Multi-target chimeric antigen receptor for bridging hematopoietic stem cell transplantation and application thereof

Abstract

The invention discloses a multi-target chimeric antigen receptor for bridging hematopoietic stem cell transplantation and application of the multi-target chimeric antigen receptor, and relates to the field of biomedicine and immunotherapy. Through a multi-target strategy, the multi-target chimeric antigen receptor disclosed by the invention not only can effectively remove T cells so as to reduce the risk of anti-graft response (GVHD) of a recipient, but also can attack other target cells so as to improve the treatment effect. The invention further provides a preparation or a medicine for bridging hematopoietic stem cell transplantation, the preparation or the medicine is used before hematopoietic stem cell transplantation, an ideal immune environment can be created for transplantation, and the transplantation success rate is further increased. Compared with the prior art, the invention provides an effective multi-target CAR-immune cell therapy, the success rate of hematopoietic stem cell transplantation can be remarkably improved, adverse reactions in the treatment process are reduced, and tumor recurrence is prevented for a long time.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and immunotherapy, and in particular to a multi-target chimeric antigen receptor for bridging hematopoietic stem cell transplantation and an application thereof. Background Art

[0002] Multi-target therapy is a strategy to improve the therapeutic effect by simultaneously targeting multiple tumor-associated antigens or signaling pathways. This therapy uses different targets to overcome the heterogeneity and immune escape mechanisms of tumor cells, thereby enhancing the immune system's ability to recognize and kill tumors. In immunotherapy, multi-target therapy can be achieved by designing multiple chimeric antigen receptors (CARs) or combining multiple immunotherapy methods (such as CAR-T cells, immune checkpoint inhibitors, etc.).

[0003] CAR-immune cell therapy is a new type of immunotherapy that uses the patient's own immune cells or the immune cells of healthy donors, and genetically modifies them to express chimeric antigen receptors (CARs), thereby enhancing the ability of immune cells to recognize and kill target cells. At present, CAR-immune cells represented by CAR-T cells have made great progress. CAR-T cell therapy has achieved remarkable results in the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) and certain types of B-cell non-Hodgkin's lymphoma (NHL).

[0004] Hematopoietic stem cell transplantation (HSCT) is a medical treatment for a variety of blood system diseases, non-malignant blood diseases, autoimmune diseases, genetic metabolic diseases, etc. HSCT can rebuild the patient's hematopoietic function and immune system by collecting hematopoietic stem cells from the patient or donor and transplanting them into the patient. This treatment method is commonly used for leukemia, lymphoma, multiple myeloma and other serious blood system diseases. HSCT is divided into autologous transplantation (self-cells) and allogeneic transplantation (donor cells). The appropriate transplantation method is selected according to the type of disease and the specific situation of the patient.

[0005] In some cases, patients may first receive CAR-immune cell therapy to eliminate refractory or recurrent tumor cells, followed by HSCT to consolidate the treatment effect and restore normal hematopoietic and immune system function. This strategy can reduce the risk of tumor recurrence and improve long-term survival. In addition, CAR-immune cell therapy can also be used as a bridging therapy for HSCT, especially while patients are waiting for a transplant donor or preparing for a transplant. CAR-immune cell therapy can effectively control disease progression and provide a time window for subsequent HSCT. For some high-risk patients with hematological malignancies, HSCT alone may not be enough to completely prevent disease recurrence. Combining CAR-immune cell therapy can further eliminate residual lesions and significantly reduce the risk of recurrence. The combination of CAR-immune cell therapy and hematopoietic stem cell transplantation has important potential and prospects in the treatment of malignant tumors, but its application requires individualized evaluation and precise treatment plans.

[0006] Before undergoing hematopoietic stem cell transplantation, patients need to undergo conditioning, which is a series of treatments designed to remove diseased cells in the body, suppress the patient's own immune system, and prepare for the transplantation of new cells. Conditioning usually includes high-dose chemotherapy and / or radiotherapy to destroy cancerous or abnormal cells in the patient's bone marrow and reduce the risk of rejection. The specific choice of conditioning regimen will be determined based on the patient's disease type, physical condition, and treatment goals to ensure that the transplanted hematopoietic stem cells can successfully colonize and rebuild the patient's hematopoietic system.

[0007] High-dose chemotherapy and radiotherapy are often used in pretreatment, which can cause serious toxic and side effects, including nausea, vomiting, hair loss, oral ulcers, etc., and in severe cases may lead to infection and bleeding. In addition, the patient's immune system will be completely suppressed during the pretreatment process, increasing the risk of infection and other complications, and may even cause immune-related complications such as graft-versus-host disease (GVHD). Pretreatment may also damage the patient's organ function, such as the heart, lungs, liver and other organs, increasing the difficulty of recovery after transplantation and the possibility of complications. In addition, pretreatment may also lead to long-term psychological and emotional problems, including anxiety, depression, etc., affecting the patient's quality of life and mental health. Therefore, developing precise targeted pretreatment regimens as a method of pretreatment for hematopoietic stem cell transplantation to reduce potential hazards and side effects is of great significance for the treatment of patients.

[0008] Although existing tumor immunotherapy technologies, such as single-target CAR-immune cell therapy, have achieved certain success in treating certain types of tumors, there are still some problems:

[0009] 1. Host immune rejection reaction: Existing allogeneic CAR-immune cell therapy can cause rejection reaction of the host immune system, affecting the therapeutic effect.

[0010] 2. Hematotoxicity: CAR-immune cell therapy can cause significant hematotoxicity, affecting the patient's blood system.

[0011] 3. Difficulty in hematopoietic stem cell transplantation: Due to the presence of host T cells and NK cells, myeloablative pretreatment is required before the hematopoietic stem cell transplantation process, which may cause side effects such as tissue damage, infection, and GVHD in the patient.

[0012] 4. Risk of recurrence: Existing CAR-immune cell therapy may not be able to effectively prevent tumor recurrence over a long period of time. Summary of the invention

[0013] Based on the deficiencies in the prior art, the present invention provides a multi-target chimeric antigen receptor and its application that can be used for bridging hematopoietic stem cell transplantation, specifically a multi-target CAR-immune cell therapy, in which one target is a T cell marker antigen (such as CD7, CD5, CD3, CD2, CD4 or CD8, etc.), and the other one or more targets target other target cells. By targeting these T cell marker antigens, the T cells in the patient's body can be effectively eliminated, creating good conditions for hematopoietic stem cell transplantation, thereby avoiding recipient-versus-graft response (GVHD).

[0014] The present invention uses a precise treatment strategy to reduce disease recurrence by bridging hematopoietic stem cell transplantation while treating tumors. This strategy can effectively reduce the risk of infection and post-transplant complications, improve the success rate of transplantation, and shorten the patient's recovery time. This can not only improve the patient's treatment effect and quality of life, but also help reduce the use of medical resources and treatment costs, which is conducive to promoting the development and clinical application of hematopoietic stem cell transplantation technology.

[0015] The technical solution of the present invention is as follows:

[0016] The present invention provides a multi-target chimeric antigen receptor that can be used for bridging hematopoietic stem cell transplantation, comprising an antigen binding domain targeting a T cell marker antigen and an antigen binding domain targeting a disease-related target cell antigen.

[0017] The target selection in the present invention is: T cell marker antigen: selectively targeting T cell marker antigens, such as CD7, CD5, CD3, CD2, CD4 or CD8, etc. These markers are surface antigens of T cells. By targeting these antigens, T cells in the patient's body can be effectively eliminated and T cell-mediated immune responses can be inhibited.

[0018] Targets of other target cells: By targeting other target cell surface antigens associated with the disease, the specificity and effectiveness of treatment can be further improved.

[0019] The disease-related target cell antigens include but are not limited to CLL-1, CD19, CD33, BCMA, α-fetoprotein, α-actinin-4, A3, A33 antigen, ART-4, B7, Ba733, BAFF-R, BAGE, BrE3, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD56, CD59, CD64, CD66a / b / c / e, C D67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD117, CD123, CD126, CD132, CD133, CD 138. CD147, CD154, CD312, CD319, CD371, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen p (CSAP), CEA, CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GPC-3, GRO-β, GPRC5D, HLA-DR, HM1.24. Human chorionic gonadotropin and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-5R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor 1, IgK, IL1RAP, Lewis Y, LMP1, KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor, MAGE, MAGE-3, MART1, MARR-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MMG49, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM--3, NCA66, NCA95, NCA90, NKG2D, pancreatic cancer mucin, PD-1, PD-L1, placental growth factor, p53, PLAGL2 , prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, ROR1, T101, SAGE, S100, survivin, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Fredenrich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bc1-2, bc1-6, Kras, oncogene markers and oncogene products. .

[0020] The target point can be a single target point sequential structure, a series structure or a parallel structure.

[0021] Specifically, it includes connecting in series or in parallel an antigen binding domain targeting a disease-related target cell antigen and an antigen binding domain targeting a T cell marker antigen.

[0022] It includes a signal peptide, an antigen binding domain targeting a disease-related target cell antigen, an antigen binding domain targeting a T cell marker antigen, a hinge region, a transmembrane region, a co-stimulatory domain and an intracellular signal transduction peptide connected in sequence.

[0023] It contains the intracellular signaling domain of the CD3ζ chain and co-stimulatory molecules (such as CD28 or 4-1BB) to activate the killing function of T cells.

[0024] Preferably, the amino acid sequence of the multi-target chimeric antigen receptor is shown in SEQ ID No. 1 to 4.

[0025] The present invention also provides a nucleic acid molecule encoding the multi-target chimeric antigen receptor. The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 5-8.

[0026] In some embodiments of the present invention, the nucleic acid molecule comprises a nucleic acid sequence encoding a promoter.

[0027] In some embodiments of the present invention, the nucleic acid molecule comprises a nucleic acid sequence encoding WPRE, and the nucleic acid sequence encoding WPRE is located downstream of the nucleic acid sequence encoding the antigen binding domain targeting the disease-associated target cell antigen and the antigen binding domain targeting the T cell marker antigen.

[0028] In some embodiments of the present invention, the nucleic acid molecule includes a nucleic acid sequence encoding a backbone vector, and the nucleic acid sequence encoding the backbone vector is located upstream of the nucleic acid sequence encoding the promoter.

[0029] The backbone vector comprises a lentiviral vector.

[0030] In some embodiments of the present invention, the nucleic acid molecule includes a 5'LTR sequence, which is located at the 5' end of the nucleic acid molecule, and includes a 3'LTR sequence, which is located at the 3' end of the nucleic acid molecule.

[0031] The nucleic acid molecule comprises, from the 5' end to the 3' end, a 5' LTR, a lentiviral vector, a nucleic acid sequence encoding a promoter, a nucleic acid sequence encoding a signal peptide, a nucleic acid sequence encoding an antigen binding domain targeting a target cell antigen associated with a disease, a nucleic acid sequence encoding a cleavage peptide, a nucleic acid sequence encoding an antigen binding domain targeting a T cell marker antigen, a nucleic acid sequence encoding WPRE, and a 3' LTR sequence.

[0032] The present invention also provides an expression vector, comprising the nucleic acid molecule.

[0033] The present invention also provides a host cell, wherein the host cell contains the expression vector, or the exogenous nucleic acid molecule is integrated into the chromosome of the host cell.

[0034] The present invention also provides a CAR-immune cell, wherein the CAR-immune cell has one or more of the following characteristics:

[0035] The CAR-immune cell expresses the multi-target chimeric antigen receptor;

[0036] The nucleic acid molecule is integrated into the genome of the CAR-immune cell;

[0037] The CAR-immune cell includes the expression vector.

[0038] The immune cells include but are not limited to T cells, NK cells, γδT cells, macrophages, NKT cells, double negative T cells, neutrophils and the like.

[0039] The preparation method may be:

[0040] Extracting immune cells from patients or healthy donors, wherein the immune cells include but are not limited to T cells, NK cells, γδT cells, macrophages, NKT cells, double negative T cells, neutrophils, etc.;

[0041] The CAR molecules are introduced into immune cells through genetic engineering technology to make them express chimeric antigen receptors (CARs) against two or more targets, which can be single target sequential structures, tandem structures or parallel structures;

[0042] Multi-target CAR-immune cells carry out targeted attacks on T cells and other target cells in the patient's body.

[0043] The present invention also provides a pharmaceutical composition that can be used for bridging hematopoietic stem cell transplantation, comprising an immunologically active component and a pharmaceutically acceptable carrier;

[0044] Wherein, the immune active component includes at least one of the multi-target chimeric antigen receptor, the nucleic acid molecule, the expression vector and the CAR-immune cell.

[0045] The present invention also provides the use of the multi-target chimeric antigen receptor, the nucleic acid molecule, the expression vector, the host cell, the CAR-immune cell and the pharmaceutical composition in the preparation of a preparation or a drug that can be used for bridging hematopoietic stem cell transplantation.

[0046] Before hematopoietic stem cell transplantation, the preparation or drug that can be used for bridging hematopoietic stem cell transplantation is used to effectively remove T cells in the patient's body to reduce or avoid recipient-versus-graft reaction (GVHD), and to prevent CAR-immune cell suicide by natural selection or direct knockout of the immune cell's own target antigen. That is, while removing the patient's T cells, the success rate of hematopoietic stem cell transplantation is promoted.

[0047] No additional chemotherapy or radiation conditioning is required during bridging hematopoietic stem cell transplantation.

[0048] The use of the multi-target chimeric antigen receptor described in any one of claims 1 to 4 in the preparation of a drug or preparation for treating or preventing a disease associated with bridging hematopoietic stem cell transplantation, wherein the drug or preparation also includes at least one of the following types of therapeutic drugs or preparations, and the target targeted by the therapeutic drug or preparation is not the same as any target in the multi-target chimeric antigen receptor described in claims 1 to 4: (a) a drug or preparation for TCR-T cell therapy, wherein the TCR-T cell is targeted at a specific disease-related target; (b) a drug or preparation for STAR-T cell therapy, wherein the STAR-T cell is targeted at a specific disease-related target; (c) a BiTEs drug preparation, wherein the BiTEs molecule binds to two specific disease-related targets; (d) an ADC drug preparation, wherein the ADC drug targets a specific disease-related antigen; (e) a monoclonal antibody drug or preparation, wherein the monoclonal antibody is targeted at a specific disease-related antigen; (f) a cancer vaccine drug or preparation, wherein the vaccine stimulates an immune response against a specific disease-related antigen.

[0049] Specifically, the above multi-target therapy forms include but are not limited to CAR-immune cells, TCR-T cells, STAR (synthetic T cell receptor and antigen receptor)-T cells, BiTEs (bispecific T cell engagers), ADC (antibody-drug conjugates) drugs, monoclonal antibodies (mAbs), cancer vaccines, etc.

[0050] The multi-target chimeric antigen receptor, the nucleic acid molecule, the expression vector, the host cell, the CAR-immune cell and the pharmaceutical composition of the present invention can also be used for genetic diseases, infectious diseases, environmental and lifestyle-related diseases, immune diseases, tumor diseases, nutritional diseases, anti-aging, plastic surgery, etc. using the multi-target therapy of the present invention.

[0051] Compared with the prior art, the present invention has the following advantages:

[0052] 1. Eliminate host T cells: By targeting T cell marker antigens, it is possible to effectively eliminate T cells in the patient's body, avoid host immune rejection reactions, and improve the success rate of hematopoietic stem cell transplantation.

[0053] 2. Targeting tumor cells: Targeting tumor cells simultaneously, enhancing the anti-tumor effect, and effectively controlling tumor growth and metastasis.

[0054] 3. Reduce blood toxicity: During CAR-immune cell therapy, multi-target design can reduce blood toxicity and free up hematopoietic niches for donor stem cell colonization.

[0055] 4. No need for myeloablative conditioning chemotherapy: The present invention does not require myeloablative conditioning chemotherapy, thereby reducing the risk of infection, and does not require the use of immunosuppressants to prevent GVHD.

[0056] 5. Long-term prevention of tumor recurrence: CAR-immune cells can persist for a long time and work together with the graft-versus-leukemia effect (GVL) to effectively prevent tumor recurrence.

[0057] Beneficial effects of the present invention:

[0058] The present invention uses a multi-target strategy, and the multi-target chimeric antigen receptor of the present invention can not only effectively eliminate T cells, thereby reducing the risk of recipient graft-versus-graft disease (GVHD), but also attack other target cells, thereby improving the treatment effect. The present invention also provides a preparation or drug that can be used to bridge hematopoietic stem cell transplantation. The use of the preparation or drug before hematopoietic stem cell transplantation can create an ideal immune environment for transplantation and further improve the success rate of transplantation. Compared with the prior art, the present invention provides an effective multi-target CAR-immune cell therapy that can significantly improve the success rate of hematopoietic stem cell transplantation, reduce adverse reactions during treatment, and prevent tumor recurrence in the long term. The multi-target CAR-immune cell therapy of the present invention provides an innovative treatment for hematopoietic stem cell transplant patients and has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 UNK1-CAR-NK cells can specifically kill target cells expressing CD5 or CLL-1 in vitro; A: expression of CD5 on CEM-C1 and Nalm6 cell lines; B: expression of CLL-1 on U937 and Nalm6 cell lines; C: killing of UNK1-CAR-NK cells and control NK cells after 1:1 co-incubation with CEM-C1 or Nalm6 cells for 4 hours and secretion level of IFN-γ in the supernatant; D: killing of UNK1-CAR-NK cells and control NK cells after 1:1 co-incubation with U937 or Nalm6 cells for 4 hours and secretion level of IFN-Y in the supernatant. The experiment was repeated three times independently, and the data are expressed as mean ± standard deviation; ** p<0.01.

[0060] Figure 2 To establish a highly usable humanized mouse model; A: Schematic diagram of the establishment of the humanized mouse model; B: On the 90th day after transplantation, the peripheral blood CD3 + T cells, CD33 + Myeloid cells, CD19 +B cells, and CD56 + Representative flow cytometry of NK cell ratio; CLL-1 expression on 937 and Nalm6 cell lines; C: Statistical graph of cells of various lineages detected in peripheral blood of mice on day 90 after transplantation. (n=5).

[0061] Figure 3 UNK1-CAR-NK cells can kill tumor cells and can be used as a pretreatment regimen for bridging hematopoietic stem cell transplantation; A: Schematic diagram of allogeneic hematopoietic stem cell transplantation in a humanized mouse tumor model; B: CD3 in the peripheral blood of NSGS-IL15 mice was detected on days 0, 93, 100, 120, and 210 after transplantation. + T cells, CD33 + Myeloid cells, CD19 + B cells, CD56 + NK cells, GFP + Statistical graph of tumor cell ratio. (n=5).

[0062] Figure 4 CD7&CD19 CAR-T cells can specifically kill target cells expressing CD7 or CD19 in vitro; A: flow cytometry was used to detect tumor cells (Nalm6 cells) expressing CD7 (Jurkat cells) and CD19, respectively, for subsequent verification of killing function; B is the killing effect of CD7&CD19 CAR-T cells and control T cells after co-incubation with Nalm6 or Jurkat cells, respectively; C is the secretion level of IFN-γ in the supernatant after co-incubation.

[0063] Figure 5 The anti-immune rejection ability of CD7&CD19 CAR-T cells against tumor cells was detected on days 0, 93, 100, 120, and 210 after transplantation. + T cells, CD33 + Myeloid cells, CD19 + B cells, CD56 + NK cells, GFP + Statistical graph of the proportion of tumor cells.

[0064] Figure 6 CD7&CD33 CAR-T cells can specifically kill target cells expressing CD7 or CD33 in vitro; A: U937 cells expressing CD33 were detected by flow cytometry for subsequent verification of killing function; B is the killing effect of CD7&CD33 CAR-T cells and control T cells after co-incubation with U937 or Jurkat cells respectively; C is the secretion level of IFN-γ in the supernatant after co-incubation.

[0065] Figure 7 CD7&CD33 CAR-T cells' ability to resist tumor cell rejection; CD3 in the peripheral blood of NSGS-IL15 mice was detected on days 0, 93, 100, 120, and 210 after transplantation. + T cells, CD33 + Myeloid cells, CD19 + B cells, CD56 + NK cells, GFP + Statistical graph of the proportion of tumor cells.

[0066] Figure 8 CD7&BCMA CAR-T cells can specifically kill target cells expressing CD7 or BCMA in vitro; A: IM9 cells expressing BCMA were detected by flow cytometry for subsequent verification of killing function; B is the killing effect of CD7&BCMA CAR-T cells and control T cells after co-incubation with IM9 or Jurkat cells respectively; C is the secretion level of IFN-γ in the supernatant after co-incubation.

[0067] Fig. 9 The anti-immune rejection ability of CD7&BCMACAR-T cells against tumor cells was detected on days 0, 93, 100, 120, and 210 after transplantation. + T cells, CD33 + Myeloid cells, CD19 + B cells, CD56 + NK cells, GFP + Statistical graph of the proportion of tumor cells. DETAILED DESCRIPTION

[0068] Example 1: Preparation of CD5&CLL-1CAR-NK (UNK1-CAR-NK) cells and their application in the treatment of myeloid tumors or T-cell malignancies

[0069] UNK1-CAR-NK cell preparation:

[0070] The CD5&CLL-1CAR sequence was gene synthesized (the sequence is shown in SEQ ID No. 5, and the encoded amino acid sequence is shown in SEQ ID No. 1), and the synthesized sequence was subcloned into the BamHI and SallI sites of the PCDH-Lenti-EF1 vector, the plasmid was extracted, and added to 293T cells for lentivirus preparation.

[0071] Take 50ml of umbilical cord blood, separate the mononuclear cells of umbilical cord blood through Ficoll cell separation medium, and separate the mononuclear cells of umbilical cord blood through CD3 +NK cells were purified from cord blood mononuclear cells by cell depletion and then enriched for CD56 + Cells were cultured and maintained in NK MACS medium (NK basal medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 1% NK MACS supplement, 5% CTS immune cell serum and a combination of 500 IU / mL IL-2 (Sihuan Shengwu, Beijing, China) and 28 ng / mL IL-15 (Miltenyi)). After 3 days of in vitro culture, NK cells were infected with lentivirus, and the prepared UNK1-CAR-NK cells continued to be cultured in vitro.

[0072] Functional validation of UNK1-CAR-NK cells:

[0073] Tumor cells were incubated with antibodies against the antigen for 30 minutes, washed twice with PBS and then placed on the machine. Flow cytometry revealed that CD5 was significantly expressed on the surface of the CEM-C1 cell line, CLL-1 was strongly expressed on the surface of the U937 cell line, and neither CD5 nor CLL-1 was expressed on the surface of the Nalm6 cell line ( Figure 1 A and B in the figure) were co-incubated with control NK and UNK1-CAR-NK cells for 4 hours, respectively, and the killing and IFN-γ secretion levels in the supernatant were detected. The results showed that UNK1-CAR-NK cells significantly killed cells expressing CD5 or CLL-1 and produced more IFN-γ ( Figure 1 AC), but had a weaker effect on Nalm6 cells that did not express either CD5 or CLL-1 ( Figure 1 C and D in ).

[0074] Construction of highly humanized mouse models:

[0075] The applicant obtained heterozygous NSGS-IL15 mice by mating NSG-SGM3 and NSG-IL15 mice introduced from Jackson, USA. + After hematopoietic stem cell engraftment, NSGS-IL15 mice support high levels of CD3 + T cells, CD33 + Myeloid progenitor cells, CD14 + Monocytes and CD56 dim+ / CD16 + This provides researchers with a highly translatable humanized mouse model to evaluate a variety of therapeutic indications and mechanisms of action.

[0076] Female or male NSGS-IL15 mice were irradiated with 1.5 Gy and then transplanted with human CD34 +Hematopoietic stem cells. On the 90th day after transplantation, the proportion of various blood cells in the peripheral blood of mice was detected by flow cytometry, and it was found that mice of both sexes had high levels of CD3 + T cells, CD33 + Myeloid cells, and CD56 + NK cells ( Figure 2 AC). The successfully established highly usable humanized mouse model provides important support for this project to evaluate the function and anti-immune rejection ability of UNK1-CAR-NK cells.

[0077] UNK1-CAR-NK cells can be used to bridge hematopoietic stem cell transplantation:

[0078] To simulate the hematopoietic stem cell transplantation process ( Figure 3 ), on day 0 (D0), NSGS-IL15 mice were irradiated with 1.5 Gy and transplanted with human HLA-A2 within 24 hours. - CD34 + Hematopoietic stem cells successfully constructed HLA-A2 - In the genotype humanized mouse model, the U937 cell line expressing CLL-1 and GFP fluorescence was injected into the tail vein on D93. The levels of hematopoietic cells and tumor cells of various lines were evaluated on D100, and it was found that tumor cells had colonized in the mice, and the expression levels of hematopoietic cells of various lines were relatively stable. On D101, UNK1-CAR-NK cells were injected into the tail vein. On D120, the tail vein blood of the mice was collected to evaluate the levels of hematopoietic cells and tumor cells of various lines. It was found that the level of tumor cells was reduced, and the hematopoietic cells of various lines except B cells were significantly reduced. On D121, the mice were re-transplanted with CD34 + Hematopoietic stem cells. D210 day evaluation of the levels of hematopoietic cells and tumor cells found A2 + All cell lines were successfully colonized, and no tumor cells were found ( Figure 3 B).

[0079] Example 2: Preparation of CD7 & CD19 CAR-T cells and their application in the treatment of B cell tumors or T

[0080] 1. Gene synthesize CD7 & CD19 CAR sequence (sequence as shown in SEQ ID No.6, encoded amino acid sequence as shown in SEQ ID No.2), subclone the synthesized sequence into the BamHI and SalI sites of PCDH-Lenti-EF1 vector, extract the plasmid, add it to 293T cells, and prepare lentivirus. Tumor cells were incubated with antibodies against antigens for 30 minutes, washed twice with PBS, and then put on the machine. Flow cytometry was used to detect tumor cells (Nalm6 cells) expressing CD7 (Jurkat cells) and CD19, respectively, for subsequent killing function verification ( Figure 4 A).

[0081] 2. Preparation of CAR-T cells: sort T cells from healthy donors, use the packaged lentivirus to infect activated T cells, change the medium after 12 hours and continue culturing to prepare CD7&CD19 CAR-T cells.

[0082] 3. Verification of the killing function of CAR-T cells: Uninfected T cells and CD7&CD19 CAR-T cells were co-incubated with tumor cells at ratios of 1:1, 2:1, 4:1 and 8:1 for 24 hours, and the killing efficiency was detected by flow cytometry.

[0083] The results showed that compared with uninfected T cells, CD7&CD19CAR-T cells can significantly kill tumor cells ( Figure 4 B).

[0084] The culture medium after incubation was collected to detect the secretion level of cytokine IFNγ. The results showed that compared with uninfected T cells, the secretion level of IFNγ was significantly increased after the action of CD7&CD19CAR-T cells ( Figure 4 C).

[0085] 4. Simulate the hematopoietic stem cell transplantation process. The process is as in Example 1. On D101, CD7 & CD19 CAR-T cells were injected into the tail vein. On D120, the tail vein blood of the mice was collected to evaluate the levels of hematopoietic cells and tumor cells of various lines. It was found that the level of tumor cells decreased and the hematopoietic cells of various lines decreased significantly. On D121, the mice were re-transplanted with CD34 + Hematopoietic stem cells. D210 day evaluation of the levels of hematopoietic cells and tumor cells found A2 + All cell lines were successfully colonized, and no tumor cells were found ( Figure 5 ).

[0086] In summary, CD7&CD19CAR-T cells can be used to bridge hematopoietic stem cell transplantation.

[0087] Example 3: Preparation of CD7&CD33 CAR-T cells and their application in the treatment of myeloid tumors

[0088] 1. Design the plasmid of CD7&CD33CAR structure, gene synthesize CD7&CD33CAR sequence (sequence as shown in SEQ ID No.7, encoded amino acid sequence as shown in SEQ ID No.3), subclone the synthesized sequence into the BamHI and SalI sites of PCDH-Lenti-EF1 vector, extract the plasmid, add it to 293T cells, and package the lentivirus. Incubate the tumor cells with the antibody for the antigen for 30 minutes, wash them with PBS twice, and then put them on the machine. Detect by flow cytometry, screen out tumor cells (U937) expressing CD33 for subsequent killing function verification ( Figure 6 A).

[0089] 2. Preparation of CAR-T cells: T cells from healthy donors were sorted, and the above-packaged lentivirus was used to infect the activated T cells. After 12 hours, the medium was changed and cultured continuously to prepare CD7&CD33 CAR-T cells.

[0090] 3. Verification of the killing function of CAR-T cells: Uninfected T cells and CD7&CD33 CAR-T cells were co-incubated with tumor cells at ratios of 1:1, 2:1, 4:1 and 8:1 for 24 hours, and the killing efficiency was detected by flow cytometry.

[0091] The results showed that compared with uninfected T cells, CD7&CD33 CAR-T cells can significantly kill tumor cells ( Figure 6 B).

[0092] The culture medium after incubation was collected to detect the secretion level of cytokine IFNγ. The results showed that compared with uninfected T cells, the secretion level of IFNγ was significantly increased after the action of CD7&CD33 CAR-T cells ( Figure 6 C).

[0093] 4. Simulate the hematopoietic stem cell transplantation process. The process is as in Example 1. On D101, CD7 & CD33 CAR-T cells were injected into the tail vein. On D120, the tail vein blood of the mice was collected to evaluate the levels of hematopoietic cells and tumor cells. It was found that the level of tumor cells was reduced, and the hematopoietic cells of all lines except B cells were significantly reduced. On D121, CD34 was re-transplanted into the mice. + Hematopoietic stem cells. D210 day evaluation of the levels of hematopoietic cells and tumor cells found A2 + All cell lines were successfully colonized, and no tumor cells were found ( Figure 7 ).

[0094] In summary, CD7&CD33 CAR-T cells can be used to bridge hematopoietic stem cell transplantation.

[0095] Example 4: Preparation of CD7&BCMACAR-T cells and their application in the treatment of plasma cell tumors

[0096] 1. Design a plasmid with CD7 & BCMACAR structure, synthesize CD7 & BCMACAR sequence (sequence as shown in SEQ ID No.8, encoded amino acid sequence as shown in SEQ ID No.4), subclone the synthesized sequence into the BamHI and SalI sites of PCDH-Lenti-EF1 vector, extract the plasmid, add it to 293T cells, and package the lentivirus. Incubate the tumor cells with the antibody for the antigen for 30 minutes, wash twice with PBS, and then put them on the machine. Detect by flow cytometry to screen out tumor cells expressing BCMA for subsequent killing function verification ( Figure 8 A).

[0097] 2. Preparation of CAR-T cells: sort T cells from healthy donors, use the packaged lentivirus to infect activated T cells, change the medium after 12 hours and continue culturing to prepare CD7&BCMACAR-T cells.

[0098] 3. Verification of the killing function of CAR-T cells: Uninfected T cells and CD7&BCMACAR-T cells were co-incubated with tumor cells at ratios of 1:1, 2:1, 4:1 and 8:1 for 24 hours, and the killing efficiency was detected by flow cytometry.

[0099] The results showed that compared with uninfected T cells, CD7&BCMACAR-T cells can significantly kill tumor cells ( Figure 8 B).

[0100] The culture medium after incubation was collected to detect the secretion level of cytokine IFNγ. The results showed that compared with uninfected T cells, the secretion level of IFNγ was significantly increased after the action of CD7&BCMACAR-T cells ( Figure 8 C).

[0101] 4. Simulate the hematopoietic stem cell transplantation process. The process is the same as in Example 1. On D101, CD7&BCMACAR-T cells were injected into the tail vein. On D120, the tail vein blood of the mice was collected to evaluate the levels of hematopoietic cells and tumor cells. It was found that the level of tumor cells was reduced, and the hematopoietic cells of all lines except B cells were significantly reduced. On D121, CD34 + Hematopoietic stem cells. D210 day evaluation of the levels of hematopoietic cells and tumor cells found A2 + All cell lines were successfully colonized, and no tumor cells were found ( Fig. 9 ).

[0102] In summary, CD7&BCMACAR-T cells can be used to bridge hematopoietic stem cell transplantation.

Claims

1. A multi-target chimeric antigen receptor for bridging hematopoietic stem cell transplantation, characterized in that: It includes an antigen-binding domain targeting a T cell marker antigen and an antigen-binding domain targeting a disease-associated target cell antigen.

2. The multi-target chimeric antigen receptor for bridging hematopoietic stem cell transplantation according to claim 1, characterized in that: The T cell marker antigens include CD7, CD5, CD3, CD2, CD4, and CD8; The disease-related target cell antigens include CLL-1, CD19, CD33, BCMA, α-fetoprotein, α-actinin-4, A3, A33 antigen, ART-4, B7, Ba733, BAFF-R, BAGE, BrE3, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD56, CD59, CD64, CD66a / b / c / e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD117, CD123, CD126, CD132, CD133, CD138, CD147, CD154, CD312, CD319, CD371, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen p, CEA, CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GPC-3, GRO-β, GPRC5D, HLA-DR, HM1.

24. Human chorionic gonadotropin and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor, HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-5R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor 1, IgK, IL1RAP, Lewis Y, LMP1, KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor, MAGE, MAGE-3, MART1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MMG49, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, NKG2D, pancreatic cancer mucin, PD-1, PD-L1, placental growth factor, p53, PLAGL2 , prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, ROR1, T101, SAGE, S100, survivin, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Fredenrich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, Kras, oncogene markers and oncogene products. .

3. The multi-target chimeric antigen receptor for bridging hematopoietic stem cell transplantation according to claim 1, characterized in that: The method comprises connecting in series or in parallel an antigen binding domain targeting a target cell antigen associated with a disease and an antigen binding domain targeting a T cell marker antigen.

4. The multi-target chimeric antigen receptor for bridging hematopoietic stem cell transplantation according to claim 1, characterized in that: The amino acid sequences of the multi-target chimeric antigen receptor are shown in SEQ ID No. 1-4.

5. A nucleic acid molecule encoding the multi-target chimeric antigen receptor according to any one of claims 1 to 4.

6. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 5.

7. A host cell, characterized in that The host cell contains the expression vector as claimed in claim 6, or the exogenous nucleic acid molecule as claimed in claim 5 is integrated into the chromosome of the host cell.

8. A CAR-immune cell, characterized in that: The CAR-immune cell has one or more of the following characteristics: the CAR-immune cell expresses the multi-target chimeric antigen receptor described in any one of claims 1 to 4; The nucleic acid molecule of claim 5 is integrated into the genome of the CAR-immune cell; The CAR-immune cell comprises the expression vector of claim 6.

9. The CAR-immune cell according to claim 8, characterized in that The CAR molecules are introduced into immune cells through genetic engineering technology to obtain the CAR-immune cells, so that the CAR-immune cells express chimeric antigen receptors targeting two or more targets. The target point is a single target point sequential structure, a series structure or a parallel structure.

10. The CAR-immune cell according to claim 8, characterized in that: The immune cells in the CAR-immune cells are selected from immune cells of patients or healthy donors, wherein the immune cells include T cells, NK cells, γδT cells, macrophages, NKT cells, double negative T cells, and neutrophils.

11. A pharmaceutical composition for bridging hematopoietic stem cell transplantation, characterized in that: comprising an immunologically active component and a pharmaceutically acceptable carrier; Wherein, the immune active component includes at least one of the multi-target chimeric antigen receptor described in any one of claims 1 to 4, the nucleic acid molecule described in claim 5, the expression vector described in claim 6, and the CAR-immune cell described in any one of claims 8 to 10.

12. Use of the multi-target chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host cell according to claim 7, the CAR-immune cell according to any one of claims 8 to 10, and the pharmaceutical composition according to claim 11 in the preparation of a preparation or drug for bridging hematopoietic stem cell transplantation.

13. Use of the multi-target chimeric antigen receptor according to any one of claims 1 to 4 in the preparation of a drug or preparation for treating or preventing a disease associated with bridging hematopoietic stem cell transplantation, wherein the drug or preparation further comprises at least one of the following types of therapeutic drugs or preparations, wherein the target of the therapeutic drug or preparation is not the same as any target in the multi-target chimeric antigen receptor according to claims 1 to 4: (a) A drug or preparation for TCR-T cell therapy, wherein the TCR-T cell is directed against a specific disease-related target; (b) a drug or preparation for STAR-T cell therapy, wherein the STAR-T cells are directed against a specific disease-related target; (c) BiTEs pharmaceutical preparations, wherein the BiTEs molecules bind to two specific disease-related targets; (d) ADC drug preparations, wherein the ADC drug targets specific disease-associated antigens; (e) a monoclonal antibody drug or preparation, wherein the monoclonal antibody is directed against a specific disease-associated antigen; (f) Cancer vaccine drugs or formulations, wherein the vaccine stimulates an immune response against a specific disease-associated antigen.

14. Use of the multi-target chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host cell according to claim 7, the CAR-immune cell according to any one of claims 8 to 10, and the pharmaceutical composition according to claim 11 in the preparation of a preparation or medicine for treating any of the following diseases: Genetic diseases, infectious diseases, environmental and lifestyle related diseases, immune diseases, tumor diseases, nutritional diseases, anti-aging, plastic surgery.

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