Chimeric antigen receptor, immune cell and application thereof
By developing chimeric antigen receptors that dual-target BCMA and CD19 to transform NK cells, the problems of large side effects, high drug resistance and high cost of CAR-T cell therapy have been solved, and a blood tumor treatment effect with low side effects, sustained efficacy and low cost has been achieved.
Patent Information
- Application Number
- CN202510798790.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-09-16
AI Technical Summary
Existing CAR-T cell therapy has problems in treating blood cancers, such as severe side effects, high drug resistance, high cost, and poor effectiveness against solid tumors. Although CAR-NK cell therapy has advantages, it still needs improvement.
Develop a chimeric antigen receptor that dual-targets BCMA and CD19 to transform NK cells and enhance their ability to recognize and attack cancer cells. It includes antigen-binding domains, co-stimulatory molecules, and CD3ζ activation signaling domains, and prepares CAR-NK cells through lentiviral transduction.
It has achieved the treatment of blood tumors with lower side effects, less immune rejection reactions, strong and sustained efficacy, and low cost, and has shown significant anti-tumor activity both in vitro and in vivo experiments.
Abstract
Description
Technical Field
[0001] The present application relates to the field of cell immunotherapy technology, and specifically to chimeric antigen receptors, immune cells and their applications. Background Art
[0002] Traditional treatments for blood tumors such as leukemia, lymphoma, and multiple myeloma include chemotherapy, radiotherapy, and hematopoietic stem cell transplantation, but some patients still face problems of drug resistance or relapse. In recent years, chimeric antigen receptor cell therapy (CAR) has made breakthroughs in malignant tumors (such as acute lymphoblastic leukemia and diffuse large B-cell lymphoma). The current main cell therapies include CAR-T cell therapy and CAR-NK cell therapy. The principles of the two cell therapies are similar, both of which are to genetically modify T cells or NK cells to enhance their recognition and attack on cancer cells. Among them, chimeric antigen receptors usually include the following parts: Extracellular recognition domain: can specifically recognize antigens on the surface of cancer cells. Transmembrane domain: fixes the receptor to the cell membrane of immune cells. Intracellular signal transduction domain: used to activate the killing function of immune cells.
[0003] CAR-NK cell therapy is an emerging cell therapy with the following advantages: lower side effects, lower risk of immune rejection, independence from human leukocyte pairing, lower drug resistance, stronger broad-spectrum anti-tumor effects, and lower manufacturing and treatment costs.
[0004] CAR-T cell therapy is one of the earliest new tumor treatments. Although it has made significant progress in the treatment of certain types of blood cancers (such as acute lymphoblastic leukemia and diffuse large B-cell lymphoma) and has shown strong therapeutic potential, it also has some shortcomings. Clinically, it has been found to cause side effects such as cytokine release syndrome, neurotoxicity, and immunosuppression in patients. In addition, it has poor therapeutic effects on some solid tumors and causes tumor antigen escape. In addition, CAR-T cell therapy is very complex and time-consuming, and as the treatment progresses, the problem of drug resistance becomes increasingly prominent, and the cost of treatment is also very expensive. Summary of the Invention
[0005] Based on this, one embodiment of the present application provides a chimeric antigen receptor that dual-targets BCMA and CD19, or an immune cell containing the chimeric antigen receptor, or a use thereof.
[0006] The technical solution is as follows:
[0007] A chimeric antigen receptor comprising an antigen binding domain comprising a single domain antibody (scFv) targeting BCMA and / or a single chain antibody (scFv) targeting CD19.
[0008] In one embodiment, the chimeric antigen receptor further comprises a connecting peptide, a co-stimulatory molecule and a CD3ζ activation signaling domain, wherein the co-stimulatory molecule comprises CD28 and / or 4-1BB.
[0009] In one embodiment, the chimeric antigen receptor includes, from N-terminus to C-terminus, an antigen binding domain, a connecting peptide, a co-stimulatory molecule and a CD3ζ activation signal domain.
[0010] A nucleic acid molecule encoding the chimeric antigen receptor.
[0011] An expression cassette comprising the nucleic acid molecule.
[0012] The recombinant vector contains the nucleic acid molecule or the expression cassette.
[0013] An immune cell expressing the chimeric antigen receptor, containing the nucleic acid molecule, containing the expression cassette or containing the recombinant vector.
[0014] In one embodiment, the immune cells include NK cells.
[0015] A pharmaceutical composition comprising the immune cells and, optionally, one or more pharmaceutically acceptable carriers.
[0016] Use of the immune cells or the pharmaceutical composition in preparing a product for preventing or treating BCMA and / or CD19 target-related diseases, optionally, the related diseases include tumors and autoimmune diseases.
[0017] Use of the immune cells or the pharmaceutical composition in preparing a product for preventing or treating tumors; optionally, the tumors include blood tumors and solid tumors.
[0018] In one embodiment, the immune cells or the pharmaceutical composition are used to kill BCMA and / or CD19-positive tumor cells.
[0019] In one embodiment, the immune cell or the pharmaceutical composition is used to inhibit the proliferation of BCMA and / or CD19 positive tumor cells.
[0020] In one embodiment, the immune cell or the pharmaceutical composition is used to inhibit the growth of BCMA and / or CD19 positive tumors.
[0021] A method for treating blood tumors with fewer side effects, fewer immune rejection reactions, longer-lasting efficacy, and lower cost, comprising the following steps: treating with the immune cells.
[0022] Compared with traditional technologies, this application has the following beneficial effects:
[0023] Experiments have shown that the CAR-NK cells prepared in this application have strong in vitro activity and transfection efficiency. In vitro cell experiments have shown that CAR-NK cells have a certain killing effect on blood tumors. In addition, a human xenograft tumor mouse model was constructed, and the in vivo experimental effect of CAR-NK cell therapy was detected using a small animal living imaging system. The results showed that CAR-NK cell therapy can also play a therapeutic role in xenograft tumor mouse models. DETAILED DESCRIPTION
[0024] To make the above-mentioned objects, features, and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail below. The following description sets forth many specific details to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.
[0026] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.
[0027] The NK cells used in this application were obtained from peripheral blood mononuclear cells by magnetic bead negative sorting. The CD19-CAR and BCMA-CAR vectors were designed using lentivirus, and the dual CAR expression units were connected by T2A self-cleavage peptides, placed under the same promoter, and infected 293T cells to produce high-titer lentivirus. The activated NK cells and lentivirus were then placed together in a 37°C biochemical incubator for culture, and cell screening and functional verification experiments were performed after culture.
[0028] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.
[0029] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.
[0030] Example 1 Isolation and purification of NK cells
[0031] Collect peripheral blood: Take 10-20mL of peripheral blood, followed by density gradient centrifugation: Dilute the blood sample 1:1 in PBS. Slowly add the diluted blood to a test tube containing density gradient centrifugation fluid (such as Ficoll-Paque), taking care not to mix. Centrifuge at 800g for 20-30 minutes, collect the PBMC layer: wash twice with PBS, and centrifuge at 300g for 10 minutes to remove residual Ficoll. Prepare a magnetic bead separation system (such as Miltenyi Biotec's NK cell isolation kit): According to the kit instructions, add the corresponding antibodies and magnetic beads, and mix and incubate with PBMC. Pass the mixture through the magnetic column, and unlabeled NK cells will flow out. Collect the outflow portion, which usually contains highly pure NK cells. Wash the cells twice with PBS to remove residual reagents. Prepare the culture medium: Use RPMI-1640 medium, add 10% FBS and 1% penicillin-streptomycin. The purified NK cells are seeded into culture flasks / culture plates, and the cell density is usually 1-2x 10 6 cells / mL.
[0032] Add cell activation factors: Add an appropriate amount of IL-2 (e.g., 1000 IU / mL) or other NK cell activation factors to promote NK cell proliferation and activation. Culture the cells in a 37°C, 5% CO2 incubator.
[0033] During culture: Change the culture medium every 2-3 days to maintain cell viability. Regularly measure cell density and viability, and perform cell counts and viability tests as needed. Flow cytometric analysis: NK cell purity is assessed using flow cytometry, typically using CD56 and CD3 antibody markers. Functional validation: NK cell activity is verified through cytotoxicity assays or other functional assays.
[0034] Example 2 Construction of CD19-CAR and BCMA-CAR vectors and preparation of CAR-NK cells
[0035] Construction of CD19-CAR vector: CD19-CAR usually includes: single-chain antibody (scFv) targeting CD19, linker peptide, co-stimulatory molecule (such as CD28 or 4-1BB), and an internal CD3ζ activation signal domain.
[0036] The above sequence was synthesized and cloned into a suitable plasmid vector (such as pCDNA3.1 or other suitable plasmid).
[0037] Construction of BCMA-CAR vector: BCMA-CAR also consists of scFv (specific for BCMA), a connecting peptide, a co-stimulatory molecule, and a CD3ζ signaling domain. The above BCMA-CAR sequence was synthesized and cloned into a separate plasmid vector.
[0038] The constructed CD19-CAR and BCMA-CAR vectors are transfected into suitable viral packaging cells (such as HEK293T cells) along with packaging plasmids. Three plasmids are typically required: one for the target vector (CD19-CAR or BCMA-CAR), one for the packaging plasmid containing the adenovirus or lentivirus, and one for the viral envelope plasmid. A transfection reagent (such as PEI) is used to transfect the plasmids into the packaging cells. After transfection, the cells are cultured for 24-48 hours, during which time the viral product will be secreted into the culture medium. The culture supernatant is collected and the cells are removed by centrifugation to obtain the viral supernatant. The prepared viral supernatant is mixed with NK cells and electroporated to enhance viral entry into T cells. Following transduction, the cells are cultured for an additional 48 hours to ensure CAR expression. Finally, flow cytometry is used to analyze whether the NK cells express both the CD19-CAR and BCMA-CAR on their surface.
[0039] If necessary, magnetic bead sorting or other methods can be used to sort out dual CAR-positive NK cells (BCMA CD19CAR-NK cells) for further experiments.
[0040] Example 3 In vivo anti-tumor function detection of dual CAR-positive NK cells
[0041] A human primary tumor cell xenograft model was established, and the dual-CAR-positive NK cells prepared in Example 2 were used to treat the human primary tumor cell xenograft model. The therapeutic effect was observed using an in vivo imaging system.
[0042] Establishment of human primary tumor cell xenograft model:
[0043] Obtain tumor samples from patients. Ethics committee approval should be obtained and informed consent should be obtained from the patients.
[0044] Tumor tissue processing: The collected tumor tissue was processed under sterile conditions. Blood and impurities were removed by washing with PBS. The tumor tissue was cut into small pieces, typically approximately 1-3 mm each, avoiding excessive crushing to preserve the tissue structure.
[0045] Select appropriate immunodeficient mice, such as NOD / SCID mice, nude mice (Balb / c nude mice), or NSG mice (NOD-scid IL2rgnull). These mice lack functional T cells, B cells, and natural killer cells and are able to accept xenografts of human tumors. Mice are usually 8-12 weeks old to ensure their health. Anesthesia is usually performed on the mice, usually using isoflurane gas anesthesia or a combination of ketamine and diazepam. Use sterile surgical instruments to prepare the mice for tumor transplantation surgery. Mix the cut tumor tissue pieces with Matrigel (or other matrix gel) to enhance the survival and growth of tumor cells. Use a sterile syringe to subcutaneously inject the tumor pieces into the back of the mouse or other suitable location. Each mouse can be injected with 1-2 tumor tissue pieces, and the total amount injected is usually 100-200μL.
[0046] Tumor tissue can be digested into a single-cell suspension by trypsinization and centrifugation. The cell suspension can be transplanted into mice via subcutaneous injection, intraperitoneal injection, or other methods.
[0047] Postoperative Care: After transplantation, carefully monitor the health of the mice to ensure they have recovered from anesthesia and are able to eat and move normally. Ensure the mice are housed in a sterile environment to avoid infection. Measure tumor size with a caliper every 2-3 days. Analyze the tumor microenvironment using methods such as immunohistochemistry and flow cytometry to analyze the infiltration of immune cells in the tumor and the expression of tumor-related markers.
[0048] Once the tumor reaches the predetermined experimental endpoint (such as the tumor volume exceeds the specified maximum volume, or the mouse's health condition deteriorates significantly), it can be humanely killed using cervical dislocation or an overdose of anesthetics. The tumor tissue is removed and further histological analysis (such as H&E staining, immunohistochemistry staining, Western Blot, etc.) is performed to evaluate the tumor's morphological characteristics, proliferation, apoptosis and other markers. A metastasis model is established, and the primary tumor and its distant metastatic sites (such as lungs, liver, bones, etc.) can be removed to analyze the metastatic lesions and evaluate the tumor's metastatic potential. Record and analyze data such as tumor growth curves, pathological changes in tumor tissue, and treatment effects.
[0049] Use appropriate statistical methods (such as t-test, analysis of variance, etc.) to process the experimental data and evaluate the differences between different treatment groups.
[0050] Dual CAR-positive NK cells can significantly inhibit tumor growth, kill tumors, and have a longer-lasting inhibitory effect.
[0051] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0052] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims, and the specification may be used to interpret the content of the claims.
Claims
1. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises an antigen-binding domain, which comprises a single-chain antibody targeting BCMA and / or a single-chain antibody targeting CD19.
2. The chimeric antigen receptor according to claim 1, wherein The chimeric antigen receptor further comprises a connecting peptide, a costimulatory molecule and a CD3ζ activation signaling domain, wherein the costimulatory molecule comprises CD28 and / or 4-1BB.
3. A nucleic acid molecule, characterized in that It encodes the chimeric antigen receptor according to claim 1 or 2.
4. An expression cassette, characterized in that Containing the nucleic acid molecule according to claim 3.
5. A recombinant vector, characterized in that Contains the nucleic acid molecule according to claim 3 or contains the expression cassette according to claim 4.
6. An immune cell, characterized in that A method of expressing the chimeric antigen receptor according to claim 1 or 2, containing the nucleic acid molecule according to claim 3, containing the expression cassette according to claim 4, or containing the recombinant vector according to claim 5.
7. The immune cell according to claim 6, characterized in that The immune cells include NK cells.
8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the immune cells according to claim 6 or 7, and optionally, further comprises one or more pharmaceutically acceptable carriers.
9. Use of the immune cell according to claim 6 or 7 or the pharmaceutical composition according to claim 8 in the preparation of a product for preventing or treating BCMA and / or CD19 target-related diseases, optionally wherein the related diseases include tumors and autoimmune diseases.
10. Use of the immune cell according to claim 6 or 7 or the pharmaceutical composition according to claim 8 in the preparation of a product for preventing or treating tumors; optionally, the tumor includes a blood tumor and a solid tumor.