Bispecific chimeric antigen receptor (CAR) and preparation method thereof

Abstract

The present invention relates to a bispecific chimeric antigen receptor (CAR) and a preparation method thereof. The present invention first provides a bispecific chimeric antigen receptor targeting BCMA and CD7, having the following structure: S-BCMA scFv-CD7 scFv-H-TM-C-CD3ζ, wherein BCMA scFv is a BCMA antigen binding domain; CD7 scFv is a CD7 antigen binding domain; S is a signal peptide; H is a hinge region; TM is a transmembrane region; C is a co-stimulatory factor; CD3ζ is a CD3ζ intracellular signaling domain, and the chimeric antigen receptor can simultaneously bind to both BCMA and CD7 anti-tumor targets, which can significantly improve targeting and effectiveness; knocking out the immune checkpoint inhibitor gene in T cells may prevent tumor cells from triggering immune escape by specifically binding to the relevant immune checkpoint inhibitor site. The present invention selects the PD-1 / PD-L1 gene site that is most widely studied and applied for knocking out, which can greatly inhibit the tumor immune escape phenomenon. CAR-T cells are prepared by introducing the above-mentioned chimeric antigen receptor into T cells. The CAR-T cells can significantly inhibit the proliferation of leukemia cells, regulate the expression levels of cytokines such as IL-12, TNFα, and TGF-β, inhibit tumor growth in animal models, prolong the animal's survival cycle, and exert a synergistic anti-tumor effect.

Description

Technical field:

[0001] The present invention belongs to the field of tumor immunotherapy and specifically provides a bispecific chimeric antigen receptor (CAR) and a preparation method thereof. Background technology:

[0002] Malignant tumors are the leading threat to human health. Their diagnosis and treatment remain a hot topic and a challenge both domestically and internationally. Despite the continuous development and improvement of surgical procedures, radiotherapy techniques, and new chemotherapeutic agents, achieving a long-term prognosis for various malignancies remains elusive, making the search for new anti-tumor approaches both urgent and significant. Among known malignancies, multiple myeloma (MM) is a clonal proliferation of bone marrow plasma cells. MM accounts for 10% of all hematologic malignancies and is the second most common hematologic malignancy after malignant lymphoma. Myeloma patients present with monoclonal immunoglobulins or their fragments in their blood and urine, leading to impaired function in target organs such as bone marrow hematopoiesis, kidneys, and bones. Clinical manifestations include anemia, bone pain and osteolytic lesions, renal insufficiency, and recurrent infections. Currently, myeloma treatment primarily relies on chemotherapy and stem cell transplantation. However, chemotherapy has significant side effects, severely impacting patients' quality of life. Furthermore, the selection of cell donors, patient age, and post-transplant complications such as graft-versus-host disease and relapse remain significant challenges in hematopoietic stem cell transplantation. Therefore, new treatments are urgently needed to improve the prognosis of myeloma patients.

[0003] Among the research on novel cancer treatments, tumor immunotherapy is gaining increasing attention. Research on anti-tumor immunity and the tumor microenvironment has provided a new foundation for tumor immunotherapy, which includes adoptive immunotherapy and targeted drug therapy. In 1988, American researchers Rosenberg et al. first proposed the concept of adoptive immunotherapy. This involves culturing and activating a patient's own immune cells, such as T lymphocytes and natural killer (NK) cells, in vitro. These cells are then infused back into the patient using growth factors, enabling these immune effector cells to kill tumor cells, thereby achieving the goal of treating the tumor. Although this therapy has achieved promising results in some tumors, including melanoma, it has been less effective against most malignancies, primarily due to the restriction of the major histocompatibility complex (MHC) that significantly reduces the ability of T lymphocytes to directly recognize and kill tumors. Against this backdrop, chimeric antigen receptor (CAR)-modified T lymphocytes, characterized by their high specificity, persistence, and cytotoxic activity, have come into the spotlight. The basic structure of a CAR consists of a tumor-associated antigen (TAA) binding region (usually derived from the scFv segment of the monoclonal antibody antigen-binding region), an extracellular hinge region, a transmembrane region, and an intracellular immunoreceptor tyrosine-based activation motif (ITAM). This structure can break through the constraints of the MHC and can specifically target tumor cells through the extracellularly expressed antigen-binding domain to launch an attack, thereby exerting efficient and long-lasting anti-tumor activity.

[0004] CAR-T cells have gone through four generations of structure. The first-generation CAR had a simple structure and no costimulatory signaling domain, which resulted in the inability of T cells to be fully activated, manifested as an inability to expand in vivo to a level that meets clinical needs, and unsatisfactory therapeutic effects; the second-generation CAR added a costimulatory domain (CD28 or 4-1BB), which significantly increased the duration of action of CAR-T cells; the third-generation CAR added two costimulatory domains at the same time, making CAR-T cells more active in vivo, but some studies have suggested that third-generation CAR may cause the threshold value of T cells to respond to stimulation to be lowered, inducing more severe cytokine release syndrome; the fourth-generation CAR-T cells, also known as TRUCK (T-cells redirected for universal cytokine killing)-T cells, contain an activated T cell nuclear factor transcriptional corresponding element, which enables CAR-T cells to secrete specific cytokines within the tumor infiltration range to modify the tumor microenvironment and promote other immune cells to participate in the immune response. In current clinical trials, CD19 CAR-T cells can achieve a CR rate of over 80% in the treatment of B-ALL patients, a response rate of 52%-83% in B-cell lymphoma, and a certain response rate in the treatment of CLL.

[0005] However, while CAR-T cell therapy brings satisfactory remission rates, the related adverse reactions it produces have also attracted attention in current clinical treatment. The toxic and side effects of CAR-T cell therapy mainly include: 1) Insertional mutation: CAR-T cell therapy technology inserts an exogenous DNA fragment into T cells, which has a certain risk of causing a second tumor; 2) Off-target effect: That is, while CAR-T cells recognize tumor-specific antigens and kill tumor cells, they may also affect normal tissues that express the same antigen in trace amounts, which is called off-target effect; 3) Inflammatory response: CAR-T cells are activated under antigen stimulation and secrete a large amount of inflammatory factors, forming inflammatory reactions such as cytokine release syndrome and tumor lysis syndrome; 4) Neurotoxicity; 5) B cell hypoplasia: This is the expected result of CD19 CAR-T cell therapy. While killing tumor cells, it also kills normal CD19-expressing B cells, thereby leading to B cell dysfunction or even B cell exhaustion. Among these adverse reactions, cytokine release syndrome (CRS) is the most common, with reported overall and severe CRS rates of 58%-97% and 15%-39%, respectively. Its mechanism of action is that CAR-T cells bind to antigens, triggering widespread immune activation and the release of large amounts of cytokines, leading to a systemic inflammatory response, also known as a cytokine storm. Clinically, the initial symptom of CRS is usually fever, with other reactions including flu-like symptoms, fatigue, headache, and myalgia. It can also affect various organ systems, causing nausea, vomiting, diarrhea, rash, hypoxia, pulmonary edema, hypotension, previously unseen arrhythmias, heart failure, liver failure, and abnormal coagulation parameters. Severe cases can even lead to life-threatening shock, capillary leak, hypoxia requiring mechanical ventilation, malignant arrhythmias, and end-organ dysfunction.

[0006] It can be seen that in the current research field of chimeric antigen receptors, on the one hand, it is necessary to develop new structures and select appropriate targets to improve the targeting and effectiveness of anti-tumor drugs; on the other hand, it is necessary to adopt appropriate measures to control and manage the occurrence of serious adverse reactions and ensure the safety of treatment.

[0007] BCMA (B cell maturation antigen), also known as CD269, is a member of the tumor necrosis factor receptor (TNF-receptor) superfamily. It is located on the short arm of human chromosome 16 (16p13.13) and consists of three exons and two introns. BCMA is a 184-amino acid, 20.2-kDa type III transmembrane glycoprotein with a terminal motif containing six conserved cysteine ​​residues. BCMA is undetectable in immature B cells and is rarely expressed in hematopoietic stem cells and the normal blood system. However, BCMA expression has been identified in nearly all multiple myeloma (MM) cells, making it an important biomarker for disease activity and prognosis in MM. At present, a variety of antibody drugs and chimeric antigen receptor immune cells have been developed targeting the BCMA target in domestic and foreign research. In order to improve the selectivity of tumor treatment, researchers have begun to focus on the study of bispecific antibodies or chimeric antigen receptors targeting BCMA. For example, WO2019067677A1 discloses a bispecific chimeric antigen receptor targeting CD307E and BCMA, CN109485734A discloses a bispecific chimeric antigen receptor targeting BCMA and CD19, and HK40050871A discloses a bispecific antibody targeting BCMA and CD3.

[0008] While the aforementioned products and therapies have achieved certain therapeutic effects, their targeting and effectiveness still need to be enhanced. Screening for chimeric antigen receptors targeting BCMA, and identifying which other known and / or unknown anti-tumor antigen recognition molecules the antigen-binding domain can combine with to form a strong and effective bispecific anti-tumor structure, thereby providing a safe and effective means of treating tumors, have become research hotspots and priorities in the field of tumor immunotherapy. Furthermore, with the revelation of the phenomenon of tumor immune escape, how to prevent it and how to shut down the immune checkpoint inhibitor pathway in chimeric antigen receptor immune cells are also receiving increasing attention within the industry. Summary of the invention:

[0009] To solve the above technical problems, the present invention provides a bispecific chimeric antigen receptor, which comprises the following structure: S-BCMA scFv-CD7 scFv-H-TM-C-CD3ζ, wherein "-" is a connecting peptide or peptide bond; S is a signal peptide; H is a hinge region; TM is a transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is an intracellular signal transduction sequence; BCMA scFv comprises the heavy chain CDR regions shown in SEQ ID NOs: 1-3 and the light chain CDR regions shown in SEQ ID NOs: 4-6; CD7 scFv comprises the heavy chain CDR regions shown in SEQ ID NOs: 7-9 and the light chain CDR regions shown in SEQ ID NOs: 10-12.

[0010] The prior art has reported a variety of potential anti-tumor targets that can form bispecific antibodies or chimeric antigen receptors with BCMA, including but not limited to CD19, CD20, CD3, PD-1 / PD-L1, etc. However, there is still no consensus on which anti-tumor target BCMA is more effective in combination with and has more advantages in preparing chimeric antigen receptor immune cells, and it is difficult to draw a clear conclusion. Therefore, in the present invention, a BCMA-targeted antigen binding domain with a novel structure was screened and obtained, which was combined with antigen-binding molecules targeting different anti-tumor targets to form a bispecific chimeric antigen receptor structure. The inhibitory effect on multiple myeloma was verified respectively, and it was found that the bispecific chimeric antigen receptor targeting BCMA and CD7 had the highest anti-tumor activity, thereby providing a new research approach and ideas for the development of anti-tumor drugs based on BCMA antigens.

[0011] Furthermore, the BCMA scFv comprises a heavy chain variable region as set forth in SEQ ID NO: 13 and a light chain variable region as set forth in SEQ ID NO: 14; the heavy chain variable region or light chain variable region in the scFv sequence may also be a molecule that maintains a high degree of identity with the above amino acid sequence and has the same function, wherein the identity comprises 80-99%, preferably 85%, 90%, 95%, 98%, or 99%.

[0012] Furthermore, the CD7 scFv includes a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 16; the heavy chain variable region or light chain variable region in the scFv sequence may also be a molecule that maintains a high degree of identity with the above amino acid sequence and has the same function, and the identity includes 80-99%, preferably 85%, 90%, 95%, 98%, or 99%.

[0013] Furthermore, the co-stimulatory signal molecule is selected from one or more of CD27, CD28, 4-1BB, CD11c, ITGB1, OX40, CD30, CD40, ICOS, CD11b, and ITGAX, preferably CD28 and / or 4-1BB, and more preferably CD28.

[0014] Furthermore, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 17; the chimeric antigen receptor amino acid sequence may also be a molecule that maintains a high degree of identity with the above amino acid sequence and has the same function, and the identity includes 80-99%, preferably 85%, 90%, 95%, 98%, or 99%.

[0015] Provided is a nucleic acid molecule encoding the above-mentioned bispecific chimeric antigen receptor.

[0016] Furthermore, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 18; it can also be a molecule that maintains a high degree of identity with SEQ ID NO: 18 and has the same function, and the identity includes 80-99%, preferably 85%, 90%, 95%, 98%, or 99%.

[0017] Provided is a T cell, wherein the T cell expresses the above-mentioned bispecific chimeric antigen receptor.

[0018] Furthermore, the T cells are T cells with PD-1 gene knocked out.

[0019] Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule belonging to the immunoglobulin superfamily and a membrane protein with 288 amino acid residues. Its ligand is PD-L1. PD-1 and PD-L1 bind to initiate programmed cell death in T cells, allowing tumor cells to gain immune escape function. This is also an important molecular mechanism for tumors to resist the killing effects of immune cells such as T cells and NK cells. In order to combat the phenomenon of tumor immune escape, PD-1 / PD-L1 antibodies have been developed, such as Opdivo (nivolumab) and Keytruda (pembrolizumab). In CAR-T cell research, some researchers have proposed that knocking out or interfering with PD-1 / PD-L1 gene expression in T cells can improve anti-tumor activity. Based on this teaching, the present invention compares the anti-tumor activity of CAR-T cells before and after PD-1 gene knockout, demonstrating that knocking out the PD-1 gene in dual-characteristic CAR-T cells targeting BCMA and CD7 can further improve anti-tumor activity and enhance tumor killing effects.

[0020] Provided is a use of a bispecific chimeric antigen receptor or corresponding CAR-T cell in the preparation of a tumor drug. The tumors include, but are not limited to, lymphoma, leukemia, multiple myeloma, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, and Hodgkin's lymphoma; multiple myeloma is preferred.

[0021] Beneficial effects

[0022] The present invention provides a novel bispecific chimeric antigen receptor targeting BCMA and CD7, which can significantly enhance the killing effect on myeloma cells, enrich the research on bispecific or multispecific antibodies or chimeric antigen receptors based on BCMA targets, and provide a new research basis and application basis for the screening of combination targets; further, knocking out the immune checkpoint PD-1 gene in T cells can eliminate tumor immune escape and enhance the killing effect on myeloma cells; in in vivo experiments, it can significantly inhibit the tumor growth rate, promote the expression of anti-tumor immune factors, trigger the body's immune anti-tumor mechanism, and exert a synergistic anti-tumor effect. Description of the drawings:

[0023] Figure 1 :Schematic diagram of the chimeric antigen receptor structure

[0024] Figure 2 :The effects of different CAR-T cells on the survival rate of myeloma cells;

[0025] Figure 3 :The effect of immune checkpoint inhibitor gene knockout on the anti-tumor activity of CAR-T cells;

[0026] Figure 4 : Tumor volume change diagram;

[0027] Figure 5 :The change of IL-12 expression level;

[0028] Figure 6 :The change of TNF-α expression level;

[0029] Figure 7 : Changes in TGF-β expression levels; Specific implementation method:

[0030] The following non-limiting examples can help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any form. All technologies implemented based on the above content of the present invention should fall within the scope of protection claimed in this application.

[0031] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0032] Example 1: Chimeric Antigen Receptor Design

[0033] In this example, a bispecific antibody containing an anti-BCMA target was constructed, and the sequence diagram is shown in FIG. Figure 1 As shown in the figure, its structure is S-BCMA scFv-CDx scFv-H-TM-C-CD3ζ, wherein BCMA scFv is the antigen binding domain targeting BCMA; CDxscFv is the antigen binding domain targeting CDx, specifically including CD3scFv, CD5 scFv, CD7 scFv, CD19 scFv, and CD20scFv; "-" is a connecting peptide or peptide bond; S is a signal peptide; H is a hinge region; TM is a transmembrane domain; C is a co-stimulatory signal molecule; and CD3ζ is an intracellular signal transduction sequence.

[0034] The antigen-binding domain sequences for each target were obtained by the inventors in previous studies. Taking a bispecific chimeric antigen receptor targeting BCMA and CD7 as an example, the BCMA scFv includes the heavy chain CDR regions set forth in SEQ ID NOs: 1-3 and the light chain CDR regions set forth in SEQ ID NOs: 4-6; the CD7 scFv includes the heavy chain CDR regions set forth in SEQ ID NOs: 7-9 and the light chain CDR regions set forth in SEQ ID NOs: 10-12; the BCMA scFv includes the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 14; and the CD7 scFv includes the heavy chain variable region set forth in SEQ ID NO: 15 and the light chain variable region set forth in SEQ ID NO: 16. The amino acid sequence of the BCMA-CD7 chimeric antigen receptor is set forth in SEQ ID NO: 17, and its nucleotide sequence is set forth in SEQ ID NO: 18.

[0035] Example 2: T cell preparation

[0036] In this example, density gradient centrifugation was used to obtain peripheral blood mononuclear cells (PBMCs) from patients. The specific steps included:

[0037] Use a blood collection tube containing anticoagulant to draw 20 mL of human peripheral blood into a centrifuge tube and centrifuge at 2000 rpm for 10 minutes; collect the upper plasma, add an equal volume of pre-warmed physiological saline to the remaining blood cell pellet, resuspend it, and mix thoroughly; take another centrifuge tube, gently add the mixed blood cell pellet to the surface of the lymphocyte separation solution at a volume of 1:1, and centrifuge at 18000 rpm for 25 minutes; carefully aspirate the white lymphocyte layer; transfer the buffy coat layer to a new centrifuge tube, add PBS to 45 mL, centrifuge at 1500 rpm for 5 minutes, and wash twice; add an appropriate amount of RPMI1640 (containing 10% FBS) complete culture medium to resuspend the cells and count them.

[0038] T cells were enriched using CD4 magnetic beads and CD8 magnetic beads, and the obtained T cells were placed in fresh culture medium, and 100 U / mL human IL-7 and 200 U / mL human IL-15 were added. The purified T cells were cultured in culture medium at 37°C and 5% CO2.

[0039] Example 3: T cell PD-1 gene knockout

[0040] Based on the sequence structure of the human PD-1 gene and referring to the existing research content of related CRISPR gene editing tools, the nucleotide sequence shown in SEQ ID NO.19 was selected as the sgRNA of the PD-1 gene. The PD-1 sgRNA nucleic acid sequence was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and inserted into a standard vector and ligated to the CRISPR / CAS9 expression vector pX330A to obtain the pX330A-TIGIT vector and the pX330A-PD-1 vector.

[0041] The pX330A-PD-1 vector was electroporated into T cells. The specific steps included: taking 1×10 7 T cells were added, 500 μL of electroporation buffer was added to resuspend the cells, and the cells were pipetted up and down evenly; pX330A-PD-1 vector plasmid (10 ug) was added to the cell suspension, and the cells were pipetted up and down evenly, and the cells were transferred to a sterile and clean electroporation cup, and the parameters were set to 300 V, 10 ms, and two electric shock operations were performed; after the electric shock was completed, the electroporation cup was placed on ice and incubated for 10 minutes to allow the nucleic acid to fully enter the cells; the electroporation cup was removed from the ice, the cells were transferred out of the electroporation cup, filtered and counted, and inoculated into fresh DMEM culture medium according to a certain cell density, and cultured in a 37°C, 5% CO2 incubator.

[0042] On the 2nd to 3rd day after electroporation, the expression of PD-1 in T cells was detected by flow cytometry. The PD-1 gene knockout efficiency = (expression level in the control group - expression level in the experimental group) / expression level in the control group × 100%. The results showed that the PD-1 knockout efficiency of T cells reached 97.2%.

[0043] Example 4: CAR-T cell preparation

[0044] The chimeric antigen receptor gene fragment provided by the present invention was introduced into the psb1576 vector, which was then introduced into competent cells. The plasmid was extracted using a plasmid extraction kit (purchased from Axygen), and sequencing was performed to verify the correctness of the target gene sequence. Positive clone plasmids were delivered to Bosheng Jianke Cell Technology Co., Ltd. for synthesis of lentiviral vectors.

[0045] The T cells prepared in Example 2 and the gene knockout T cells prepared in Example 3 were activated separately, added to fresh culture medium, and cultured at 37°C, 5% CO2 for 10 days. The cells were collected by centrifugation. The cells were resuspended in fresh culture medium and the cell density was adjusted to 1×10 6 cells / mL, various lentiviruses were added to 24-well culture plates at an MOI of 10, mixed well, and cultured in a 37°C, 5% CO2 incubator. Fresh culture medium was replaced after 48-96 hours of culture. The transduction efficiency of CAR-T cells was determined by PCR, and the positive rate was over 95%, which met the experimental requirements.

[0046] Example 5: Killing effect of CAR-T cells on tumor cells

[0047] To verify the tumor killing effect of CAR-T cells, the present invention selected the human multiple myeloma cell line RPMI8226 (purchased from ATCC, USA) as the research object.

[0048] 5.1 Tumor cell culture

[0049] Remove the cells from liquid nitrogen and resuscitate RPMI 8226 cells at a volume of 1 × 10 6 Cells were inoculated into RPMI 1640 complete medium (containing 10% FBS) at 100 μg / mL and cultured aseptically in a 37°C, 5% CO2 cell incubator. Cells were counted and passaged every 36-72 hours, and the cell status was observed. Subsequent experiments were performed when the cell viability reached above 80%.

[0050] 5.2 CAR-T cell co-culture

[0051] CAR-T cells and the above tumor cells were harvested separately, washed three times with sterile PBS, and then mixed with tumor cells at a ratio of 1:1 to adjust the cell density to 1×10 6 The cells were plated in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 hours. Sterile PBS solution was used as a negative control.

[0052] 5.3 MTT assay to detect the tumor killing efficiency of different CAR-T cells

[0053] After 48 hours of co-culture of CAR-T cells and tumor cells, the supernatant was centrifuged and 20 μL of MTT was added to each well for 4 hours. The MTT was then discarded and 150 μL of DMSO was added to each well. The cells were shaken for 10 minutes. A blank control well was also set. The blank control well was zeroed and the absorbance (OD) at 490 nm was measured using a microplate reader. Relative cell viability (cell viability) was calculated as follows: cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0054] The results are as follows Figure 2 As shown, the bispecific CAR-T cells provided in the present invention can effectively kill myeloma cells, but surprisingly, compared with the more reported combinations of BCMA targets with CD3, CD19, and CD20, the BCMA-targeted antigen binding domain provided in the present invention appears to be more effective in killing myeloma cells in combination with CD7. In incubation with bispecific CAR-T cells targeting BCMA and CD7, the survival rate of RPMI 8226 cells was the lowest. The combination of BCMA with CD3 or CD5 also showed an anti-tumor effect that was significantly higher than the combination of CD19 or CD20, indicating that the combination of BCMA and CD7 targets can exert a more synergistic anti-tumor effect.

[0055] 5.4 MTT assay to detect the tumor killing efficiency of PD-1 gene knockout CAR-T cells

[0056] In order to further improve the anti-tumor effect and inhibit tumor immune escape, in this example, the target BCMA and CD7 chimeric antigen receptors were constructed into PD-1 gene knockout T cells to obtain ΔPD-1-BCMA-CD7 CAR-T cells (the construction method is the same as in Example 4). ΔPD-1BCMA-CD7 CAR-T cells and BCMA-CD7 CAR-T cells were co-cultured with the human multiple myeloma cell line RPMI 8226, and the cell viability was detected by MTT assay (the method is the same as in Sections 5.1-5.3).

[0057] The results are as follows Figure 3 As shown in the results, after knocking out the PD-1 gene, the anti-tumor activity of CAR-T cells was further enhanced, indicating that the tumor immune escape mechanism was effectively curbed, providing a new practical basis for the development of long-lasting and highly effective anti-tumor methods.

[0058] Example 6 Inhibitory Effects of CAR-T Cells on Tumors in Vivo

[0059] 6.1 Animal Model Preparation and Drug Administration

[0060] Resuscitate and culture RPMI 8226 cells using the same method as in Section 5.1. Take healthy BALB / c nude mice of similar weight, which are 6-8 weeks old, and dilute the multiple myeloma RPMI 8226 to 1×10 6 300 μL of the above cell solution was injected into the armpit of nude mice until the tumor volume increased to 100 mm 3 The experimental animals were randomly divided into three groups and injected with 1×10 6 △PD-1BCMA-CD7 CAR-T cells and BCMA-CD7 CAR-T cells were used, and an equal volume of normal saline was used as the control group.

[0061] 6.2 Tumor Volume Detection

[0062] The first administration time was recorded as week 0, and the tumor volume was detected every week. The specific method was as follows: using a vernier caliper to measure the long diameter (L) and short diameter (W) of the tumor, according to the formula V = (W 2 × L) / 2 to calculate the tumor volume, and the test was conducted for 3 weeks.

[0063] like Figure 4 As shown, the CAR-T cells provided in the present invention can significantly inhibit the increase in myeloma tumor volume and inhibit the growth process of the tumor. Compared with the normal saline control group, the tumor volume was reduced by about half after 3 weeks of treatment with CAR-T cells. The tumor inhibition effect of CAR-T cells with PD-1 gene knockout was more obvious, and the degree of tumor inhibition seemed to be higher than that of cell-level experiments, indicating that this gene-modified CAR-T cell can activate the body's immune system in in vivo experiments and exert a long-lasting and efficient tumor inhibition effect.

[0064] 6.4 Detection of IL-12, TNFα, and TGF-β levels in blood

[0065] Three weeks after administration, blood was collected from the nude mice's orbital vein and centrifuged at 3000 rpm for 15 minutes. The upper serum layer was removed and placed in a centrifuge tube and stored at -80°C until use. ELISA kits (purchased from Wuhan Boster Biotechnology Co., Ltd.) were used to measure the levels of IL-12, TNF-α, and TGF-β in the nude mouse serum.

[0066] Interleukin-12 (IL-12) is a cytokine with a wide range of biological activities. It is mainly produced by activated inflammatory cells and can exert anti-tumor effects by regulating innate immunity, acquired immunity and inhibiting tumor angiogenesis. The main ways in which IL-12 participates in the anti-tumor process include: (1) IL-12 can enhance the tumor killing effect of cytotoxic lymphocytes such as CD8+T cells and natural killer cells (NK); (2) IL-12 stimulates immune cells to produce IFN-γ and other secondary and tertiary pro-inflammatory cytokines, which can have direct toxicity to tumor cells and / or can inhibit tumor growth by inhibiting tumor angiogenesis; (3) IL-12 can enhance the body's tumor antigen-specific immunity by inducing or increasing the response capacity of Th1 cells and cytotoxic T lymphocytes (CTL); (4) IL-12 can also increase the production of opsonized and complement-binding IgG antibodies by stimulating Th1 reactions, thereby enhancing anti-tumor activity. In this embodiment, Figure 5 As shown in the results, after treatment with CAR-T cells, the IL-12 level in nude mice was significantly increased, especially in the △PD-1BCMA-CD7 CAR-T treatment group, the IL-12 level increased by nearly 3 times, indicating that this type of CAR-T cell can activate the anti-tumor mechanism in the body by mobilizing the IL-12 secretion pathway and inhibit tumor growth.

[0067] Tumor Necrosis Factor α (TNFα) is an inflammatory cytokine that can coordinate the production of other cytokines, cell survival and death to coordinate tissue homeostasis. In the body, TNFα is released by activated macrophages and other types of cells such as CD4+ T cells, neutrophils, mast cells, etc., and can induce inflammatory responses and cell apoptosis. Studies have shown that TNFα is associated with the occurrence and development of various tumors such as lung cancer, gastric cancer, colorectal cancer, breast cancer, leukemia, etc., and that in vivo injection of TNFα can inhibit tumor growth to a certain extent. In this embodiment, it was found through testing (such as Figure 6 As shown in the figure, after CAR-T cell treatment, the expression level of TNFα in the serum of nude mice increased. This trend was particularly obvious in the ΔPD-1BCMA-CD7 CAR-T treatment group, indicating that the CAR-T cells provided by the present invention can effectively regulate the secretion ability of TNFα.

[0068] Transforming growth factor-β (TGF-β) is a cytokine isolated from the serum-free culture medium of embryonic fibroblast cell lines transformed by mouse sarcoma virus. It has stimulatory or inhibitory effects on a variety of cells, and can induce the transformation of normal rat renal fibroblasts into tumor cells, so it is named transforming growth factor. The TGF-β / Smad signaling pathway is closely related to the occurrence, development, and metastasis of tumors, but its mechanism of action is also quite complex, and even exhibits different functions at different stages. For example, studies have shown that TGF-β has dual manifestations in the occurrence of tumors. In the early stage, TGF-β acts as an inhibitory factor, which prevents cells from dividing from the early / middle G1 phase to the S phase by inducing the expression of two genes encoding cell cycle-dependent kinase inhibitors p15 and p21, thereby inhibiting the growth and proliferation of tumor cells; when the tumor progresses to the middle and late stages, TGF-β becomes a tumor-promoting factor. In this example, the expression level of TGF-β in nude mouse serum was detected, and the results are as follows: Figure 7 As shown in the figures, there was no significant difference in TGF-β expression between the control group and the treatment group, indicating that the CAR-T cells provided in the present invention did not effectively participate in the regulation of TGF-β expression.

[0069] In summary, the present invention provides a novel chimeric antigen receptor targeting BCMA and CD7. After being introduced into T cells to construct CAR-T cells, it can effectively kill multiple myeloma cells. This killing effect is even more effective after knocking out the PD-1 gene. In in vivo experiments, it can significantly inhibit tumor growth and activate other immune regulatory mechanisms in the body by regulating the expression levels of IL-12 and TNFα, exerting a synergistic anti-tumor effect, providing new ideas for the development of related anti-tumor drugs or therapies.

[0070] While the invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims. Sequence Listing <110> Jiangsu Montpelier Biotechnology Co., Ltd. <120> Bispecific chimeric antigen receptor (CAR) and preparation method thereof <160> 19 <170> SIPOSequenceListing 1.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <400> 1 Leu Thr Thr Glu Pro Phe 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Val Thr Ala Glu Cys Ser Trp Met Glu 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <400> 3 Glu Glu Leu Asp Ser Ser Asn Tyr Lys Leu Gln Trp Pro 1 5 10 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <400> 4 Ser Val Glu Ala Met Pro Ser 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <400> 5 Thr Cys Thr Gln Leu Met Ala Pro 1 5 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <400> 6 Tyr Ser Asn Ala Met Trp Ser Ser Pro Lys Ala Ala 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <400> 7 Leu Leu Glu Ala Tyr 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <400> 8 Tyr Gln Gln Leu Asn Glu Met Trp 1 5 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <400> 9 Arg Glu His Ala Gly Gly Leu Asp Ala Tyr Ala Ser Ala 1 5 10 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <400> 10 Gln Gln Ser Leu Lys Ser Pro 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <400> 11 Ala Gly Arg Leu Val 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <400> 12 Pro Lys Val Ser Val Gln Ala Ser Asn Tyr Pro 1 5 10 <210> 13 <211> 444 [[ID=ID=19]]<212> PRT <213> Artificial Sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Leu Thr 20 25 30 Thr Glu Pro Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala Arg Ile Val Thr Ala Glu Cys Ser Trp Met Glu Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser 65 70 75 80 Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Glu Leu 85 90 95 Asp Ser Ser Asn Tyr Lys Leu Gln Trp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Thr Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Leu Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 14 <211> 212 <212> PRT <213> Artificial Sequence <400> 14 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Ser Val Glu Ala Met Pro Ser Trp Val Gln 20 25 30 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Thr Cys Thr Gln 35 40 45 Leu Met Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Thr Gly 50 55 60 Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala 65 70 75 80 Glu Tyr Tyr Cys Tyr Ser Asn Ala Met Trp Ser Ser Pro Lys Ala Ala 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala 100 105 110 Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn 115 120 125 Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val 130 135 140 Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu 145 150 155 160 Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser 165 170 175 Tyr Leu Ser Leu Thr Thr Glu Gln Trp Lys Ser His Arg Ser Tyr Ser 180 185 190 Cys Gln Val Thr His Glu Gln Ser Thr Val Glu Lys Thr Val Ala Pro 195 200 205 Thr Glu Cys Ser 210 <210> 15 <211> 440 <212> PRT <213> Artificial Sequence <400> 15 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Leu Leu 20 25 30 Glu Ala Tyr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Gln Gln Leu Asn Glu Met Trp Arg Val Thr Ile Ser Val Asp 50 55 60 Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala 65 70 75 80 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Glu His Ala Gly Gly Leu 85 90 95 Asp Ala Tyr Ala Ser Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190 Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Thr Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315 320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 370 375 380 Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 16 <211> 208 <212> PRT <213> Artificial Sequence <400> 16 Ser Tyr Val Leu Thr Gln Pro Pro Ser Pro Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gln Gln Ser Leu Lys Ser Pro Trp Tyr Gln 20 25 30 Gln Pro Pro Gly Gln Ala Pro Val Val Val Val Tyr Ala Gly Arg Leu 35 40 45 Val Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala 50 55 60 Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Val Tyr Tyr 65 70 75 80 Cys Pro Lys Val Ser Val Gln Ala Ser Asn Tyr Pro Phe Gly Gly Gly 85 90 95 Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr 100 105 110 Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu 115 120 125 Thr Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp 130 135 140 Lys Gly Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro 145 150 155 160 Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu 165 170 175 Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr 180 185 190 His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 195 200 205 <210> 17 <211> 1592 <212> PRT <213> Artificial Sequence <400> 17 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Pro Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Asn Leu Thr Thr Glu Pro Phe Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Arg Ile Val Thr Ala Glu Cys Ser Trp 65 70 75 80 Met Glu Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr 85 90 95 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110 Ala Arg Glu Glu Leu Asp Ser Ser Asn Tyr Lys Leu Gln Trp Pro Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 225 230 235 240 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280 285 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Thr Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415 Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys Leu Thr Val Asp Lys 420 425 430 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455 460 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 465 470 475 480 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 485 490 495 Thr Val Thr Leu Thr Cys Ser Val Glu Ala Met Pro Ser Trp Val Gln 500 505 510 Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Thr Cys Thr Gln 515 520 525 Leu Met Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Thr Gly 530 535 540 Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala 545 550 555 560 Glu Tyr Tyr Cys Tyr Ser Asn Ala Met Trp Ser Ser Pro Lys Ala Ala 565 570 575 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala 580 585 590 Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn 595 600 605 Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val 610 615 620 Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu 625 630 635 640 Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser 645 650 655 Tyr Leu Ser Leu Thr Thr Glu Gln Trp Lys Ser His Arg Ser Tyr Ser 660 665 670 Cys Gln Val Thr His Glu Gln Ser Thr Val Glu Lys Thr Val Ala Pro 675 680 685 Thr Glu Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 690 695 700 Gly Gly Ser Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 705 710 715 720 Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 725 730 735 Ser Leu Leu Glu Ala Tyr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 740 745 750 Glu Trp Ile Gly Tyr Gln Gln Leu Asn Glu Met Trp Arg Val Thr Ile 755 760 765 Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val 770 775 780 Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg Glu His Ala 785 790 795 800 Gly Gly Leu Asp Ala Tyr Ala Ser Ala Trp Gly Gln Gly Thr Leu Val 805 810 815 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 820 825 830 Pro Ser Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 835 840 845 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 850 855 860 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 865 870 875 880 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 885 890 895 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 900 905 910 Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 915 920 925 Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 930 935 940 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 945 950 955 960 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 965 970 975 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 980 985 990 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 995 1000 1005 Leu His Gln Thr Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 1010 1015 1020 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 1025 1030 1035 1040 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 1045 1050 1055 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 1060 1065 1070 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 1075 1080 1085 Asn Asn Tyr Asp Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 1090 1095 1100 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 1105 1110 1115 1120 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 1125 1130 1135 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Gly Ser 1140 1145 1150 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Leu Thr Gln 1155 1160 1165 Pro Pro Ser Pro Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys 1170 1175 1180 Gln Gln Ser Leu Lys Ser Pro Trp Tyr Gln Gln Pro Pro Gly Gln Ala 1185 1190 1195 1200 Pro Val Val Val Val Tyr Ala Gly Arg Leu Val Gly Ile Pro Glu Arg 1205 1210 1215 Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg 1220 1225 1230 Val Glu Ala Gly Asp Glu Ala Val Tyr Tyr Cys Pro Lys Val Ser Val 1235 1240 1245 Gln Ala Ser Asn Tyr Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1250 1255 1260 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1265 1270 1275 1280 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Thr Cys Leu Ile Ser Asp 1285 1290 1295 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Gly Asp Ser Ser Pro 1300 1305 1310 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 1315 1320 1325 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 1330 1335 1340 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 1345 1350 1355 1360 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Thr Thr Thr Pro Ala Pro 1365 1370 1375 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 1380 1385 1390 Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 1395 1400 1405 Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 1410 1415 1420 Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg 1425 1430 1435 1440 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 1445 1450 1455 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 1460 1465 1470 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 1475 1480 1485 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 1490 1495 1500 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 1505 1510 1515 1520 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 1525 1530 1535 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 1540 1545 1550 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 1555 1560 1565 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 1570 1575 1580 His Met Gln Ala Leu Pro Pro Arg 1585 1590 <210> 18 <211> 4776 <212> DNA <213> Artificial Sequence <400> 18 atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60 ccggaagtgc agctggtgga aagcggcggc ggcctggtgc agccgccggg cagcctgcgc 120 ctgagctgcg cggcgagcgg ctttaccttt aacctgacca ccgaaccgtt ttgggtgcgc 180 caggcgccgg gcaaaggcct ggaatgggtg gcgcgcattg tgaccgcgga atgcagctgg 240 atggaacgct ttaccattag ccgcgatgat agcaaaaaca gcctgtatct gcagatgaac 300 agcctgaaaa ccgaagaatac cgcggtgtat tattgcgcgc gcgaagaact ggatagcagc 360 aactaaac tgcagtggcc gtggggccag ggcaccctgg tgaccgtgag cagcgcgagc 420 accaaaggcc cgagcgtgtt tccgctggcg ccgtgcagcc gcagcaccag cgaaagcacc 480 gcggcgctgg gctgcctggt gaagattat tttccggaac cggtgaccgt gagctgggaac 540 agcggcgcgc tgaccagcgg cgtgcatacc tttccggcgg tgctgcagag cagcggcctg 600 tatagcctga gcagcgtggt gaccgtgccg agcagcagcc tgggcaccaa aacctatacc 660 tgcaacgtgg atcataacc gagcaacacc aaagtggata aacgcgtgga aagcaaatat 720 ggcccgccgt gcccgccgtg cccggcgccg gaagcggcgg gcggcccgag cgtgtttctg 780 tttccgccga aaccgaaaga taccctgatg attagccgca ccccggaagt gacctgcgtg 840 gtggtggatg tgagccagga agatccggaa gtgcagttta actggtatgt ggatggcgtg 900 gaagtgcata acgcgaaaac caaaccgcgc gaaacagt ttaacagcac ctatcgcgtg 960 gtgagcgtgc tgaccgtgct gcatcaggat tggctgaacg gcaccgaata taaatgcaaa 1020 gtgagcaaca aaggcctgcc gagcagcatt gaaaaaacca ttagcaaagc gaaaggccag 1080 ccgcgcgaac cgcaggtgta taccctgccg ccgagccagg aagaaatgac caaaaaccag 1140 gtgagcctga cctgcctggt gaaaggcttt tatccgagcg atattgcggt ggaatgggaa 1200 agcaacggcc agccggaaaa caactataaa accaccccgc cggtgctgga tagcgatggc 1260 agctttctgc tgtatagcaa actgaccgtg gataaaagcc gctggcagga aggcaacgtg 1320 tttagctgca gcgtgatgca tgaagcgctg cataaccatt atacccagaa aagcctgagc 1380 ctgagcctgg gcaaaggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 1440 cagaccgtgg tgacccagga accgagcctg accgtgagcc cgggcggcac cgtgaccctg 1500 acctgcagcg tggaagcgat gccgagctgg gtgcagcaga aaccgggcca ggcgccgcgc 1560 ggcctgattg gcacctgcac ccagctgatg gcgccgggca ccccggcgcg ctttagcggc 1620 agcctgaccg gcggcaaagc ggcgctgacc ctgagcggcg tgcagccgga agatgaagcg 1680 gaatattatt gctatagcaa cgcgatgtgg agcagcccga aagcggcgtt tggcggcggc 1740 accaaactga ccgtgctggg ccagccgaaa gcggcgccga gcgtgaccct gtttccgccg 1800 agcagcgaag aactgcaggc gaacaaagcg accctggtgt gcctgattag cgatttttat 1860 ccgggcgcgg tgaccgtggc gtggaaagcg gatagcagcc cggtgaaagc gggcgtggaa 1920 accaccaccc cgagcaaaca gagcaacaac aaatatgcgg cgagcagcta tctgagcctg 1980 accaccgaac agtggaaaag ccatcgcagc tatagctgcc aggtgaccca tgaacagagc 2040 accgtggaaa aaaccgtggc gccgaccgaa tgcagcggcg gcggcggcag cggcggcggc 2100 ggcagcggcg gcggcggcag ccagctgcag ctgcaggaaa gcggcccggg cctggtgaaa 2160 ccgagcgaaa ccctgagcct gacctgcacc gtgagcggcg gcagcattag cctgctggaa 2220 gcgtattgga ttcgccagcc gccgggcaaa ggcctggaat ggattggcta tcagcagctg 2280 aacgaaatgt ggcgcgtgac cattagcgtg gataccagca aaaaccagtt tagcctgaaa 2340 ctgagcagcg tgaccgcggc ggataccgcg gtgtattatt gcgcgcgccg cgaacatgcg 2400 ggcggcctgg atgcgtatgc gagcgcgtgg ggccagggca ccctggtgac cgtgagcagc 2460 gcgagcacca aaggcccgag cgtgtttccg ctggcgccga gcagccgcag caccagcgaa 2520 agcaccgcgg cgctgggctg cctggtgaaa gattattttc cggaaccggt gaccgtgagc 2580 tggaacagcg gcgcgctgac cagcggcgtg catacctttc cggcggtgct gcagagcagc 2640 ggcctgtata gcctgagcag cgtggtgacc gtgccgagca gcagcctggg caccaaaacc 2700 tatacctgca acgtggatca taaaccgagc aacaccaaag tggataaacg cgtggaaagc 2760 aaatatggcc cgccgtgccc gccgtgcccg gcgccggaag cggcgggcgg cccgagcgtg 2820 tttctgtttc cgccgaaacc gaaagatacc ctgatgatta gccgcacccc ggaagtgacc 2880 tgcgtggtgg tggatgtgag ccaggaagat ccggaagtgc agtttaactg gtatgtggat 2940 ggcgtggaag tgcataacgc gaaaaccaaa ccgcgcgaag aacagtttaa cagcacctat 3000 cgcgtggtga gcgtgctgac cgtgctgcat cagacctggc tgaacggcaa agaataataaa 3060 tgcaaagtga gcaacaaagg cctgccgagc agcattgaaa aaaccattag caaagcgaaa 3120 ggccagccgc gcgaaccgca ggtgtatacc ctgccgccga gccaggaaga aatgaccaaa 3180 aaccaggtga gcctgacctg cctggtgaaa ggcttttatc cgagcgatat tgcggtggaa 3240 tgggaaagca acggccagcc ggaaaacaac tatgatacca ccccgccggt gctggatagc 3300 gatggcagct ttttctgta tagccgcctg accgtggata aaagccgctg gcaggaaggc 3360 aacgtgttta gctgcagcgt gatgcatgaa gcgctgcata accattatac ccagaaaagc 3420 ctgagcctga gcctgggcaa aggcggcggc ggcagcggcg gcggcggcag cggcggcggc 3480 ggcagcagct atgtgctgac ccagccgccg agcccgagcg tggcgccggg ccagaccgcg 3540 cgcattacct gccagcagag cctgaaaagc ccgtggtatc agcagccgcc gggccaggcg 3600 ccggtggtgg tggtgtatgc gggccgcctg gtgggcattc cggaacgctt tagcggcagc 3660 aacagcggca acaccgcgac cctgaccatt agccgcgtgg aagcgggcga tgaagcggtg 3720 tattattgcc cgaaagtgag cgtgcaggcg agcaactatc cgtttggcgg cggcaccaaa 3780 ctgaccgtgc tgggccagcc gaaagcggcg ccgagcgtga ccctgtttcc gccgagcagc 3840 gaagaactgc aggcgaacaa agcgaccctg acctgcctga ttagcgattt ttacggggc 3900 gcggtgaccg tggcgtggaa aggcgatagc agcccggtga aagcgggcgt ggaaaccacc 3960 accccgagca aacagagcaa caacaaatat gcggcgagca gctatctgag cctgaccccg 4020 gaacagtgga aaagccatcg cagctatagc tgccaggtga cccatgaagg cagcaccgtg 4080 gaaaaaaccg tggcgccgac cgaatgcagc accaccaccc cggcgccgcg cccgccgacc 4140 ccggcgccga ccattgcgag ccagccgctg agcctgcgcc cggaagcgtg ccgccccggcg 4200 gcgggcggcg cggtgcatac ccgcggcctg gattttgcgt gcgatattta tatttgggcg 4260 ccgctggcgg gacctgcgg cgtgctgctg ctgagcctgg tgattaccct gtattgccgc 4320 agcaaacgca gccgcctgct catagacgat tatatgaaca tgaccccgcg ccgcccgggc 4380 ccgacccgca aacattatca gccgtatgcg ccgccgcgcg attttgcggc gtatcgcagc 4440 cgcgtgaaat ttagccgcag cgcggatgcg ccggcgtatc agcagggcca gaaccagctg 4500 tataacgaac tgaacctggg ccgccgcgaa gaatatgatg tgctggataa acgccgcggc 4560 cgcgatccgg aaatgggcgg caaaccgcgc cgcaaaaacc cgcaggaagg cctgtataac 4620 gaactgcaga aagataaaat ggcggaagcg tatagcgaaa ttggcatgaa aggcgaacgc 4680 cgccgcggca aaggccatga tggcctgtat cagggcctga gcaccgcgac caaagatacc 4740 tatgatgcgc tgcatatgca ggcgctgccg ccgcgc 4776 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <400> 19 gaggaccgca gccagcc 17

Claims

1. A bispecific chimeric antigen receptor targeting BCMA and CD7, characterized in that: The chimeric antigen receptor includes the following structure: S-BCMA scFv-CD7 scFv-H-TM-C-CD3ζ, wherein BCMA scFv is an antigen binding domain targeting BCMA; CD7 scFv is an antigen binding domain targeting CD7; "-" is a connecting peptide or peptide bond; S is a signal peptide; H is a hinge region; TM is a transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is an intracellular signaling sequence; BCMA scFv includes heavy chain CDR1-3 regions as shown in SEQ ID NOs: 1-3, respectively, and light chain CDR1-3 regions as shown in SEQ ID NOs: 4-6, respectively; CD7 scFv includes heavy chain CDR1-3 regions as shown in SEQ ID NOs: 7-9, respectively, and light chain CDR1-3 regions as shown in SEQ ID NOs: 10-12, respectively.

2. The bispecific chimeric antigen receptor according to claim 1, wherein The co-stimulatory signal molecule is CD28.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the bispecific chimeric antigen receptor according to any one of claims 1-2.

4. A T cell, characterized in that The T cell expresses the bispecific chimeric antigen receptor according to any one of claims 1-2.

5. The T cell according to claim 4, characterized in that The T cells are T cells with PD-1 gene knocked out.

6. Use of the bispecific chimeric antigen receptor according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, or the T cell according to any one of claims 4 to 5 in the preparation of a drug for treating a tumor, wherein the tumor is myeloma.

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