistent, with a lower tumor fluorescence value than that of the G3 group (B7H3 CAR7) at Day 36.

[0234] Therefore, by reducing the hydrolysis of IL15Rα in the IL15 / IL15Rα complex, the persistence and tumor killing function of CAR-Vδ2T cells can be enhanced to some extent.

[0235] In combination with the above embodiments, the inventors found and confirmed that the IL15 / IL15Rα complex structure (B7H3 CAR4 / B7H3 CAR6) expressing only the sushi domain of the extracellular region of IL15Rα has no biological activity, and has no difference in function with the structure (B7H3 CAR1) not expressing the IL15 / IL15Rα complex. The IL15 / IL15Rα complex has biological activity only when there is a specific amino acid fragment between the sushi domain and the transmembrane region of IL15Rα, and the difference in the amino acid fragment affects the activity of the IL15 / IL15Rα complex. Among them, the IL15 / IL15Rα complex structure of IL15Rα (73aa) (B7H3 CAR5 / B7H3 CAR7) is the most optimal. Based on this, the inventors constructed a functional enhanced membrane-bound IL15 / IL15Rα complex, effectively improved the persistence and killing function of γδT, and significantly improved the inhibition effect on tumors.

[0236] In addition, the inventors also confirmed that the IL15 / IL15Rα complex with the hydrolysis site of IL15Rα deleted can significantly promote the proliferation of γδT cells in the absence of cytokines and the killing of target cells, and significantly prolong the persistence of γδT in mice and enhance its anti-tumor effect.

[0237] In summary, the present application relates to the following technical solutions:

[0238] 1. An IL15 / IL15Rα complex in a membrane-bound form, comprising IL15 and IL15Rα, wherein,

[0239] The IL15 comprises IL15 wild type and IL15 mutant,

[0240] The IL15Rα comprises, from N terminus to C terminus, (1) IL15Rα sushi domain (65aa), (2) a region between the IL15Rα sushi domain and the IL15Rα transmembrane region, (3) IL15Rα transmembrane region, and (4) IL15Rα intracellular region, wherein (1) IL15Rα sushi domain (65aa) and (2) a region between the IL15Rα sushi domain and the IL15Rα transmembrane region are extracellular regions,

[0241] The region (2) between the IL15Rα sushi domain and the IL15Rα transmembrane region is an amino acid sequence naturally existing between the IL15Rα sushi domain and the IL15Rα transmembrane region, or a combined fragment of the amino acid sequence naturally existing between the IL15Rα sushi domain and the IL15Rα transmembrane region and other natural or engineered amino acid fragments.

[0242] 2. The IL15 / IL15Ra complex according to claim 1, wherein the region between the (2) IL15Ra sushi domain and the IL15Ra transmembrane region is a full-length amino acid sequence naturally present between the IL15Ra sushi domain and the IL15Ra transmembrane region, or is a truncated fragment or a combination of truncated fragments of the full-length amino acid sequence.

[0243] 3. The IL15 / IL15Ra complex according to claim 1, wherein the IL15 / IL15Ra complex is IL15 / IL15Ra (truncated).

[0244] 4. The IL15 / IL15Ra complex according to any one of claims 1 to 3, wherein the amino acid sequence of the extracellular region of IL15Ra is selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 27, the amino acid sequence represented by SEQ ID NO: 28, the amino acid sequence represented by SEQ ID NO: 29, the amino acid sequence represented by SEQ ID NO: 30, the amino acid sequence represented by SEQ ID NO: 31, the amino acid sequence represented by SEQ ID NO: 25, the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence represented by SEQ ID NO: 33, the amino acid sequence represented by SEQ ID NO: 34, the amino acid sequence represented by SEQ ID NO: 35, and combinations thereof.

[0245] 5. The IL15 / IL15Ra complex according to any one of claims 1 to 4, wherein the amino acid sequence of the extracellular region of IL15Ra is selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 28, the amino acid sequence represented by SEQ ID NO: 31, the amino acid sequence represented by SEQ ID NO: 32, the amino acid sequence represented by SEQ ID NO: 34.

[0246] 6. The IL15 / IL15Ra complex according to any one of claims 1 to 5, wherein

[0247] the DNA sequence of the IL15 wild type is represented by SEQ ID NO: 5, and the amino acid sequence is represented by SEQ ID NO: 23,

[0248] the DNA sequence of the IL15 mutant is represented by SEQ ID NO: 8, and the amino acid sequence is represented by SEQ ID NO: 24.

[0249] 7. The IL15 / IL15Ra complex of any one of claims 1-6, wherein the IL15 / IL15Ra complex has an amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37.

[0250] 8. The IL15 / IL15Ra complex of any one of claims 1-7, wherein the other natural or engineered amino acid fragment is an amino acid fragment that provides a linkage between the two polypeptide fragments.

[0251] The other natural or engineered amino acid fragment is a GGGGS flexible linker, or a hinge region of IgG.

[0252] 9. The IL15 / IL15Ra complex of claim 1 or 2, wherein the hydrolysis site of IL15Ra is deleted from the amino acid sequence of IL15Ra.

[0253] 10. The IL15 / IL15Ra complex of claim 9, wherein the serine at position 181 is deleted from the amino acid sequence of IL15Ra.

[0254] 11. The IL15 / IL15Ra complex of claim 10, wherein the amino acid sequence is set forth in SEQ ID NO: 49.

[0255] 12. A nucleic acid molecule encoding the IL15 / IL15Ra complex of any one of claims 1-11.

[0256] 13. A vector comprising the nucleic acid molecule of claim 12.

[0257] 14. The vector of claim 13, which is a lentivirus vector, a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector.

[0258] 15. A cell comprising the IL15 / IL15Ra complex of any one of claims 1-11, the nucleic acid molecule of claim 12, or the vector of claim 13 or 14.

[0259] 16. The cell of claim 15, wherein the cell comprises an autologous or allogeneic T cell, a B cell, an NK cell, a macrophage, a monocyte, a dendritic cell, a neutrophil, a basophil, an eosinophil, a mast cell, an NK-T cell, a MAIT cell, a hematopoietic stem cell, an embryonic stem cell, an induced pluripotent stem cell and a cell derived therefrom, a red blood cell.

[0260] The T cell comprises an alpha beta T cell, a gamma delta T cell, a regulatory T cell.

[0261] 17. The cell of claim 16, wherein the γδ T cell comprises a δ1, δ2, δ3, δ4, and δ5 T cell.

[0262] 18. A cell preparation comprising the IL15 / IL15Ra complex of any one of claims 1-11, the nucleic acid molecule of claim 12, the vector of claim 13 or 14, or the cell of any one of claims 15-17.

[0263] 19. The cell preparation of claim 18, wherein the cell preparation is a CAR-γδ T cell preparation.

[0264] 20. The cell preparation of claim 18 or 19, wherein the cell preparation expresses a CAR comprising an antigen binding domain that targets B7H3, CD19.

[0265] 21. The cell preparation of claim 18 or 19, wherein the CAR structure comprises an optional signal peptide, a polypeptide that binds a tumor cell membrane antigen, a hinge region, a transmembrane region, and an immune active cell activation signaling region, wherein the immune active cell activation signaling region further comprises a costimulatory domain and a signaling domain.

[0266] 22. A pharmaceutical composition comprising:

[0267] the IL15 / IL15Ra complex of any one of claims 1-11, the nucleic acid molecule of claim 12, the vector of claim 13 or 14, the cell of any one of claims 15-17, or the cell preparation of claims 18-21, and

[0268] a pharmaceutically acceptable excipient as needed.

[0269] 23. Use of the IL15 / IL15Ra complex of any one of claims 1-11, the nucleic acid molecule of claim 12, the vector of claim 13 or 14, the cell of any one of claims 15-17, the cell preparation of claims 18-21, or the pharmaceutical composition of claim 22, in the manufacture of a medicament for preventing or treating a disease, including a malignant / benign hematological tumor, a malignant / benign solid tumor, an autoimmune disease, a bacterial infection, a fungal infection, a viral infection, a parasitic infection, a bone growth abnormality, an allogeneic transplant, a transplant rejection, aging.

[0270] 24. The use of claim 23, wherein the disease comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, renal tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome; systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, multiple sclerosis, Sjogren's syndrome, ankylosing spondylitis, autoimmune glomerulonephritis, autoimmune hepatitis, idiopathic leukopenia, psoriasis, vitiligo, mixed connective tissue disease, autoimmune hemolytic anemia, autoimmune disease of the thyroid, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis; epidemic cerebrospinal meningitis, bacterial dysentery, whooping cough, scarlet fever, acute bacterial conjunctivitis, typhoid fever, diphtheria, tuberculosis, plague, cholera; candidiasis, aspergillosis, cryptococcosis, histoplasmosis, mucormycosis, paracoccidioidomycosis, sporotrichosis, dermatophytosis; viral hepatitis, poliomyelitis, hand-foot-and-mouth disease, influenza, measles, rubella, roseola infantum, varicella, smallpox, herpes simplex virus infection, foot-and-mouth disease, mumps, hemolytic-uremic syndrome, human papillomavirus infection, rabies, cytomegalovirus infection, acquired immunodeficiency syndrome, epidemic hemorrhagic fever, yellow fever, Rift Valley fever, Argentine hemorrhagic fever, Lassa fever, Marburg hemorrhagic fever, Ebola hemorrhagic fever, dengue fever, and dengue hemorrhagic fever; amebiasis, helminthiasis, nematodiasis, malaria, leishmaniasis, ascariasis, enterobiasis, ancylostomiasis, trichuriasis, fascioliasis, paragonimiasis, schistosomiasis, filariasis; Alzheimer's disease.

[0271] 25. A method of treating or preventing a disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the cell preparation of claims 18-21, or the pharmaceutical composition of claim 22.

[0272] 26. The method of claim 25, wherein the disease comprises malignant / benign hematological tumor, malignant / benign solid tumor, autoimmune disease, bacterial infection, fungal infection, viral infection, parasitic infection, abnormal bone growth, allogeneic transplantation, transplant rejection, aging.

[0273] 27. The method of claim 26, wherein the disease comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, renal tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome; systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, multiple sclerosis, Sjogren’s syndrome, ankylosing spondylitis, autoimmune glomerulonephritis, autoimmune hepatitis, idiopathic leukopenia, psoriasis, vitiligo, mixed connective tissue disease, autoimmune hemolytic anemia, autoimmune disease of the thyroid, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture’s syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre syndrome; epidemic cerebrospinal meningitis, bacterial dysentery, whooping cough, scarlet fever, acute bacterial conjunctivitis, typhoid fever, diphtheria, tuberculosis, plague, cholera; candidiasis, aspergillosis, cryptococcosis, histoplasmosis, mucormycosis, paracoccidioidomycosis, sporotrichosis, dermatophytosis; viral hepatitis, poliomyelitis, hand-foot-and-mouth disease, influenza, measles, rubella, infantile acute exanthema, varicella, smallpox, herpes simplex virus infection, foot-and-mouth disease, mumps, hemolytic-uremic syndrome, human papillomavirus infection, rabies, cytomegalovirus infection, acquired immunodeficiency syndrome, epidemic hemorrhagic fever, yellow fever, Rift Valley fever, Argentine hemorrhagic fever, Lassa fever, Marburg hemorrhagic fever, Ebola hemorrhagic fever, dengue fever, and dengue hemorrhagic fever; amebiasis, helminthiasis, nematodiasis, malaria, leishmaniasis, ascariasis, enterobiasis, ancylostomiasis, clonorchiasis, paragonimiasis, schistosomiasis, filariasis; Alzheimer’s disease.

[0274] Biosequence list

[0275] In the DNA sequence, the underlined single line portion is the signal peptide DNA sequence, the underlined dotted line portion is the linker peptide DNA sequence, and the underlined double line portion is the mutated DNA sequence.

[0276] SEQ ID NO: 1: DNA sequence 1 (Anti-B7H3 scFv nucleotide sequence, structure: VH-linker peptide-VL)

[0277] SEQ ID NO: 2: DNA Sequence 2 (B7H3 CAR Hinge and Transmembrane Region Nucleotide Sequence)

[0278] SEQ ID NO: 3: DNA Sequence 3 (B7H3 CAR Costimulatory Domain CD28 Nucleotide Sequence)

[0279] SEQ ID NO: 4: DNA Sequence 4 (B7H3 CAR Intra-cellular Activation Domain CD3 zeta Nucleotide Sequence)

[0280] SEQ ID NO: 5: DNA Sequence 5 (IL15 Nucleotide Sequence)

[0281] SEQ ID NO: 6: DNA Sequence 6 (IL15R a Nucleotide Sequence)

[0282] SEQ ID NO: 7: DNA Sequence 7 (Linker Peptide Nucleotide Sequence between IL15 and IL15R a)

[0283] SEQ ID NO: 8: DNA Sequence 8 (IL15 (N72D) Nucleotide Sequence)

[0284] SEQ ID NO: 9: DNA Sequence 9 (IL15R a (65 aa) Extracellular Region Nucleotide Sequence)

[0285] SEQ ID NO: 10: DNA Sequence 10 (IL15R a (66 aa) Extracellular Region Nucleotide Sequence)

[0286] SEQ ID NO: 11: DNA Sequence 11 (IL15R a (73 aa) Extracellular Region Nucleotide Sequence)

[0287] SEQ ID NO: 12: DNA Sequence 12 (IL15R a (77 aa) Extracellular Region Nucleotide Sequence)

[0288] SEQ ID NO: 13: DNA Sequence 13 (IL15R a (86 aa) Extracellular Region Nucleotide Sequence)

[0289] SEQ ID NO: 14: DNA sequence 14 (IL15Ra (102 aa) extracellular region nucleotide sequence)

[0290] SEQ ID NO: 15: DNA sequence 15 (IL15Ra (sushi) + linker nucleotide sequence)

[0291] SEQ ID NO: 16: DNA sequence 16 (IL15Ra (DeEXON3) extracellular region nucleotide sequence)

[0292] SEQ ID NO: 17: DNA sequence 17 (IL15Ra (DeEXON4) extracellular region nucleotide sequence)

[0293] SEQ ID NO: 18: DNA sequence 18 (IL15Ra (DeEXON5) extracellular region nucleotide sequence)

[0294] SEQ ID NO: 19: DNA sequence 19 (IL15 / IL15Ra (73 aa) complex nucleotide sequence)

[0295] SEQ ID NO: 20: DNA sequence 20 (IL15(N72D) / IL15Ra (73 aa) complex nucleotide sequence)

[0296] SEQ ID NO: 21: DNA sequence 21 (P2A nucleotide sequence)

[0297] SEQ ID NO: 22: Anti-B7H3 scFv (amino acid sequence 1)

[0298] SEQ ID NO: 23: IL15 (amino acid sequence 2)

[0299] SEQ ID NO: 24: IL15(N72D) (amino acid sequence 3)

[0300] SEQ ID NO: 25: IL15Ra extracellular region (amino acid sequence 4):

[0301] SEQ ID NO: 26: IL15Ra (65 aa) extracellular region (amino acid sequence 5)

[0302] SEQ ID NO: 27: IL15Ra (66 aa) extracellular region (amino acid sequence 6):

[0303] SEQ ID NO: 28: IL15Ra (73 aa) extracellular region (amino acid sequence 7):

[0304] SEQ ID NO: 29: IL15Ra (77 aa) extracellular region (amino acid sequence 8):

[0305] SEQ ID NO: 30: IL15Ra (86 aa) extracellular region (amino acid sequence 9):

[0306] SEQ ID NO: 31: IL15Ra (102 aa) extracellular region (amino acid sequence 10):

[0307] SEQ ID NO: 32: IL15Ra (DeEXON3) extracellular region (amino acid sequence 11):

[0308] SEQ ID NO: 33: IL15Ra (DeEXON4) extracellular region (amino acid sequence 12):

[0309] SEQ ID NO: 34: IL15Ra (DeEXON5) extracellular region (amino acid sequence 13):

[0310] SEQ ID NO: 35: IL15Ra ((sushi + (G4S)3) extracellular region (amino acid sequence 14):

[0311] SEQ ID NO: 36: IL15 / IL15Ra (73 aa) complex (amino acid sequence 15):

[0312] SEQ ID NO: 37: IL15(N72D) / IL15Ra (73 aa) complex (amino acid sequence 16):

[0313] SEQ ID NO:38: P2A (amino acid sequence 17):

[0314] SEQ ID NO:39: Sense sequence of sequencing primer

[0315] SEQ ID NO:40: Antisense sequence of sequencing primer

[0316] SEQ ID NO:41: Sense sequence of IL15(N72D) mutation primer

[0317] SEQ ID NO:42: Antisense sequence of IL15(N72D) mutation primer

[0318] SEQ ID NO:43: Sense sequence of IL15Ra(65aa) truncation primer

[0319] SEQ ID NO:44: Antisense sequence of IL15Ra(65aa) truncation primer

[0320] SEQ ID NO:45: Sense sequence of IL15Ra(73aa) truncation primer

[0321] SEQ ID NO:46: Antisense sequence of IL15Ra(73aa) truncation primer

[0322] SEQ ID NO:47: IL15Ra full length (amino acid sequence 18):

[0323] SEQ ID NO:48: DNA sequence 22 (IL15(N72D) / IL15Ra(73aa) deletion S181 nucleotide sequence):

[0324] SEQ ID NO:49: IL15(N72D) / IL15Ra(73aa) deletion S181 (amino acid sequence 19):

[0325] SEQ ID NO:50: Sense sequence of B7H3 CAR16 primer

[0326] SEQ ID NO: 51: Antisense sequence of B7H3 CAR16 primer

[0327] SEQ ID NO: 52: Transmembrane domain of IL15Ra deletion S181

[0328] SEQ ID NO: 53: Linker peptide

[0329] SEQ ID NO: 54: Signal peptide nucleotide sequence of complex

[0330] SEQ ID NO: 55:

[0331] SEQ ID NO: 56: EXON3

[0332] SEQ ID NO: 57: EXON4

[0333] SEQ ID NO: 58: EXON5

Claims

1. An IL15 / IL15Ra complex in a membrane-bound form, comprising IL15 and IL15Ra, wherein, the IL15 comprises IL15 wild type and IL15 mutant, the IL15Ra comprises, from N-terminus to C-terminus, (1) IL15Ra sushi domain (65 aa), (2) a region between IL15Ra sushi domain and IL15Ra transmembrane region, (3) IL15Ra transmembrane region, and (4) IL15Ra intracellular region, wherein (1) IL15Ra sushi domain (65 aa) and (2) the region between IL15Ra sushi domain and IL15Ra transmembrane region are extracellular regions, the region between IL15Ra sushi domain and IL15Ra transmembrane region is a naturally occurring amino acid sequence between IL15Ra sushi domain and IL15Ra transmembrane region, or a combined fragment of the naturally occurring amino acid sequence and other naturally occurring or engineered amino acid fragment.

2. The IL15 / IL15Ra complex of claim 1, wherein, the region between IL15Ra sushi domain and IL15Ra transmembrane region is a full-length amino acid sequence of the naturally occurring amino acid sequence between IL15Ra sushi domain and IL15Ra transmembrane region, or a truncated fragment or a combination of truncated fragments of the full-length amino acid sequence.

3. The IL15 / IL15Ra complex of claim 1, wherein, the amino acid sequence of the extracellular region of IL15Ra is selected from the group consisting of the amino acid sequence of SEQ ID NO: 27, the amino acid sequence of SEQ ID NO: 28, the amino acid sequence of SEQ ID NO: 29, the amino acid sequence of SEQ ID NO: 30, the amino acid sequence of SEQ ID NO: 31, the amino acid sequence of SEQ ID NO: 25, the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 33, the amino acid sequence of SEQ ID NO: 34, the amino acid sequence of SEQ ID NO: 35, and combinations thereof; alternatively, the amino acid sequence of the extracellular region of IL15Ra is selected from the group consisting of the amino acid sequence of SEQ ID NO: 28, the amino acid sequence of SEQ ID NO: 31, the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 34; alternatively, the amino acid sequence of the IL15 / IL15Ra complex is the sequence of SEQ ID NO: 36 or the sequence of SEQ ID NO:

37.

4. A nucleic acid molecule encoding the IL15 / IL15Ra complex of claims 1-3.

5. A vector comprising the nucleic acid molecule of claim 4, preferably a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

6. A cell comprising the IL15 / IL15Ra complex of any one of claims 1-3, the nucleic acid molecule of claim 4, or the vector of claim 5, preferably, the cell comprises an autologous or allogeneic T cell, B cell, NK cell, macrophage, monocyte, dendritic cell, neutrophil, basophil, eosinophil, mast cell, NK-T cell, MAIT cell, hematopoietic stem cell, embryonic stem cell, induced pluripotent stem cell, red blood cell, The T cell comprises an alpha beta T cell, a gamma delta T cell, a regulatory T cell, and the gamma delta T cell comprises a delta 1, delta 2, delta 3, delta 4, and delta 5 T cell.

7. A cell preparation comprising the IL15 / IL15Ra complex of any one of claims 1-3, the nucleic acid molecule of claim 4, the vector of claim 5, or the cell of claim 6.

8. The cell preparation of claim 7, wherein, The cell preparation is a CAR-gamma delta T cell preparation, preferably, the cell preparation expresses a CAR comprising an antigen binding domain targeting B7H3, CD19.

9. A pharmaceutical composition comprising: the IL15 / IL15Ra complex of any one of claims 1-3, the nucleic acid molecule of claim 4, the vector of claim 5, the cell of claim 6, or the cell preparation of claim 6 or 7, and a pharmaceutically acceptable excipient as needed.

10. Use of the IL15 / IL15Ra complex of any one of claims 1 to 3, the nucleic acid molecule of claim 4, the vector of claim 5, the cell of claim 6, the cell preparation of claim 6 or 7, or the pharmaceutical composition of claim 7 for the manufacture of a medicament for the prevention or treatment of a disease, including malignant / benign hematological tumors, malignant / benign solid tumors, autoimmune diseases, bacterial infections, fungal infections, viral infections, parasitic infections, bone growth abnormalities, allogeneic transplantation, transplant rejection, aging, preferably, the disease includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, renal tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protrusion, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma or myelodysplastic syndrome; systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, multiple sclerosis, Sjogren's syndrome, ankylosing spondylitis, autoimmune glomerulonephritis, autoimmune hepatitis, idiopathic leukopenia, psoriasis, vitiligo, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmunity, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis; epidemic cerebrospinal meningitis, bacterial dysentery, whooping cough, scarlet fever, acute bacterial conjunctivitis, typhoid fever, diphtheria, tuberculosis, plague, cholera; candidiasis, aspergillosis, cryptococcosis, histoplasmosis, mucormycosis, paracoccidioidomycosis, sporotrichosis, dermatophytosis; viral hepatitis, poliomyelitis, hand-foot-and-mouth disease, influenza, measles, rubella, infantile acute exanthema, varicella, smallpox, herpes simplex virus infection, foot-and-mouth disease, mumps, hemolytic-uremic syndrome, human papillomavirus infection, rabies, cytomegalovirus infection, acquired immunodeficiency syndrome, epidemic hemorrhagic fever, yellow fever, Rift Valley fever, Argentine hemorrhagic fever, Lassa fever, Marburg hemorrhagic fever, Ebola hemorrhagic fever, dengue fever and dengue hemorrhagic fever; amebiasis, helminthiasis, nematodiasis, malaria, leishmaniasis, ascariasis, enterobiasis, ancylostomiasis, trichuriasis, liver fluke disease, lung fluke disease, blood fluke disease, filariasis; Alzheimer's disease.

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