B7-H3 chimeric antigen receptor modified Vdelta1 T cell and application thereof

Abstract

The invention provides a Vdelta1T cell modified by a B7-H3 chimeric antigen receptor and application of the Vdelta1T cell. Specifically, the invention provides a CAR-V delta 1T cell targeting B7-H3, and the CAR-V delta 1T cell shows an outstanding anti-tumor effect on solid tumors in in-vivo and in-vitro functional verification. Meanwhile, the invention finds that the combined expression of IL-2 can improve the durability of the cytotoxic function of the CAR-Vdelta1T cell, and has an application prospect in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a B7-H3 chimeric antigen receptor-modified Vδ1 T cell and an application thereof. Background Art

[0002] Currently, chimeric antigen receptor-modified CAR-T cell therapy developed based on αβT cells has made significant clinical progress in the treatment of hematological tumors, but the clinical progress for solid tumors is relatively slow. This is closely related to factors such as the homing barriers of CAR-αβT cells in solid tumors, the inhibition of CAR-αβT cells by the solid tumor microenvironment, and the high antigenic heterogeneity of solid tumors. Therefore, there is an urgent need to develop a new cell therapy to improve the clinical efficacy of solid tumors.

[0003] Chimeric antigen receptor-modified T cell therapy (CAR-T) developed based on αβT cells has achieved significant clinical efficacy in the treatment of malignant blood tumors derived from B cells and plasma cells. The approval of the first CD19-CAR-T product by the FDA in 2017 also marked the revolutionary success of cell immunotherapy. However, the clinical progress of CAR-T in the treatment of solid tumors is relatively slow, which is closely related to factors such as the homing barrier of CAR-T cells to solid tumors, the inhibition of CAR-T cells by the tumor microenvironment, and the high antigen heterogeneity of solid tumors. In addition, the inherent limitations of autologous CAR-T cell products also limit their application, such as long preparation cycle, high cost, and customized process that increases the risk of cell preparation. In view of this, in order to overcome the challenges of solid tumors on the one hand and the limitations of autologous CAR-T products on the other hand, more allogeneic products based on innate immune cells are being explored, such as: γδT cells, NK cells, macrophages, NKT cells, etc.

[0004] γδT cells are different from other innate immune cells (such as NK cells). γδT cells are considered to be a bridge between innate and adaptive immune responses, and play an important role in tumor immune surveillance. First, unlike αβT, γδT cells recognize tumor antigens independently of the formation of MHC complexes, indicating that γδT cell therapy will not lead to graft-versus-host disease (GVHD); in addition, γδT cells express abundant natural toxicity receptors (NCRs) on their surface, such as high expression of NKG2D, NKp30, NKp44, etc., so that γδT cells can kill various tumor cells by recognizing ligands on the surface of the cells through surface receptors; and in order to further enhance the function of γδT, γδT cells are modified with CAR fragments, and CAR is used to specifically bind to tumor surface antigens to produce toxicity. The rich tumor antigen recognition mechanism gives γδT cells a broad spectrum of anti-tumor properties. In addition, more importantly, γδT cells can effectively infiltrate a variety of tumors, which is closely related to clinical effects.

[0005] γδT cells account for 1%-5% of lymphocytes in peripheral blood, and the γ and δ chains that make up TCR are relatively rich. Among them, the common subtype in peripheral blood is Vδ1, which accounts for about 80% of total γδT cells and is the most common subtype, while Vδ1 accounts for a very low proportion of total cells. So far, most studies have focused on Vδ1 cells because Vδ1 cells can be activated by phosphorylated antigens (PAgs), such as isopentenyl pyrophosphate (IPP) accumulated in tumor cells, and (E)-4-hydroxy-3-methyl-2-enyl pyrophosphate (HMBPP) present in bacterial, fungal and protozoan pathogens. Vδ1 cells are not only rare in peripheral blood, but also more complicated to separate and activate in vitro, making them difficult to mass produce for clinical applications. In addition, due to the inherent antiviral properties of γδT cells, the transduction efficiency of lentiviral vectors to prepare CARs is very low, which has become a global technical barrier to the development of CAR-γδT. Vδ1 T cells are mainly found in epithelial and mucosal tissues, naturally highly express chemokine receptors and chemokines, have stronger homing ability, and their infiltration in solid tumor tissues has been shown to be closely related to good patient prognosis. The TCR of Vδ1 cells is highly sensitive to antigen recognition and can broadly recognize phospholipid antigens induced by tumor stress signals.

[0006] At present, there is no mature Vδ1 T-based CAR-T therapy in the existing technology, and new CAR-Vδ1 T cells and related treatment methods need to be developed in this field. Summary of the invention

[0007] The purpose of the present invention is to provide a B7-H3 chimeric antigen receptor modified Vδ1 T cell and its application.

[0008] In a first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the structure of the chimeric antigen receptor is shown in the following formula I:

[0009] L-scFv-H-TM-C-CD3ζ-IL2(I)

[0010] in,

[0011] L is none or a signal peptide sequence;

[0012] scFv is a scFv targeting B7-H3;

[0013] H is the hinge region;

[0014] TM is the transmembrane domain;

[0015] C is a co-stimulatory signal molecule;

[0016] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0017] IL2 is none or interleukin 2 (IL-2) coding sequence;

[0018] Each "-" is a connecting peptide or a peptide bond connecting the above elements.

[0019] In another preferred embodiment, the scFv has a light chain variable region and a heavy chain variable region targeting B7-H3, and the heavy chain variable region has the following complementary determining region CDR:

[0020] HCDR1:GTFFSTY(SEQ ID NO.2),

[0021] HCDR2: LSGGT (SEQ ID NO. 3),

[0022] HCDR3: RYGHDHYYAMDY (SEQ ID NO.4); and

[0023] The light chain variable region has the following complementarity determining regions CDR:

[0024] LCDR1:AASSSVSSSYLH(SEQ ID NO.6)

[0025] LCDR2: STSNLAS (SEQ ID NO. 7)

[0026] LCDR3: QYHRSPWT (SEQ ID NO. 8).

[0027] In another preferred embodiment, the scFv has a heavy chain variable region as shown in SEQ ID NO.1 and / or a light chain variable region as shown in SEQ ID NO.2.

[0028] In another preferred embodiment, the antibody heavy chain variable region and the antibody light chain variable region are connected via a connecting peptide.

[0029] In another preferred embodiment, the amino acid sequence of the connecting peptide is as shown in (G4S)n, wherein n is 2-6, preferably 3-4.

[0030] In another preferred embodiment, the scFv amino acid sequence is shown as SEQ ID NO.9.

[0031] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0032] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0033] In another preferred embodiment, the amino acid sequence of L is shown as SEQ ID NO.10.

[0034] In another preferred embodiment, the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0035] In another preferred embodiment, the TM is a transmembrane domain of a protein selected from the following group: ICOS, CD8, CD28, CD3epsilon, CD45, CD4, CD5, CD9, CD16, B7-H3, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0036] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: ICOS (CD278), 4-1BB (CD137), Dap10, CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, CDS, ICAM-1, LFA-1 (CD11a / CD18), NKG2D, GITR, TLR2, or a combination thereof.

[0037] In another preferred embodiment, the chimeric antigen receptor has a hinge region derived from Fc, and its amino acid sequence is shown in SEQ ID NO.11.

[0038] In another preferred embodiment, the chimeric antigen receptor has a hinge region and / or a transmembrane domain derived from CD8.

[0039] In another preferred embodiment, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO.12.

[0040] In another preferred embodiment, the chimeric antigen receptor has a transmembrane domain and / or a co-stimulatory signal molecule derived from CD28.

[0041] In another preferred embodiment, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO.13.

[0042] In another preferred embodiment, the chimeric antigen receptor has a co-stimulatory signal molecule derived from 4-1BB, and its amino acid sequence is shown in SEQ ID NO.14.

[0043] In another preferred embodiment, the chimeric antigen receptor has a costimulatory signal molecule derived from DAP10, and its amino acid sequence is shown in SEQ ID NO.15.

[0044] In another preferred embodiment, the chimeric antigen receptor has a transmembrane domain and / or a co-stimulatory signal molecule derived from ICOS.

[0045] In another preferred embodiment, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO.16.

[0046] In another preferred embodiment, the amino acid sequence of CD3ζ is shown as SEQ ID NO.17.

[0047] In another preferred embodiment, the amino acid sequence of IL2 is shown as SEQ ID NO.18.

[0048] In another preferred embodiment, IL2 is linked to the chimeric antigen receptor via a self-cleavage element at the N-terminus.

[0049] In another preferred embodiment, the self-cleaving element comprises a 2A peptide, preferably a T2A peptide.

[0050] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.19, 20, 21, 22 or 23.

[0051] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.19 or 23.

[0052] In a second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the chimeric antigen receptor (CAR) described in the first aspect of the present invention.

[0053] In another preferred embodiment, the nucleic acid molecule is isolated.

[0054] In the third aspect of the present invention, a vector is provided, wherein the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0055] In another preferred embodiment, the vector is selected from the following group: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or a combination thereof.

[0056] In another preferred embodiment, the vector is selected from the following group: plasmid, viral vector.

[0057] In another preferred embodiment, the vector is in the form of a virus particle.

[0058] In another preferred embodiment, the vector is a lentiviral vector.

[0059] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the third aspect of the present invention or a chromosome in which the exogenous nucleic acid molecule described in the second aspect of the present invention is integrated or expresses the CAR described in the first aspect of the present invention.

[0060] In another preferred embodiment, the host cells include eukaryotic cells and prokaryotic cells.

[0061] In another preferred embodiment, the host cell includes Escherichia coli.

[0062] In the fifth aspect of the present invention, an engineered immune cell is provided, wherein the immune cell expresses the CAR described in the first aspect of the present invention.

[0063] In another preferred embodiment, the engineered immune cells express interleukin 2 (IL-2).

[0064] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0065] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0066] In another preferred embodiment, the cells include T cells and NK cells.

[0067] In another preferred embodiment, the T cells are Vδ1 T cells.

[0068] In the sixth aspect of the present invention, a preparation is provided, which contains the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the immune cell described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0069] In another preferred embodiment, the preparation is a liquid preparation.

[0070] In another preferred embodiment, the preparation is in the form of an injection.

[0071] In another preferred embodiment, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0072] In another preferred embodiment, the preparation further comprises a second anti-tumor active ingredient, preferably a second antibody or a chemotherapeutic agent.

[0073] In another preferred embodiment, the chemotherapeutic agent is selected from the following group: docetaxel, carboplatin, or a combination thereof.

[0074] In the seventh aspect of the present invention, there is provided a use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the immune cell described in the fifth aspect of the present invention, or the preparation described in the sixth aspect of the present invention for preparing a drug or preparation for preventing and / or treating cancer or tumors.

[0075] In another preferred embodiment, the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.

[0076] In another preferred embodiment, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0077] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

[0078] In another preferred embodiment, the tumor is a B7-H3 positive tumor, preferably a tumor with high expression of B7-H3.

[0079] In another preferred embodiment, the B7-H3 positive tumor is selected from the following group: neuroblastoma, lung cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, prostate cancer, brain glioma, or a combination thereof.

[0080] In the eighth aspect of the present invention, a kit for preparing the host cell described in the fourth aspect of the present invention is provided, the kit comprising a container, and the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention located in the container.

[0081] In a ninth aspect of the present invention, a method for preparing an engineered immune cell is provided, wherein the immune cell expresses the CAR according to the first aspect of the present invention, and the method comprises the following steps:

[0082] (a) providing immune cells to be modified; and

[0083] (b) transducing the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention into the immune cell, thereby obtaining the engineered immune cell.

[0084] In another preferred embodiment, the engineered immune cells are CAR-T cells or CAR-NK cells.

[0085] In another preferred embodiment, the method further comprises the step of testing the function and effectiveness of the obtained engineered immune cells.

[0086] In another preferred embodiment, the engineered immune cells are CAR-Vδ1 T cells.

[0087] In the tenth aspect of the present invention, a method for treating a disease is provided, comprising administering an appropriate amount of the vector described in the third aspect of the present invention, the immune cell described in the fifth aspect of the present invention, or the preparation described in the sixth aspect of the present invention to a subject in need of treatment.

[0088] In another preferred embodiment, the disease is cancer or tumor.

[0089] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 AF show the steps and methods for in vitro expansion of Vδ1 T cells. Figure 1 A shows a schematic diagram of the process of preparing CAR-Vδ1T cells from healthy donor PBMCs in vitro; Figure 1 B shows the purity of the prepared Vδ1T cells detected by flow cytometry (CD3+Vδ1T+); Figure 1 C shows the remnants of αβT cells in the prepared Vδ1T cells; Figure 1D shows the in vitro Vδ1 cell expansion fold, with an average expansion fold of 10997; Figure 1 E shows the flow cytometry detection of Vδ1T cell phenotypes, and the memory phenotypes were distinguished by CD45RA and CD27 protein expression (Tnaive: CD45RA+CD27+, TCM: CD27+CD45RA-, TEM: CD27-CD45RA-, TEMRA: CD27-CD45RA+); Figure 1 F shows the transfection efficiency of CAR lentivirus in Vδ1T cells prepared from different donors.

[0091] Figure 2A -G shows the in vitro functional validation of B7-H3-CAR-Vδ1T constructed with different CAR structures. Figure 2A Schematic diagram of the B7-H3-CAR structure containing different costimulatory molecules is shown; Figure 2B The expression of surface molecules CD69, CD25 and PD1 and the release of cytokines IL-2, GranzymeB and TNF-α in the supernatant of the co-incubation of the four CAR-Jurkat cells with the B7-H3-Raji cell line were shown; Figure 2C The positive rates of Vδ1T cells transfected with four CAR lentiviruses under the same MOI conditions (MOI = 10) are shown; Figure 2D The results show that the four CAR-Vδ1T cells can kill B7-H3-Raji cells in vitro (E:T=3:1, T=24h) and inhibit the growth of natural solid tumor cell lines (PC-3, DU145, E:T=1:1, T=48h); Figure 2E The release of cytokines (IFN-γ, TNF-α, and Granzyme B) in the supernatant of the four B7-H3-CAR-Vδ1T cells co-incubated with the above tumor cell lines is shown; Figure 2F The loss of B7-H3 antigen on the surface of target cells after co-incubation of Vδ1T and four CAR-Vδ1T with B7-H3-Raji cells (E:T=1:1, T=16h) was shown; Figure 2G The figure shows the killing detection of target cells by four types of CAR-Vδ1T cells following repeated stimulation of B7-H3-Raji cells (the effector-target ratio was 5:1, and target cells were supplemented every 24 hours).

[0092] Figure 3A -H showed that co-expression of IL-2 could significantly improve the functional persistence of B7-H3-CAR-Vδ1T cells in vitro. Figure 3A It was shown that the addition of exogenously added cytokine IL-2 can significantly improve the anti-tumor persistence of CAR-Vδ1T cells; Figure 3B The changes in CAR-Vδ1T positivity before and after repeated antigen stimulation are shown; Figure 3C The schematic diagram of the structure of AQ-CAR is shown, which is based on AG-CAR and co-expresses the secretory cytokine IL-2 (T2A connection); Figure 3D The positive rate detection of two CAR-Vδ1T cells prepared in vitro (top) and the release of cytokine IL-2 in the supernatant of single culture (bottom) are shown; Figure 3E The absolute value statistics of the two CAR-Vδ1T cells in persistent killing of target cells B7-H3-Raji are shown (the effector-target ratio was initially set to 3:1, 2*10^5 target cells were added every 24 hours, and the number of repeated antigen stimulations was set to 5 times); Figure 3F The released contents of cytokines (IFN-γ, IL-2, GranzymeB and TNF-α) in the co-incubation supernatant after each antigen repeated stimulation are shown; Figure 3G The two CAR-Vδ1 T cells showed the inhibitory effects on the growth of various natural solid tumor cells; Figure 3H The release levels of cytokines (IFN-γ, IL-2, Granzyme B and TNF-α) in the supernatant of two CAR-Vδ1T cells co-cultured with various natural solid tumor cells are shown.

[0093] Figure 4A -D shows that the B7-H3-CAR-Vδ1T cells of the present invention have no tumor-promoting characteristics and have a lower HVGR. Figure 4A The release of IL-17A and VEGF in the supernatant of Vδ1T, AQ-CAR-Vδ1T, and AQ-CAR-T cells cultured alone until D9 and D16 is shown; Figure 4B The release of IL-17A and VEGF in the co-culture supernatant after repeated stimulation of Vδ1T, AQ-CAR-Vδ1T, and AQ-CAR-T cells by target cell B7-H3-Raji is shown (E:T=5:1. T=24h, the number of repeated antigen stimulations was set to 5 times); Figure 4C It shows that activated T cells and NK cells express CD69 and CD137 molecules on their surfaces; Figure 4D The in vitro clearance effect of activated T and NK cells on AQ-CAR-Vδ1T cells was shown (E:T=1:5, 1:1, 3:1, T=24h).

[0094] Figure 5A -K showed that AQ-CAR-Vδ1T cells exhibited significant in vivo anti-tumor efficacy against multiple solid tumor models. Figure 5A The experimental scheme of establishing pancreatic cancer (SW1990) model subcutaneously in mice is shown; Figure 5B The tumor volume of mice in each experimental group was monitored, and this value was the average tumor volume of 6 mice; tumor tissues of mice in each group were collected and photographed 35 days after administration; Figure 5C The infiltration of CD3+ cells in the tumor tissues of two mice in the Vδ1T and AQ-CAR-Vδ1T groups that had no therapeutic effect was detected; Figure 5D The experimental scheme of establishing subcutaneous neuroblastoma (SK-N-AS) model in mice is shown; Figure 5E The tumor growth dynamics of mice in each experimental group are shown. This value is the average tumor volume of 6 mice. Tumor tissues of mice in each group were collected and photographed 20 days after administration. Fig. 5F The infiltration of CD3+ cells in the tumor tissue of one mouse with no therapeutic effect in the Vδ1T and AQ-CAR-Vδ1T groups was detected; Figure 5G The experimental scheme of establishing a subcutaneous lung cancer (A549) model in mice is shown; Figure 5H The tumor growth dynamics of mice in each experimental group are shown. This value is the average tumor volume of 6 mice. Tumor tissues of mice in each group were collected and photographed 37 days after administration. Fig.5I Shown is the re-establishment of a mouse subcutaneous neuroblastoma model (SK-N-AS); Figure 5J The tumor growth volume monitoring of each group of mice in the experiment is shown. This value is the average tumor volume of 5 mice. Tumor tissues of mice in each group were collected and photographed 15 days after administration. Figure 5K The flow cytometry analysis was performed on the CD3+ cell infiltration and CAR-Vδ1 cell positivity in the tumor tissues of the remaining two mice in the AQ-CAR-Vδ1T group.

[0095] Fig. 6A -C shows that AQ-CAR-Vδ1T cells exhibit good safety in vivo. Fig. 6A The figure shows the real-time monitoring of the weight of mice during the experiment for three solid tumor mouse models (from left to right: pancreatic cancer model, neuroblastoma model and lung cancer model); Figure 6B It shows that for the neuroblastoma model, at the end point of the experiment (20 days after administration), mouse tissues (heart, liver, spleen, lung, and kidney) were collected for immunohistochemical analysis, and their tissues were pathologically analyzed and CD3+ cell infiltration was detected; Figure 6C The figure shows that for the pancreatic cancer model (top) and the lung cancer model (bottom), mouse tissues (heart, liver, spleen, lung, kidney and bone marrow) in the CAR-Vδ1T group were collected at the experimental endpoint, and the infiltration of CD3+ cells in each tissue was detected by flow cytometry.

[0096] Fig. 7A -F shows that co-expression of IL-2 significantly upregulated genes and signaling pathways related to anti-tumor, homing, and proliferation in AQ-CAR-Vδ1T cells. Fig. 7A Dimensionality reduction and clustering analysis of CAR-Vδ1T cells are shown; Figure 7BDetermination of subpopulation cell types based on gene expression is shown; Figure 7C The percentage of each subpopulation in the total cells in both groups of cells is shown; Fig.7D A heatmap display of the gene expression differences between the two groups as a whole is shown; Fig. 7E A heat map display of the differences in gene expression of the same subpopulations in the two groups is shown; Figure 7F The signaling pathways that were upregulated in the AQ group compared to the two groups as a whole (based on GSEA analysis) are shown.

[0097] Fig. 8A -E shows the in vitro functional validation of B7-H3-CAR-Vδ1T constructed with different CAR structures. Fig. 8A The detection of B7-H3 antigen expression on the surface of various natural solid tumor cells is shown; Figure 8B The construction of the overexpression cell line B7-H3-Raji is shown; Figure 8C Four B7-H3-CAR-jurkat cell line constructions are shown; Fig.8D It shows the percentage of CAR-Vδ1T cells and target cells in the total cells after each antigen stimulation with B7-H3-Raji tumor antigen stimulation; Fig. 8E The in vivo neuroblastoma mouse experimental design is shown. IL-2 is injected intraperitoneally (injected once every other day, injection dose 2000 IU / mouse); tumor growth kinetics of each group of mice in the experiment are monitored.

[0098] Fig.9A -C shows that jurkat-AQ-CAR cells can be specifically activated by B7-H3 antigen. Fig.9A The construction of the overexpression cell line jurkat-AQ-CAR and the release of cytokine IL-2 in the supernatant are shown; Fig. 9B The expression of surface molecules CD69 and PD1 of the two CAR-Jurkat cells after co-incubation with the B7-H3-Raji cell line is shown; Fig. 9C The release of cytokines IL-2, GranzymeB, and TNF-α in the supernatant after in vitro activation is shown.

[0099] Fig. 10A -B shows that the proliferation ability of AQ-CAR-Vδ1T cells is stronger than that of AG-CAR-Vδ1T cells. Fig. 10A The absolute statistics of the number of two types of CAR-Vδ1T cells and the percentage of total cells after each repeated antigen (B7-H3-Raji) stimulation are shown; Fig. 10BThe CFSE dilution method is shown: two types of CAR-Vδ1T cells were repeatedly stimulated three times with B7-H3-Raji cells in vitro (E:T=1:1, T=24h), and the surface fluorescence values ​​of CAR-Vδ1T cells were detected by flow cytometry every 24h, and 2*10^5 target cells were added at the same time.

[0100] Fig.11 It was shown that AQ-CAR-Vδ1T cells exhibited significant in vivo anti-tumor efficacy against a variety of solid tumor models. The tumor volume and weight of each group of mice were counted at the end of the experiment; Fig.11 A is a pancreatic cancer model; Fig.11 B is a neuroblastoma model; Fig.11 C is a lung cancer model.

[0101] Fig. 12A -Showed that Vδ1T cells themselves express multiple proteins associated with anti-tumor activity. Fig. 12A The results of flow cytometry analysis of multiple protein profiles on the surface of Vδ1T cells (left) and CAR-Vδ1T cells (right) harvested on D19 are shown; Fig. 12B The results of surface protein detection of Vδ1T cells sorted from the same donor on day D0 are shown. DETAILED DESCRIPTION

[0102] After extensive and in-depth research, the inventors have constructed a CAR-Vδ1T cell targeting B7-H3 for the first time. The results of transcriptome sequencing and mass spectrometry flow cytometry show that the B7-H3-CAR-Vδ1T cell of the present invention has significant high expression of anti-tumor related genes and proteins, and has shown anti-tumor effects on solid tumors in in vitro and in vivo functional verification, and basically does not release the tumor-promoting cytokine IL-17A. When IL-2 is co-expressed, the persistence of B7-H3-CAR-Vδ1T cells is improved, and they mainly home to solid tumor tissues, with good safety. On this basis, the present invention is completed.

[0103] the term

[0104] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0105] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0106] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0107] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0108] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0109] γδ T cells

[0110] γδT cells can be divided into two main subpopulations based on their δ chain usage preference, among which Vδ2+ cells prefer to co-express with Vγ9 chain, while Vδ2- cells can pair with a range of Vγ chains. Vδ1T subtype cells are the main γδT cells in peripheral blood circulation. Vδ1T cells have natural anti-tumor activity and strong ability to infiltrate the microenvironment of solid tumors. Previous studies have shown that γδT cells play an important role in cancer treatment, and γδT cells in tumors are the most favorable prognostic indicator in various cancers. Vδ1T cells can also act as specialized APCs after activation and play an important role in enhancing immune responses.

[0111] B7-H3

[0112] B7-H3 is an abnormal O-glycosylated antigen formed by the loss of COSMC gene function. It is highly abnormally expressed on the surface of various tumor cells such as breast cancer, gastric cancer, lung cancer, and pancreatic cancer, but is not expressed or expressed at a low level in normal tissues. Therefore, B7-H3 antigen is an ideal target for immunotherapy of many solid tumors. Studies have shown that 5E5 CAR-αβT cells targeting B7-H3 antigen have very effective anti-tumor effects on T cell leukemia and pancreatic cancer in vitro and in animal models in vivo, indicating that immunotherapy targeting B7-H3 antigen is very feasible for the treatment of tumor diseases.

[0113] Chimeric Antigen Receptor (CAR)

[0114] The design of CARs has gone through the following process: The first generation of CAR has only one intracellular signal component CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause short-term T cell proliferation and less cytokine secretion, but cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical efficacy. The second generation of CARs introduces a co-stimulatory molecule such as CD28, 4-1BB, OX40, and ICOS based on the original structure. Compared with the first generation of CARs, the function is greatly improved, further enhancing the persistence of CAR-T cells and the ability to kill tumor cells. On the basis of the second generation of CARs, some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and developed into the third and fourth generation of CARs.

[0115] The chimeric antigen receptor (CAR) of the present invention is a second-generation CAR, including an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain including co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands. In one embodiment, the CAR of the present invention comprises a co-stimulatory signal molecule from source CD28.

[0116] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0117] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting B7-H3. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells to not grow, be prompted to die or be affected in other ways, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain.

[0118] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the scFv comprises an antibody that specifically recognizes B7-H3, preferably a humanized single-chain antibody.

[0119] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0120] The CAR cells of the present invention can exert a significantly enhanced anti-tumor effect in the presence of IL-2, therefore, the CAR of the present invention can be co-expressed with IL-2 to improve the therapeutic effect. In one embodiment, the IL-2 coding sequence can be connected to the CAR sequence of the present invention by a self-cleaving element (such as T2A) so that it can be co-expressed in the cell. In addition, cells can also be modified in other ways to express IL-2, and such methods are well known to those skilled in the art.

[0121] Carrier

[0122] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0123] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0124] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0125] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0126] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0127] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0128] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0129] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0130] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0131] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0132] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0133] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0134] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0135] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0136] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0137] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0138] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0139] preparation

[0140] The present invention provides a preparation containing the CAR-T cells of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×104 -1×10 7 cells / ml.

[0141] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0142] Therapeutic applications

[0143] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the tumor cell marker B7-H3, synergistically activate T cells, and induce a T cell immune response, thereby significantly improving its efficiency in killing tumor cells.

[0144] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cell of the present invention to the mammal.

[0145] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach has a very low probability of graft-versus-host disease, and antigens are recognized by T cells in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0146] In one embodiment, the CAR-T cells of the present invention can undergo a robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-B7-H3 CAR-T cells cause a specific immune response to cells that are positive for anti-B7-H3.

[0147] Although the data disclosed herein specifically discloses a lentiviral vector comprising an anti-B7-H3 scFv, hinge and transmembrane regions, an intracellular co-stimulatory signal molecule, and a CD3ζ signaling domain, the present invention should be construed to include any number of variations to each of the construct components.

[0148] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors.Cancers may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors.The types of cancers treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas.Also include adult tumors / cancers and childhood tumors / cancers.

[0149] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0150] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

[0151] In a preferred embodiment, the treatable cancer is a B7-H3 positive tumor, such as neuroblastoma, lung cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, prostate cancer, brain glioma, etc.

[0152] The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0153] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0154] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0155] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0156] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0157] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0158] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0159] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6The T cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0160] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0161] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, simultaneously or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0162] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10The modified T cells of the present invention (e.g., CAR-T cells) are administered to the patient, for example, by intravenous infusion.

[0163] The main advantages of the present invention include:

[0164] (a) The CAR-Vδ1T cells of the present invention have strong specific cytotoxic effects on a variety of tumor cells in vitro.

[0165] (b) The present invention found that IL-2 can enhance the cytotoxicity of CAR-Vδ1T cells and improve the persistence of cells.

[0166] (c) In experimental animals, the CAR-Vδ1T cells of the present invention have no obvious toxic side effects and do not secrete the tumor-promoting factor IL-17A.

[0167] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0168] Materials and Methods

[0169] Tumor cell line culture

[0170] Raji cells (ATCC source) were transfected with lentivirus to construct a B7-H3-overexpressing Raji cell line, and the B7-H3-Raji cell line was cultured in RPMI 1640 medium containing 10% FBS. The culture conditions of natural solid tumor cell lines are as follows: PC-3 (from ATCC), DU145 (from ATCC) and HGC-27 (from Nanjing Kebai Company) were all cultured in RPMI1640 medium containing 10% FBS; the culture conditions of SK-N-AS (from Nanjing Kebai Company) were DMEM+10% FBS+1% NonEssential Amino Acids (NEAA); HuH-7 (from Nanjing Kebai Company), MDA-MB-231 (from Nanjing Kebai Company) and SW1990 (from ATCC) cells were all cultured in DMEM medium containing 10% FBS; A549 (from Nanjing Kebai Company) was cultured in F12K medium containing 10% FBS; U-138MG (from Nanjing Kebai Company) was cultured in MEM+10% FBS+1% NonEssential Amino Acids (NEAA)+1mM Sodium Pyruvate.

[0171] Preparation of CAR-Vδ1T cells from peripheral blood

[0172] Single blood samples from healthy donors were purchased from Bokang Company. PBMCs were extracted by Ficoll gradient centrifugation. Vδ1T cells in PBMCs were sorted using the Anti-TCR Vδ1 magnetic bead sorting kit (Miltenyi Biotec). The sorted Vδ1T cells were placed in a 24-well plate for activation and transfected with CAR lentivirus 48 hours later. The cells were cultured in vitro and collected on D19. The in vitro culture conditions for CAR-Vδ1T cells were Optivitro T medium (ExcellBio) + 200IU / ml IL-2 (nearshore) + 20ng / ml IL-21 (nearshore). In addition to the addition of cytokines during the culture process, irradiated aAPC cells were added on D5 and D17, with a supplement ratio of 2:1 (aAPC: Vδ1T).

[0173] Flow cytometry

[0174] Anti-human CD3 antibody (BD) and anti-human Vδ1 antibody (eBioscience TM) The purity of the prepared Vδ1T cells was detected by flow cytometry. The antibody was diluted 1:100 and stained at room temperature for 20 minutes. After staining, it was washed twice with 1ml PBS (3000rpm, 3min), and finally resuspended with 300ul PBS for detection; except that the dilution factor of Fc antibody was 1:1000, the operation methods of other antibodies were the same. The expression of B7-H3 antigen on the surface of tumor cells was detected by anti-human B7-H3 antibody (MiltenyiBiotec); the positive rate of B7-H3-CAR was detected by anti-B7-H3-ScFv antibody (Suzhou Aikangde Company) or anti-human Fc antibody (Jackson, 1:1000 dilution); the cell phenotype was stained with anti-human CD45RA antibody (BD) and anti-human CD27 antibody (BD). Vδ1T cells infiltrating in tumor tissues were detected with anti-human CD3 antibody (BD). All samples were detected on a flow cytometer (ACEA / Novocyte), and some data were processed using FlowJo software.

[0175] CAR-jurkat in vitro activation assay

[0176] First, Jurkat cells were transfected with lentivirus containing different CAR fragments to construct different CAR-Jurkat cells. Before CAR-Jurkat cells were co-incubated with target cells B7-H3-Raji, the target cells were stained with CFSE (37°C, 15 min). After staining, they were washed once with 1 ml PBS. Then, CAR-Jurkat cells were added according to the effector-target ratio of 1:1, and the co-incubation system was 1 ml. The co-incubation medium was RPMI 1640 with 10% FBS. After 24 hours, the cells were collected, and the surface expression of CAR-Jurkat cells was detected by flow cytometry using anti-human CD25 (BD), anti-human CD69 (BD) and anti-human PD1 (BD) antibodies. The co-incubation supernatant was collected and the cytokine release was detected using a CBA kit.

[0177] In vitro toxicity test

[0178] In the cytotoxicity experiment, before CAR-Vδ1T cells were co-incubated with B7-H3-Raji cells, B7-H3-Raji cells were first stained with CFSE (37°C, 15min), and then washed with 1ml PBS; then different numbers of effector cells were added according to the different effector-target ratios set; cells were collected after 24h, and apoptosis reagents 5ul7-AAD antibody and 10ul Annexin V (Multi Sciences Biotech, Hangzhou, China) were added to each group, incubated at room temperature in the dark for 5min, and early apoptosis and late apoptosis of target cells were detected and counted by flow cytometry. At the same time, the co-incubation supernatant was collected for subsequent cytokine detection.

[0179] Antigen repeat stimulation experiment

[0180] In the antigen repeated stimulation system, the target cell B7-H3-Raji cells were still stained with CFSE. The number of groups was set according to the number of antigen stimulations during the first co-incubation and plating. Every 24 hours, one group of cells was collected and stained with 7-AAD antibody and Annexin V, and the target cell apoptosis was detected by flow cytometry. At the same time, the positive rate of CAR-Vδ1T cells was detected with anti-human FC antibody (Jackson, 1:1000 dilution), and the absolute value and percentage of CAR-Vδ1T cells were also counted. The remaining groups continued to add the same number of CFSE-labeled target cells as in the first plating. The next step is to repeat the test and add tumor cells every 24 hours, and the number of repeated stimulations is the number of antigen stimulations.

[0181] CFSE dilution method

[0182] Before co-incubation with target cells B7-H3-Raji, CAR-Vδ1T cells were stained with CFSE, the number of cells was added according to the set effect-target ratio, and the number of groups was set according to the number of target cell additions. After 48 hours, the CFSE fluorescence on the surface of CAR-Vδ1T cells was detected by flow cytometry, and the remaining groups were supplemented with the same number of tumor cells B7-H3-Raji as the first time, and repeated 3 times. The faster the CAR-Vδ1T cells proliferate under tumor antigen stimulation, the faster the CFSE fluorescence value on its surface is diluted, and the weaker the fluorescence value.

[0183] In vitro solid tumor cell growth inhibition assay (RTCA)

[0184] In vitro, the RTCA instrument (ACEA / xCELLigence) was used to monitor the inhibitory effect of CAR-Vδ1T cells on the growth of solid tumor cells in real time. The tumor cells (5000-10000) were plated in a well plate with a chip the day before, and the system was 100ul. After the tumor cells grew for 24h, different numbers of effector cells CAR-Vδ1T and control cells were added according to the effect-target ratio. The final co-incubation system was set to 200ul, and the monitoring time was set. The monitoring process was carried out in a 37°C, 5% CO2 incubator.

[0185] Cytokine detection

[0186] In this study, all cytokines released from the co-incubation supernatant were detected by flow cytometry using a cytokine CBA kit (BD), and the specific operation was carried out according to the product instructions. The types of cytokines detected included IL2, TNF-α, IFN-γ, and Granzyme B.

[0187] In vivo experiments

[0188] The mice used in this study were all 5-6 weeks old immune-deficient NPG mice. The solid tumor mouse models involved were all inoculated with tumor cells subcutaneously, and the effector cells were all injected intravenously once. There were 3 groups: 1: PBS group; 2: Vδ1T group; 3: CAR-Vδ1T group. The number of tumor cells inoculated in the pancreatic cancer SW1990 model was 1*10^6 / mouse, and 50% of matrix gel was added to assist tumor formation; the total number of effector cells infused back after D7 days of inoculation was 2*10^7 / mouse, including 8*10^6 CAR-positive cells / mouse; the number of tumor cells inoculated in the neuroblastoma SK-N-AS model was 3*10^6 / mouse, and 50% of matrix gel was added to assist tumor formation; the total number of effector cells infused back after D10 days of inoculation was 2*10^7 / mouse, including 1*10^7 CAR-positive cells / mouse; the number of tumor cells inoculated in the lung cancer A549 model was 1*10^6 / mouse; the total number of effector cells infused back after D19 days of inoculation was 2*10^7 / mouse, including 1.4*10^7 CAR-positive cells / mouse. All mouse models monitored the tumor volume and mouse weight regularly throughout the experiment, and observed the growth status of the mice.

[0189] Immunohistochemistry

[0190] For the SK-N-AS neuroblastoma model, other tissues of mice in the PBS and CAR-Vδ1T groups were collected at the end of the experiment, including heart, liver, spleen, lung and kidney, for immunohistochemical detection. The specific operation included paraffin embedding of tissues, sectioning, and then staining with anti-human CD3 antibody.

[0191] Mass cytometry

[0192] First, we determined the types of surface proteins of Vδ1T and CAR-Vδ1T to be detected, and sent the Vδ1T and CAR-Vδ1T cells cultured to the end point (D19 days) for testing respectively. The mass spectrometry flow cytometry was completed by a third-party testing company (Protin). Mass cytometry is a flow technology that uses the principle of mass spectrometry to perform multi-parameter detection on single cells. Cells are labeled with metal label antibodies and passed through the ICP mass spectrometry detection device one by one. Then, various metal labels in each cell are quantitatively detected, and the content of each target protein in each cell is obtained.

[0193] Single-cell sequencing

[0194] After AG-CAR-Vδ1T and AQ-CAR-Vδ1T cells were repeatedly stimulated 4 times by B7-H3-Raji cells in vitro, tumor cells were added every 24 hours. After the stimulation, the cell suspension was collected and sent to a third party for single-cell sequencing (Xingeyuan Company). Seurat v3.1.2 was used for quality control, dimensionality reduction and clustering. The cell type of each subpopulation was determined based on gene expression. CeleScope v1.5.2 with default parameters was used to process the original reads to generate gene expression profiles. The genes mainly included genes closely related to cell functions (proliferation, cytotoxicity, chemotaxis, etc.). At the same time, the GSEA analysis method was used to analyze and compare related signaling pathways (T cell proliferation, activation, killing, chemotaxis, etc.).

[0195] Statistical analysis

[0196] GraphPad Prism was used for statistical analysis (V8.01), and T-test was used to analyze the statistical significance of each group (*P<0.01, **P<0.005, ***P<0.0005, ****P<0.0001, ns: not significant, unpaired Student's t-test).

[0197] Example 1 Successfully expanded Vδ1T cells with excellent properties in vitro

[0198] A method for large-scale preparation of Vδ1T cells using PBMC from healthy donors was successfully established in vitro. Figure 1 A), the entire expansion cycle is 19 days. At the end of cell culture, we tested the purity of Vδ1T cells prepared from PBMC cells of 6 different healthy donors, and the purity was more than 95% ( Figure 1 B), the residual proportion of αβT cells is less than 5% ( Figure 1C), indicating that the purity of the expanded Vδ1T cells is very high; the average expansion multiple of Vδ1T cells is 10997 times ( Figure 1 D) (5800-28113 times), and the expansion multiple was also very high. We further tested the phenotype of Vδ1T cells, and the results showed that most donor-derived Vδ1T cells were mainly Tnaive cells with a low degree of differentiation ( Figure 1 E). In addition, we used the optimized CAR lentivirus to transfect the amplified Vδ1T cells, and the transfection efficiency was also very high, with an average CAR positive rate of 63% ( Figure 1 F) (46% to 88%). The above results show that the Vδ1T cells amplified by the present invention have several excellent characteristics, including: high Vδ1T cell purity and amplification multiple, the cell subtype is mainly Tnaive cells with a low degree of differentiation, and the CAR positivity rate of CAR-Vδ1T cells is also very high.

[0199] Example 2 In vitro functional verification of B7-H3-CAR-Vδ1T constructed with different CAR structures

[0200] In order to screen the optimal B7-H3-CAR structure suitable for Vδ1T cells, four B7-H3-CARs containing different co-stimulatory molecules were constructed (the B7-H3 antibody sequence was provided by Boshengji Pharmaceutical Technology (Suzhou) Co., Ltd.). The schematic diagram of the B7-H3-CAR structure is shown in the figure Figure 2A In order to compare the functions of B7-H3-CAR with different structures, in addition to screening a variety of B7-H3 positive solid tumor cell lines as target cells ( Fig. 8A ), and also constructed a B7-H3-Raji cell line overexpressing B7-H3 using Raji cells ( Figure 8B In order to verify whether the B7-H3-CAR with four different CAR structures can be specifically activated by target cells, four B7-H3-CAR-jurkat cell lines were first constructed by lentiviral transfection ( Figure 8C ), and then the four B7-H3-CAR-jurkat cells were incubated with B7-H3-Raji cells respectively. The results showed that compared with the control group, after the four CAR-jurkat cells were co-incubated with target cells, the cell surface activation proteins CD69, CD25 and immune checkpoint PD1 molecules were specifically increased to varying degrees. At the same time, significant specific release of cytokines IL-2, Granzyme B and TNF-α was also detected in the supernatant incubated with target cells ( Figure 2B The above results indicate that the four B7-H3-CARs with different structures can be specifically activated by B7-H3-positive target cells.

[0201] Then, the four prepared B7-H3-CAR lentiviruses were used to transfect Vδ1T cells under the same MOI condition (MOI = 10) to prepare B7-H3-CAR-Vδ1T cells. The results showed that the positive rates of CARs with different structures varied greatly (15% to 69%). The transfection efficiency of AG structure was the highest, while the transfection efficiency of AI and GH was lower ( Figure 2C ), which indicates that different CAR structures will affect the transfection efficiency of CAR-Vδ1T cells. Compared with Vδ1T cells not transfected with CAR, the four B7-H3-CAR-Vδ1T cells have obvious specific killing effects on B7-H3-Raji cell lines overexpressing B7-H3 and tumor cell lines naturally expressing B7-H3 (PC-3, Du145). Figure 2D ); In addition, the release of cytokines in the co-incubation supernatant in the above experiment was also analyzed. The results also showed that compared with Vδ1T cells not transfected with CAR, the anti-tumor active cytokines IFN-γ and TNF-a in the supernatant incubated with B7-H3-CAR-Vδ1T and target cells were released to varying degrees ( Figure 2E Moreover, neither the Vδ1T cells that were not transfected with CAR nor the B7-H3-CAR-Vδ1T cells were incubated with B7-H3-Raji cells, which resulted in no significant downregulation of the B7-H3 antigen on the target cells ( Figure 2F ), indicating that the probability of tumor immune escape due to antigen downregulation after treatment with CAR-Vδ1T cells is relatively low.

[0202] Finally, in order to further compare the in vitro killing persistence of B7-H3-CAR-Vδ1T cells with different CAR structures, B7-H3-CAR-Vδ1T was repeatedly stimulated with B7-H3-Raji cells. It was found that compared with CAR-Vδ1 with other structures, AG-CAR-Vδ1T had relatively strong killing ability. However, no matter which structure of CAR-Vδ1T, after four times of repeated antigen stimulation, the in vitro killing ability of target cells was significantly reduced ( Figure 2G ), indicating that the CAR-Vδ1T cells constructed with the traditional CAR structure have a weak ability to kill target cells persistently, and the CAR structure needs to be further optimized to improve the persistence of CAR-Vδ1T cells in vitro.

[0203] Example 3 Co-expression of IL-2 can significantly improve the persistence of the anti-tumor effect of B7-H3-CAR-Vδ1T cells in vitro

[0204] The above results show that the anti-tumor persistence of CAR-Vδ1T cells in vitro is not ideal. In order to further improve the anti-tumor persistence of AG-CAR-Vδ1T cells, cytokines IL-2 and IL-15 were exogenously added in the killing experiment of repeated antigen stimulation. The results show that the anti-tumor persistence of AG-CAR-Vδ1T cells can be significantly improved in the group with the addition of cytokine IL-2 alone and the group with the addition of IL-2 and IL-15 at the same time. After 5 times of repeated antigen stimulation, the absolute value of tumor cell killing can still be maintained at more than 80% ( Figure 3A ), and the effect of the group with IL-2 added alone was not inferior to that of the group with cytokine IL-2 and IL-15 added at the same time. In addition, with repeated antigen stimulation, the proportion of CAR-Vδ1T cells in the group with cytokine IL-2 added showed an upward trend, and the proportion of tumor cells showed a downward trend ( Fig.8D ); and the CAR-Vδ1T positive rate did not decrease with repeated antigen stimulation ( Figure 3B ). The results showed that the addition of exogenous cytokine IL-2 can significantly enhance the in vitro anti-tumor persistence of CAR-Vδ1T cells. However, unlike the in vitro results, in the mouse experiment, it was found that neither the CAR-Vδ1T group alone nor the group with intraperitoneal injection of cytokine IL-2 (injected once every other day, each injection dose of 20,000 IU / mouse) produced obvious in vivo therapeutic effects ( Fig. 8E ), which is speculated to be related to the fact that intraperitoneal injection of IL-2 has a short half-life in vivo and is degraded quickly, making it insufficient to improve the persistence of CAR-Vδ1T cell function.

[0205] Next, a CAR structure that can co-express the secretory cytokine IL-2, namely AQ-CAR ( Figure 3C ), enhancing the anti-tumor effect of B7-H3-CAR-Vδ1T cells in vivo by secreting IL-2.

[0206] First, the AQ-CAR-jurkat cell line overexpressing AQ-CAR was constructed by lentiviral transfection, and IL-2 secreted in the culture supernatant of AQ-CAR-jurkat cells was also detected. Fig.9A ), indicating that CAR-jurkat cells with AQ-CAR structure can normally express IL-2. AQ-CAR-jurkat can also be activated by specific binding to B7-H3 antigen, and compared with AG-CAR-jurkat, AQ-CAR-jurkat cells are not overactivated by the secretion of cytokine IL-2 ( Fig. 9B , Fig. 9C In addition, the secretion of cytokine IL-2 can also be detected in the culture supernatant of AQ-CAR-Vδ1T cells prepared in vitro ( Figure 3D), the in vitro killing experiment of repeated antigen stimulation showed that compared with AG-CAR-Vδ1T, the in vitro anti-tumor persistence of AQ-CAR-Vδ1T cells co-expressing IL-2 was significantly improved ( Figure 3E ), the anti-tumor active cytokines IL-2, IFN-γ, Granzyme B and TNF-α in the co-incubation supernatant were also higher than those in the AG-CAR-Vδ1T group, and IL-2 was only detectable in the AQ-CAR-Vδ1T group ( Figure 3F ); In addition, with repeated antigen stimulation, the absolute value and percentage of AQ-CAR-Vδ1T cells in the total cells were significantly higher than those in the AG-CAR-Vδ1T group ( Fig. 10A ), indicating that AQ-CAR-Vδ1T cells have stronger proliferation ability; at the same time, the CFSE dilution method also found that AQ-CAR-Vδ1T cells have stronger proliferation ability under repeated antigen stimulation than AG-CAR-Vδ1T group ( Fig. 10B ). The above results showed that compared with AG-CAR-Vδ1T cells without IL-2, AQ-CAR-Vδ1T cells co-expressing IL-2 significantly improved the sustained killing ability of tumor cells, the release of specific cytokines, and better specific proliferation ability of CAR-Vδ1T cells.

[0207] Then, the growth inhibition of AQ-CAR-Vδ1T on various solid tumor cell lines was monitored in real time in vitro using an RTCA instrument, including SK-N-AS (neuroblastoma), A549 (lung cancer), SW1990 (pancreatic cancer), MDA-MB-231 (breast cancer), HuH-7 (liver cancer), HGC-27 (gastric cancer), DU145 (prostate cancer) and U-138MG (glioma). Compared with the unmodified Vδ1T group, CAR-Vδ1T could effectively inhibit the growth of tumor cells at a low efficiency target ratio ( Figure 3G ); the functional cytokines IL-2, IFN-γ, Granzyme B and TNF-α in the co-incubation supernatant were specifically released, and the absolute value of the release in the AQ-CAR-Vδ1T group was higher than that in the AG-CAR-Vδ1T group ( Figure 3H ), indicating that the constructed AQ-CAR-Vδ1T has significant in vitro anti-tumor effects on a variety of solid tumor cell lines.

[0208] Example 4 The B7-H3-CAR-Vδ1T cells of the present invention do not have the characteristics of promoting tumors and have a lower HVGR

[0209] Some studies have reported that there is a subpopulation of γδT cells that can release IL-17A and is considered to have a tumor-promoting effect. It was also found that the production of IL-17A in tumors can enhance the production of vascular endothelial growth factor (VEGF). Therefore, in order to explore whether this subpopulation exists in AQ-CAR-Vδ1T cells, we detected the content of IL-17A and VEGF in the supernatant of AQ-CAR-Vδ1T cells cultured on D7 and D16 days, respectively. The results showed that the release of IL-17A and VEGF was basically not detected, but the release of IL-17A and VEGF was detected in the supernatant of AQ-CAR-T ( Figure 4A ), and it was also found that AQ-CAR-Vδ1T cells did not produce IL-17A under repeated antigen stimulation ( Figure 4B ), and the amount of VEGF produced by AQ-CAR-Vδ1T cells was significantly reduced compared to that of Vδ1T cells. In addition, the subsequent single-cell sequencing results of AQ-CAR-Vδ1T cells did not detect the expression of the IL-17A gene ( Fig.7D ). The above results all indicate that the prepared B7-H3-CAR-Vδ1T cells do not have tumor-promoting characteristics.

[0210] In addition, γδT cells, as allogeneic cells, may also be eliminated by activated T cells or NK cells in the host in vivo, i.e., host-versus-graft response (HVGR). In order to verify whether the CAR-Vδ1T of the present invention can be eliminated by activated T cells and NK cells in vitro, activated T cells and NK cells were first prepared, and the activated T cells and NK cells expressed CD69 and CD137 proteins on their surfaces (data from one of the donors, Figure 4C ), and then used activated T cells and NK cells to perform in vitro killing experiments on the prepared AQ-CAR-Vδ1T cells. The results showed that the overall clearance effect of activated T cells or NK cells on AQ-CAR-Vδ1T cells was relatively weak ( Figure 4D ), indicating that the HVGR response of the prepared AQ-CAR-Vδ1T cells in vivo was also relatively weak.

[0211] Example 5 AQ-CAR-Vδ1T cells exhibit excellent in vivo anti-tumor effects on various solid tumor-bearing mouse models

[0212] To further evaluate the in vivo anti-tumor effect of AQ-CAR-Vδ1T cells, mouse models of three solid tumor cell lines were established, including a pancreatic cancer model (SW1990), a neuroblastoma model (SK-N-AS), and a lung cancer model (A549). First, a subcutaneous tumor model of SW1990 pancreatic cancer was established using immunodeficient NPG mice. AQ-CAR-Vδ1T cells were infused 7 days after inoculation ( Figure 5A); The tumor volume of each group of mice was monitored regularly during the entire experiment, and it was found that compared with the control group PBS group and Vδ1T group, the AQ-CAR-Vδ1T group could effectively inhibit the growth and proliferation of tumor cells in mice. It is exciting that the tumors of 4 out of 6 mice in the AQ-CAR-Vδ1T group were completely eliminated ( Figure 5B ) indicates that the prepared AQ-CAR-Vδ1T cells have a strong anti-tumor effect in vivo. All mice were killed 35 days after administration, and the tumor volume and weight at the end of the experiment were statistically compared ( Fig.11 A). In addition, CD3 positive cells were detected in the tumor tissue cells of the two mice in the Vδ1T group and the AQ-CAR-Vδ1T group that did not show significant effects. No CD3 positive cells were detected, indicating that there was no Vδ1T infiltration in the tumor tissues of these two mice ( Figure 5C ). A neuroblastoma cell (SK-N-AS) tumor-bearing mouse model was also established, and AQ-CAR-Vδ1T cells were infused 7 days after inoculation ( Figure 5D ), the AQ-CAR-Vδ1T group also effectively inhibited the growth and proliferation of the neuroblastoma cell line SK-N-AS in vivo, and the tumor tissues of 5 tumor-bearing mice were completely cleared ( Figure 5E ), indicating that the AQ-CAR-Vδ1T cells of the present invention also have significant in vivo anti-tumor effects on neuroblastoma. At the end of the experiment, the tumor volume and weight were statistically compared ( Fig.11 B). Similarly, no Vδ1T cell infiltration was detected in the tumor tissues of mice without drug response ( Fig. 5F Finally, a subcutaneous lung cancer tumor model was established in mice using the lung cancer tumor cell line A549, and the in vivo antitumor effect of AQ-CAR-Vδ1T on the lung cancer tumor model was further evaluated ( Figure 5G ), the results also showed that AQ-CAR-Vδ1T can completely inhibit the in vivo growth of lung cancer cell line A549, and the tumors of 6 mice in the experimental group were completely eliminated ( Figure 5H ), the results were very encouraging, and statistical comparisons of tumor volume and weight were also performed at the end of the experiment ( Fig.11 C). The above results show that AQ-CAR-Vδ1T cells co-expressing IL-2 can significantly inhibit the growth and proliferation of tumor cells in pancreatic cancer-bearing mice, neuroblastoma-bearing mice, and lung cancer-bearing mice models, and exhibit a strong in vivo anti-tumor effect on solid tumors.

[0213] In order to verify whether the efficacy of AQ-CAR-Vδ1T in mice in the above animal experiments is affected by the infiltration and proliferation of CAR-Vδ1T cells, a mouse experiment with neuroblastoma SK-N-AS was designed ( Fig.5I). By real-time monitoring of the tumor size of each group of mice ( Figure 5J ), the mice were killed before the tumor completely disappeared, and the successful expansion of CAR-Vδ1T cells in the mouse tumors was detected to see if it was related to the elimination of the tumor. In the AQ-CAR-Vδ1T group, the tumors of three mice were completely eliminated, the tumor of one mouse was small (almost eliminated), and the tumor of one mouse was still growing. These two mice were killed, and the tumor tissues were taken for flow cytometry. The results showed that no Vδ1T cell infiltration was detected in the tumor cells of the mouse whose tumor continued to grow, while in another mouse whose tumor was small and almost eliminated, Vδ1T cell infiltration was detected, and the expansion of CAR-Vδ1T cells was also detected ( Figure 5K ). This result indicates that the efficacy of AQ-CAR-Vδ1T in mice is related to the infiltration and proliferation of Vδ1T cells in tumor lesions.

[0214] Example 6 AQ-CAR-Vδ1 T cells show good safety in vivo

[0215] In the above three mouse experiments, the weight of all mice was monitored in real time. During the whole experiment, the weight of mice remained stable and no symptoms of GVHD were observed ( Fig. 6A In the SK-N-AS mouse model of neuroblastoma, the organ tissues of the mice were collected at the end of the experiment for immunohistochemical detection. The immunohistochemical results showed that there were no pathological abnormalities in the mouse tissues (heart, liver, spleen, lung, and kidney), and no infiltration of CD3+ cells was detected ( Figure 6B In addition, tissue cells (heart, liver, spleen, lung, and kidney) of mice in pancreatic cancer models and lung cancer models were collected for flow cytometry. The results showed that no Vδ1T cells were detected in any tissue, indicating that Vδ1T cells would not infiltrate normal mouse tissues ( Figure 6C ). The above results show that the prepared AQ-CAR-Vδ1T cells have good safety.

[0216] Example 7 Co-expression of IL-2 significantly up-regulated anti-tumor, homing, proliferation and other related genes and related signaling pathways in AQ-CAR-Vδ1T cells

[0217] The above results show that AQ-CAR-Vδ1T cells co-expressing IL-2 show excellent anti-tumor efficacy both in vivo and in vitro. In order to further explore the reason why co-expression of IL-2 significantly enhances the anti-tumor function of CAR-Vδ1T, AG-CAR-Vδ1T and AQ-CAR-Vδ1T cells that were repeatedly stimulated with antigen (Raji-B7-H3) four times in vitro were sent for single-cell sequencing. CAR-Vδ1T cells were reduced in dimension and clustered into five different subgroups ( Fig. 7A), the cell type of each subtype was determined based on gene expression, and five subtypes were found to be related to cytotoxicity, chemotaxis, cell proliferation, and residual CD8 T cells, respectively, named Cytotoxic-gdT, CCR-gdT, MKi67-gdT, MCM-gdT, and CD8Teff (all percentages were within 3%) ( Figure 7B ).

[0218] Compared with the AG group, the proportion of two subpopulations related to cell proliferation (MKi67-gdT and MCM-gdT) in the AQ group was higher ( Figure 7C ); The heat map results of the overall gene expression differences between the two groups showed that compared with the AG group, co-expression of IL-2 could significantly upregulate cytotoxicity-related genes (GZMA, GZMB, IFNG, TNF, IFITM1, etc.) and natural toxicity receptors (NKG2D, DNAM1, NKP series, etc.) in AQ-CAR-Vδ1T cells; chemokine receptors related to homing ability (CCR series) and genes related to proliferation (MCM family genes, MKi67 series genes, etc.) were also significantly upregulated, and the absolute expression values ​​were high; in addition, genes related to immunosuppressive molecules were also upregulated, but the overall absolute expression values ​​were low; at the same time, the expression of downstream signaling pathway genes related to IL-2 production was also significantly upregulated in the AQ group ( Fig.7D ). In addition, when comparing the same subgroups between the two groups, it was also found that most of the functional enhancement genes were still more strongly expressed in the AQ group ( Fig. 7E ). The signal pathway results showed that the pathways related to T cell proliferation, activation, killing, chemotaxis, and cytokine production were upregulated in the AQ group ( Figure 7F ). Therefore, the results indicate that co-expression of IL-2 can significantly upregulate genes and related signaling pathways associated with functional enhancement, thereby improving the in vivo and in vitro efficacy of solid tumor cells.

[0219] In order to further explore the expression of proteins related to anti-tumor activity on the surface of Vδ1T cells and AQ-CAR-Vδ1 cells prepared by the present invention, the Vδ1T cells and AQ-CAR-Vδ1T cells prepared on day 19 were first sent for mass spectrometry analysis. The mass spectrometry results showed that both Vδ1T and AQ-CAR-Vδ1T cells expressed a variety of anti-tumor related proteins on their surfaces, including chemokine receptors (CXCR3, CXCR6, CCR9, etc.), natural toxicity receptors (NKG2D, DNAM1, etc.) and proteins related to the anti-tumor activity of Vδ1T cells (CD27, CD137, etc.); low expression of immunosuppressive molecules PD1 and LAG3, high expression of TIM-3 molecules ( Fig. 12A). Later, the surface proteins of Vδ1T cells from the same donor on day D0 were tested, and the results showed that the surface of the newly sorted Vδ1T cells expressed low or no expression of various proteins ( Fig. 12B ). Therefore, this result shows that the Vδ1T cells prepared by the present invention have the potential of anti-tumor function.

[0220] discuss

[0221] As a universal cell product, the core of CAR-Vδ1T cells is the persistence of anti-tumor effects, which is also the key factor in determining the efficacy of the cells. CAR-Vδ1T cells prepared with traditional CAR structures can show good anti-tumor effects in in vitro killing experiments. On this basis, in order to further improve the persistence of the anti-tumor effect of CAR-Vδ1T cells, the CAR structure was further optimized to co-express the cytokine IL-2.

[0222] Unlike traditional CAR-T cells, the release of cytokine IL-2 was not detected in the supernatant of co-incubation of CAR-Vδ1T cells prepared by traditional CAR structure with tumors. It is speculated that the reason for the unsatisfactory anti-tumor persistence of CAR-Vδ1T cells is probably due to the lack of cytokine IL-2, which leads to limited proliferation ability under repeated stimulation of tumor cells. The present invention further optimizes the traditional CAR structure so that it can secrete cytokine IL-2. The results indeed found that the persistence of optimized CAR-Vδ1T cells in killing target cells was significantly enhanced, and they showed very superior anti-solid tumor efficacy both in vivo and in vitro. Through single-cell transcriptome sequencing, it was found that co-expression of IL-2 can significantly upregulate the expression of anti-tumor, homing, proliferation and other related genes and related signal pathways in CAR-Vδ1T cells.

[0223] In addition, the flow cytometry of peripheral blood cells of mice in the above animal experiments found that the proliferation of CAR-Vδ1T cells was not detected, and the release of cytokine IL-2 was not detected. CAR-Vδ1T cells were not detected in other organs of mice, indicating that CAR-Vδ1T cells mainly home to solid tumor tissues and will not proliferate in large quantities in peripheral blood and damage other normal tissues. In addition, in vitro experiments also studied the absolute value of IL-2 released after co-incubation of different numbers of CAR-Vδ1T cells with different tumor cells (SW1990, SK-N-AS and A549). It was found that the absolute value of the release of exogenous IL-2 was also very low, and lower than the IL-2 released by traditional CAR-T cells themselves. Therefore, the possibility of side effects of IL2 in vivo is also very small, and the safety is relatively good.

[0224] The study found that in addition to the anti-tumor function, γδT cells also have a subgroup with a tumor-promoting function. This group of cells is mainly achieved by releasing the cytokine IL-17A. The produced IL-17A can increase the production of VEGF, attract regulatory T cells, neutrophils and MDSCs (bone marrow-derived suppressor cells) to migrate to the tumor site, and inhibit the anti-tumor activity of CD8 / CD4 positive T cells. Therefore, whether γδT cells are anti-tumor or tumor-promoting in tumor tissues depends on whether the released cytokines are anti-tumor factors IFN-γ or tumor-promoting factors IL-17A. The CAR-Vδ1T cells of the present invention do not release the cytokine IL-17A, whether under single culture conditions or under continuous stimulation of tumor antigens, and mainly release high concentrations of cytokines IFN-γ, indicating that the prepared CAR-Vδ1T cells are mainly anti-tumor functions and do not have tumor-promoting characteristics.

[0225] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0226] Table 1 Sequence Listing

[0227]

[0228]

[0229]

Claims

1. A chimeric antigen receptor (CAR), characterized in that The structure of the chimeric antigen receptor is shown in Formula I below: L-scFv-H-TM-C-CD3ζ-IL2(I) in, L is none or a signal peptide sequence; scFv is a scFv targeting B7-H3; H is the hinge region; TM is the transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; IL2 is none or interleukin 2 (IL-2) coding sequence; Each "-" is a connecting peptide or peptide bond connecting the above elements; The scFv has a light chain variable region and a heavy chain variable region targeting B7-H3, and the heavy chain variable region has the following complementary determining region CDR: HCDR1:GTFFSTY(SEQ ID NO.2), HCDR2: LSGGT (SEQ ID NO. 3), HCDR3: RYGHDHYYAMDY (SEQ ID NO.4); and The light chain variable region has the following complementarity determining regions CDR: LCDR1:AASSSVSSSYLH(SEQ ID NO.6) LCDR2: STSNLAS (SEQ ID NO. 7) LCDR3: QYHRSPWT (SEQ ID NO. 8).

2. The chimeric antigen receptor according to claim 1, wherein The chimeric antigen receptor has a hinge region derived from Fc, and its amino acid sequence is shown in SEQ ID NO.

11.

3. The chimeric antigen receptor according to claim 1, wherein The chimeric antigen receptor has a transmembrane domain and / or a co-stimulatory signal molecule derived from CD28.

4. The chimeric antigen receptor according to claim 1, wherein IL2 is linked to the chimeric antigen receptor via a self-cleavage element at the N-terminus.

5. The chimeric antigen receptor according to claim 1, wherein The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.19, 20, 21, 22 or 23.

6. The chimeric antigen receptor according to claim 1, wherein The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.19 or 23.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the chimeric antigen receptor (CAR) of claim 1.

8. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 7.

9. An engineered immune cell, characterized in that: The immune cells express the CAR according to claim 1.

10. The immune cell according to claim 9, characterized in that The immune cells are Vδ1T cells.

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