A bispecific chimeric antigen receptor targeting CD19 and BCMA, and related products and applications

Abstract

This invention discloses a bispecific chimeric antigen receptor targeting CD19 and BCMA, along with related products and applications. The invention creatively designs a tandem combination of BCMA single-chain antibody (scFv) and CD19 single-chain antibody (scFv). Experiments have demonstrated that bispecific chimeric antigen receptor (CAR) engineered immune cells (especially CAR-NK cells) constructed using this tandem approach, with BCMA scFv preceding CD19 scFv, exhibit significantly superior killing effects. Furthermore, they possess a broader targeting coverage, stronger anti-antigen escape ability, and better safety, demonstrating significant clinical translational prospects and industrialization value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bispecific chimeric antigen receptor targeting CD19 and BCMA, and related products and applications. Background Technology

[0002] B-cell-related diseases (including hematologic malignancies and autoimmune diseases) have become a serious threat to human health. Among them, B-cell leukemia is the most common malignant tumor in children, and the prognosis for adult patients is even worse. B-cell lymphoma has many subtypes and is highly aggressive. Although the survival time of multiple myeloma has been extended with current treatments, it is still incurable. All three types of hematologic malignancies have problems such as relapse and difficulty in treatment, and significant toxicity of current therapies. Systemic lupus erythematosus, rheumatoid arthritis, and other B-cell-related autoimmune diseases have a high incidence rate and are prone to chronic damage and disability of multiple organs. Current treatments have limitations such as limited response rates, the risk of long-term use of immunosuppression, and the high relapse rate.

[0003] Current treatments for these diseases face numerous bottlenecks: traditional standard chemotherapy, immunochemotherapy, and targeted monoclonal antibodies (such as anti-CD20 and CD38 monoclonal antibodies) can achieve partial disease control, but they are generally accompanied by severe toxic side effects and are not very effective in relapsed or refractory patients, making it difficult to overcome the dilemma of drug resistance and relapse; although CAR-T cell therapy has made breakthrough progress in related fields, it faces safety risks such as cytokine release syndrome (CRS) and neurotoxicity, as well as practical challenges such as target escape leading to treatment failure, long autologous cell preparation cycle, and high cost, which limit its clinical application scenarios.

[0004] CAR-NK therapy, as an emerging cell immunotherapy technology, has shown significant advantages: it has better safety, inducing only mild or no CRS and neurotoxicity, and is less likely to cause graft-versus-host disease (GvHD). Allogeneic NK cells have the potential to be developed into "off-the-shelf" products. At the same time, it retains the multi-killing mechanism mediated by natural cytotoxic receptors, resulting in more comprehensive killing efficacy. However, most existing CAR-NK products are designed for single targets, and have not yet solved the problem of antigen escape, which cannot address treatment failure caused by target loss, nor can they simultaneously address different coexisting types of B-cell-related diseases. Therefore, there is an urgent need in this field to overcome this core limitation through technological optimization. Summary of the Invention

[0005] In view of this, in order to solve the above-mentioned technical problems in the field, the purpose of this invention is to provide a novel bispecific target CAR-NK cell product that simultaneously targets the two key B cell markers CD19 and BCMA, aiming to address these challenges in the following ways: 1) Expanding the target range: covering the entire lineage from progenitor B cells (CD19+) to terminal plasma cells (BCMA+), with the potential to eliminate lesions in one treatment for a variety of B cell diseases; 2) Preventing antigen escape: the dual-target design acts as a "double insurance," greatly reducing the risk of treatment failure due to the loss of a single target; 3) Improving safety: utilizing the naturally safer characteristics of NK cells, it is expected to significantly reduce serious toxic side effects such as CRS while maintaining high efficacy, laying the foundation for the development of safer and more universal "off-the-shelf" cell drugs.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0007] A first aspect of the present invention provides a bispecific chimeric antigen receptor targeting CD19 and BCMA, the chimeric antigen receptor comprising an antibody targeting CD19 and an antibody targeting BCMA, wherein the antibody targeting BCMA is first and the antibody targeting CD19 is second.

[0008] The amino acid sequences of HCDR1-3 in the heavy chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:17-19, respectively.

[0009] The amino acid sequences of LCDR1-3 in the light chain variable region of the antibody targeting CD19 are shown in SEQ ID NO:20-22, respectively.

[0010] The amino acid sequences of HCDR1-3 in the heavy chain variable region of the antibody targeting BCMA are shown in SEQ ID NO:25-27, respectively.

[0011] The amino acid sequences of LCDR1-3 in the light chain variable region of the antibody targeting BCMA are shown in SEQ ID NO:28-30, respectively.

[0012] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody targeting CD19 is shown in SEQ ID NO:15;

[0013] The amino acid sequence of the light chain variable region of the antibody targeting CD19 is shown in SEQ ID NO:16;

[0014] The amino acid sequence of the heavy chain variable region of the antibody targeting BCMA is shown in SEQ ID NO:23;

[0015] The amino acid sequence of the light chain variable region of the antibody targeting BCMA is shown in SEQ ID NO:24.

[0016] Furthermore, the chimeric antigen receptor, from N-terminus to C-terminus, comprises: a signal peptide, the antibody targeting BCMA, linker 2, the antibody targeting CD19, a hinge region and a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signal transduction domain.

[0017] Furthermore, the antibody targeting BCMA is obtained by sequentially connecting the light chain variable region of the antibody targeting BCMA, linker 1, and the heavy chain variable region of the antibody targeting BCMA.

[0018] The antibody targeting CD19 is obtained by sequentially connecting the light chain variable region of the antibody targeting CD19, linker 1, and the heavy chain variable region of the antibody targeting CD19.

[0019] The hinge region and transmembrane structural domain are CD8α hinge region and transmembrane structural domain;

[0020] The co-stimulation signal structure domain is a 4-1BB co-stimulation signal structure domain;

[0021] The intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain.

[0022] Furthermore, the amino acid sequence of the antibody targeting BCMA is shown in SEQ ID NO:3;

[0023] The amino acid sequence of the antibody targeting CD19 is shown in SEQ ID NO:1;

[0024] The amino acid sequence of linker 1 is shown in SEQ ID NO:5;

[0025] The amino acid sequence of linker 2 is shown in SEQ ID NO:7;

[0026] The amino acid sequences of the CD8α hinge region and transmembrane domain are shown in SEQ ID NO:9;

[0027] The amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO:11;

[0028] The amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO:13.

[0029] In some embodiments, the linker 1 is preferably a flexible linker commonly used in the variable region of IgG antibodies, and can be selected from (GGGGS)n (where n=1-4), (GGGS)n (where n=2-5), or (GGXG)n (where X is alanine A, serine S, or threonine T, n=2-4).

[0030] In some implementations, the linker 2 may be selected from: (1) long-chain flexible linkers, such as (GGGGS)n (where n=3-6), (GGGSGG)n (where n=2-4) or (GSGGGG)n (where n=2-4), which can provide sufficient spatial distance so that the antibody targeting BCMA and the antibody targeting CD19 can independently bind to their respective antigen epitopes and avoid mutual interference; (2) composite linkers containing rigid fragments, such as (GGGGS)2-Gly-Ala-Gly-(GGGGS)2 or (GGGS)3-Pro-(GGGS)3, wherein proline (Pro) can increase the local rigidity of the linker, prevent the double antibody from overfolding, and further optimize the spatial orientation of the two antigen binding domains; (3) short peptide linkers, such as (GS)6, (GSSG)3, etc.

[0031] A second aspect of the present invention provides a nucleic acid molecule that encodes the chimeric antigen receptor described in the first aspect of the present invention.

[0032] Furthermore, the nucleotide sequences of the signal peptide, the antibody targeting BCMA, linker 2, the antibody targeting CD19, the hinge region and transmembrane domain, the co-stimulatory signaling domain, and the intracellular signal transduction domain in the chimeric antigen receptor are shown in SEQ ID NO:31, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, respectively.

[0033] A third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0034] In some embodiments, the vector is a DNA vector or an RNA vector. In other embodiments, the vector is a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector. In still other embodiments, the viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a poxvirus vector, or a herpesvirus vector.

[0035] In some embodiments, the expression vector described in this invention refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a plasmid, which is a circular double-stranded DNA loop into which additional DNA fragments can be linked. Another type of vector is a viral vector, in which additional DNA fragments can be linked to a viral genome.

[0036] The fourth aspect of the present invention provides any of the following products:

[0037] (1) An engineered immune cell expressing the chimeric antigen receptor described in the first aspect of the present invention;

[0038] (2) A pharmaceutical composition comprising the engineered immune cells;

[0039] (3) A biological agent comprising the pharmaceutical composition;

[0040] (4) A bispecific antibody, a trispecific antibody or an NK cell connector comprising a tandem combination of the antibody targeting BCMA and the antibody targeting CD19 as described in the first aspect of the present invention, wherein the antibody targeting BCMA is preceding the antibody targeting CD19.

[0041] In some embodiments, the immune cells are NK cells, T cells, B cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, or any combination thereof. In a specific embodiment of the present invention, the immune cells are NK cells, and the engineered immune cells are NK cells modified with a bispecific chimeric antigen receptor targeting CD19 and BCMA as described in the first aspect of the present invention.

[0042] In some embodiments, the engineered immune cells are immune cells that are genetically modified to stably express the bispecific chimeric antigen receptor (CAR) described in the first aspect of the present invention, preferably NK cells or T cells. These engineered immune cells are transduced via lentiviral method to introduce the CAR gene encoding the tandem combination of BCMA scFv (front) and CD19 scFv (back) into the immune cell genome, enabling the CAR molecule to be efficiently expressed on the cell surface. Its core feature lies in its ability to specifically recognize two targets, BCMA and CD19. It can precisely bind to and eliminate BCMA-positive target cells (such as MM.1S cells) and CD19-positive target cells (such as Nalm-6 cells) through a CAR-mediated specific killing mechanism, while preserving the natural killing activity of immune cells (such as NKG2D body-mediated killing of NK cells). Furthermore, this application has experimentally demonstrated that the tandem arrangement with BCMA scFv first results in higher CAR expression levels on the cell surface and stronger killing activity, which can effectively cover the complete cell lineage from CD19-positive precursor B cells to BCMA-positive terminal plasma cells, significantly reducing the risk of antigen escape.

[0043] In some embodiments, the pharmaceutical composition uses the engineered immune cells described in this invention as the active ingredient, and may also contain pharmaceutically acceptable excipients such as carriers, diluents, stabilizers, or excipients to maintain the in vitro activity, stability, and in vivo bioavailability of the engineered immune cells. Pharmaceutically acceptable excipients include, but are not limited to, cell culture-related buffers (such as PBS buffer), nutrients (such as albumin and amino acids), and protectants (such as dimethyl sulfoxide and glycerol). The selection of excipients must meet the safety standards of biopharmaceuticals, not affect the targeted killing function of the active ingredient, and not cause additional toxic side effects. This pharmaceutical composition can be formulated into a dosage form suitable for infusion according to clinical needs. Its formulation is optimized to maintain the activity and functional integrity of the engineered immune cells during storage, transportation, and infusion, providing a safe and effective form of administration for clinical treatment.

[0044] In some embodiments, the biopharmaceutical comprises the aforementioned pharmaceutical composition and is a biomedical product for disease treatment developed based on the engineered immune cells of this invention, meeting the relevant standards for the production, quality control, and clinical application of biopharmaceuticals. The preparation of this biopharmaceutical requires adherence to a strict production process, including large-scale expansion of engineered immune cells, purity screening, activity testing, aseptic processing, and formulation filling, ensuring that the quantity, purity, activity, and safety of the active cells in the product meet clinical treatment requirements. Its core advantages lie in inheriting the dual-target targeting, high killing activity, and low toxicity of engineered immune cells, while achieving product stability and consistency through standardized production processes. It can be used as an "off-the-shelf" biopharmaceutical for rapid clinical use, eliminating the need for individualized preparation. This effectively solves the problems of long preparation cycles and high costs associated with traditional cell therapies, providing a convenient and efficient clinical solution for the treatment of B-cell-related diseases.

[0045] In some implementations, the dosage forms of the biological agent include, but are not limited to, injectable suspensions and lyophilized powder for injection. Injectable suspensions can be directly administered intravenously or via other clinically applicable routes of administration. Their optimized formulation maintains the activity and stability of engineered immune cells in a liquid environment, facilitating immediate drug delivery. Lyophilized powder for injection removes moisture from the formulation through freeze-drying technology, significantly improving the product's storage stability and transport convenience. Before use, it must be rapidly reconstituted into a suspension using a dedicated reconstitution solution. Both dosage forms meet the sterility requirements, activity preservation standards, and safety and convenience requirements for clinical drug administration of biological products, and can be flexibly selected based on storage conditions, transportation distance, and clinical application scenarios.

[0046] In some embodiments, the core functional units of the bispecific antibody, trispecific antibody, or NK cell connector are all tandem combinations of the antibody targeting BCMA (BCMA scFv) and the antibody targeting CD19 (CD19 scFv) described in the first aspect of this invention, with the optimal tandem arrangement being BCMA scFv first and CD19 scFv second. Specifically, the bispecific antibody contains only this BCMA-CD19 scFv tandem combination, achieving specific recognition and killing by simultaneously binding to BCMA and CD19 antigens on the surface of target cells; the trispecific antibody can further fuse antibody fragments targeting other disease-related targets on the basis of this tandem combination, expanding the targeting range; the NK cell connector mediates the specific binding of NK cells to target cells through this tandem combination, enhancing the natural killing activity of NK cells. The product can be flexibly applied to different types of immunotherapy scenarios, does not rely on cell carriers, and has the characteristics of relatively simple preparation process and flexible use, providing diversified treatment options for B-cell related diseases.

[0047] The fifth aspect of the invention provides for any of the following applications:

[0048] (1) The application of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, or the expression vector described in the third aspect of the present invention in the preparation of engineered immune cells targeting CD19 and BCMA;

[0049] (2) The use of the engineered immune cells described in the fourth aspect of the present invention in the preparation of medicaments for treating CD19 and / or BCMA-related diseases.

[0050] Furthermore, the CD19 and / or BCMA-related diseases include B-cell malignancies or B-cell-related autoimmune diseases;

[0051] The B-cell malignant tumor is one or more of B-cell leukemia, B-cell lymphoma, and multiple myeloma;

[0052] The B-cell-related autoimmune diseases mentioned are one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, membranous nephropathy, nephritis, and multiple sclerosis.

[0053] In this invention, the CD19 and / or BCMA-related diseases are not limited to the specific disease types listed above, but include any disease related to CD19 and / or BCMA, including but not limited to: various B-cell-derived malignant proliferative diseases expressing CD19 and / or BCMA, such as precursor B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt lymphoma, etc.; autoimmune diseases mediated by abnormal B-cell activation or autoantibodies, such as Sjögren's syndrome, systemic sclerosis, autoimmune hemolytic anemia, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica spectrum disorders, etc.; and other diseases in which abnormal CD19-positive B cells or BCMA-positive plasma cells are involved in the pathogenesis.

[0054] Furthermore, the present invention provides a method for treating CD19 and / or BCMA-related diseases, the method comprising the steps of administering a therapeutically effective amount of the engineered immune cells, pharmaceutical composition or biological agent of the present invention as described above to a subject in need.

[0055] In some implementations, the "subject in need" refers to mammals, preferably humans, suffering from CD19 and / or BCMA-related diseases, including but not limited to: patients diagnosed with B-cell malignancies or B-cell-related autoimmune diseases, specifically covering B-cell leukemia (such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, etc.), B-cell lymphoma (such as diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, etc.), multiple myeloma (especially relapsed / refractory multiple myeloma), as well as patients with systemic lupus erythematosus, rheumatoid arthritis, membranous nephropathy, nephritis, and multiple sclerosis. Patients with autoimmune diseases such as chemoradiotherapy and Sjögren's syndrome, in which abnormal activation of B cells or BCMA-positive plasma cells are involved in the pathogenesis; the subjects may include those who have poor response, no response, relapse or intolerance to existing chemotherapy, immunochemotherapy, single-target CAR-T / CAR-NK therapy, anti-CD20 / CD38 monoclonal antibody and other treatment regimens, as well as treatment-naïve patients who have the presence of target cells expressing CD19 and / or BCMA (such as CD19-positive B cells, BCMA-positive plasma cells or related tumor cells) in their bodies, and who have the basic physical conditions to receive adoptive cell immunotherapy after clinical evaluation.

[0056] In some implementations, the administration refers to the process of delivering a therapeutically effective amount of the engineered immune cells, pharmaceutical composition, or biological agent of the present invention to a subject in need, with the core objective of enabling the engineered immune cells to target and kill CD19-positive and / or BCMA-positive target cells (abnormal B cells, plasma cells, or related tumor cells) in the subject, thereby achieving a therapeutic effect.

[0057] In some implementations, the administration route is primarily intravenous infusion, i.e., by introducing a pharmaceutical composition or biological agent containing the engineered immune cells described in this invention into the subject's peripheral vein. This route allows the engineered immune cells to rapidly and widely distribute throughout the bloodstream, thereby precisely identifying and binding to CD19-positive and / or BCMA-positive target cells throughout the body to exert a killing effect. This is suitable for the treatment of B-cell malignancies (such as leukemia, lymphoma, and multiple myeloma) and systemically distributed B-cell-related autoimmune diseases. In addition, depending on the subject's specific condition (such as concentrated local lesions, poor peripheral circulation, etc.), other suitable administration routes may be used after clinical evaluation, including but not limited to bone marrow infusion (directly targeting abnormal plasma cells or tumor cells in the bone marrow) and local perfusion (for locally invasive lesions). The specific administration route needs to be determined by the clinician based on the subject's disease type, severity, and physical condition.

[0058] In some implementations, the therapeutically effective dose refers to the dosage of engineered immune cells (or pharmaceutical compositions or biologics containing such cells) capable of effectively treating CD19 and / or BCMA-related diseases. This dosage must be sufficient to achieve one or more of the following therapeutic effects: inhibiting the proliferation of target cells (tumor cells expressing CD19 and / or BCMA or abnormally activated B cells); killing target cells and reducing their burden in the body; alleviating disease-related symptoms (such as bone destruction, anemia, kidney damage caused by tumors, or joint inflammation and organ damage caused by autoimmune diseases); delaying disease progression; prolonging the subject's survival; or improving their quality of life. Furthermore, this dosage must not cause unacceptable toxic side effects (such as severe cytokine release syndrome, neurotoxicity, graft-versus-host disease, etc.). The therapeutically effective dose needs to be individualized based on the specific circumstances of the subject, including but not limited to factors such as the subject's age, weight, sex, disease type, disease stage, disease severity, previous treatment history, and immune function status. It is usually measured in cell count (e.g., 1 × 10⁻⁶ cells per kilogram of body weight). 6 -1×10 8 (Engineered immune cells) can be administered via single or multiple infusions. The specific dosage and frequency of administration need to be routinely and dynamically adjusted by clinicians based on treatment response and safety monitoring results.

[0059] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0060] This invention creatively designs a tandem combination of BCMA single-chain antibody (scFv) and CD19 single-chain antibody (scFv). Experiments have demonstrated that bispecific chimeric antigen receptor (CAR) engineered immune cells (especially CAR-NK cells) constructed using this tandem approach, with BCMA scFv preceding CD19 scFv, exhibit significantly superior killing effects. Furthermore, they possess broader targeting coverage, stronger anti-antigen escape capabilities, and better safety. This invention can efficiently target various B-cell-related diseases, including CD19-positive or BCMA-positive B-cell leukemia, B-cell lymphoma, multiple myeloma, systemic lupus erythematosus, and rheumatoid arthritis, providing a superior treatment option for these diseases and demonstrating significant clinical translational prospects and industrialization value. Attached Figure Description

[0061] Figure 1 Schematic diagram of the structure of a dual-specific target CAR;

[0062] Figure 2 Flow cytometry results showing the expression of bispecific target CAR on NK cells at different time points;

[0063] Figure 3: Statistical results of the expression of bispecific target CAR on NK cells at different time points, where the vertical axis represents the positive expression rate of CAR on NK cells (in %).

[0064] Figure 4 : Results of the in vitro killing experiment of bispecific target CAR-NK cells (target cells: CD19 positive Nalm-6 cells, i.e. CD19 positive B-type acute lymphoblastic leukemia cells);

[0065] Figure 5 : Results of in vitro killing experiment of bispecific target CAR-NK cells (target cells: BCMA-positive MM.1S cells, i.e. BCMA-positive multiple myeloma cells). Detailed Implementation

[0066] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0067] Example 1: Expression of CAR with bispecific targets in NK cells

[0068] 1. Experimental Methods

[0069] (1) BCMA CAR: It is formed by sequentially connecting the signal peptide (sp), the BCMA antibody light chain variable region (BCMA-VL), linker 1, the BCMA antibody heavy chain variable region (BCMA-VH), the CD8α hinge region and transmembrane domain (CD8α hinge+TM), the 4-1BB co-stimulatory signal domain, and the CD3ζ intracellular signal transduction domain.

[0070] (2) CD19 CAR: It is formed by sequentially connecting the signal peptide (sp), the CD19 antibody light chain variable region (CD19-VL), linker 1, the CD19 antibody heavy chain variable region (CD19-VH), the CD8α hinge region and transmembrane domain (CD8α hinge+TM), the 4-1BB co-stimulatory signal domain, and the CD3ζ intracellular signal transduction domain.

[0071] (3) BCMA-CD19 dual CAR: It is formed by sequentially connecting the signal peptide (sp), the BCMA antibody light chain variable region (BCMA-VL), linker 1, the BCMA antibody heavy chain variable region (BCMA-VH), linker 2, the CD19 antibody light chain variable region (CD19-VL), linker 1, the CD19 antibody heavy chain variable region (CD19-VH), the CD8α hinge region and transmembrane domain (CD8α hinge+TM), the 4-1BB co-stimulatory signal domain, and the CD3ζ intracellular signal transduction domain.

[0072] (4) CD19-BCMA dual CAR: It is formed by sequentially connecting the signal peptide (sp), CD19 antibody light chain variable region (CD19-VL), linker 1, CD19 antibody heavy chain variable region (CD19-VH), linker 2, BCMA antibody light chain variable region (BCMA-VL), linker 1, BCMA antibody heavy chain variable region (BCMA-VH), CD8α hinge region and transmembrane domain (CD8α hinge+TM), 4-1BB co-stimulatory signal domain and CD3ζ intracellular signal transduction domain.

[0073] The structural diagram of the bispecific target CAR (BCMA-CD19 dual CAR, CD19-BCMA dual CAR) is shown below. Figure 1 As shown.

[0074] The amino acid sequence of the CD19 antibody (CD19 scFv) is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2. The amino acid sequences of the heavy chain variable region and the light chain variable region of the CD19 antibody are shown in SEQ ID NO:15-16, respectively. The amino acid sequences of HCDR1-3 in the heavy chain variable region of the CD19 antibody are shown in SEQ ID NO:17-19, respectively. The amino acid sequences of LCDR1-3 in the light chain variable region of the CD19 antibody are shown in SEQ ID NO:20-22, respectively. The amino acid sequence of the BCMA antibody (BCMA scFv) is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4. The amino acid sequences of the heavy chain variable region and the light chain variable region of the BCMA antibody are shown in SEQ ID NO:23-24, respectively. The amino acid sequences of HCDR1-3 in the heavy chain variable region of the BCMA antibody are shown in SEQ ID NO:25-27, respectively. The amino acid sequences of LCDR1-3 in the light chain variable region of the BCMA antibody are shown in SEQ ID NO:25-27, respectively. Shown in NO:28-30.

[0075] The amino acid sequence of linker 1 is shown in SEQ ID NO:5 and the nucleotide sequence is shown in SEQ ID NO:6; the amino acid sequence of linker 2 is shown in SEQ ID NO:7 and the nucleotide sequence is shown in SEQ ID NO:8; the amino acid sequence of the CD8α hinge region and transmembrane domain is shown in SEQ ID NO:9 and the nucleotide sequence is shown in SEQ ID NO:10; the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO:11 and the nucleotide sequence is shown in SEQ ID NO:12; the amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO:13 and the nucleotide sequence is shown in SEQ ID NO:14; and the amino acid sequence of the signal peptide is shown in SEQ ID NO:31 and the nucleotide sequence is shown in SEQ ID NO:32.

[0076] Based on the CARs designed above, BCMA CAR NK cells, CD19 CAR NK cells, BCMA-CD19 dual CAR NK cells, and CD19-BCMA dual CAR NK cells were constructed. The expression of CARs on the surface of NK cells was detected at different time points after lentiviral transduction. Specifically, the synthesized genes were cloned into CAR gene expression plasmids using PCR to construct the corresponding CAR lentiviral expression plasmids. The specific construction method is as follows:

[0077] (1) Packaging bispecific target CAR lentivirus using a third-generation lentivirus packaging system: 293T cells were revived in T225 cell culture flasks, passaged, and expanded; when the aggregation of 293T cells reached 85%, plasmid transfection was performed, and lentivirus was packaged; the lentivirus packaging plasmids included: transfection plasmid (expression plasmid with CAR gene cloned), helper plasmids (pMDLg / pRRE plasmid, ReV plasmid, pMD2.G plasmid), and the ratio of the above four plasmids was 2-3:1-3:1-3:0.25-1; the culture medium supernatant was harvested after 48 hours, and the lentivirus was harvested by centrifugation at 20,000 rpm.

[0078] (2) The CAR gene with bispecific target was introduced into NK cells using lentivirus and stably expressed on the surface of NK cells: NK cells were counted and 0.5 M was used for lentivirus transduction. The lentivirus transduction system was as follows: total volume 1 mL, NK culture medium 590 μL, NK cells 0.5 M, Polybrene 0.8 μL, Vectorfusion 10 μL, lentivirus 400 μL. After 12 hours, the culture medium was replaced with fresh medium and cultured for a longer period.

[0079] (3) Flow cytometry was used to detect the expression of CAR on the surface of NK cells at different time points after lentiviral transduction: After 4, 8, 12, and 14 days of culture following lentiviral transduction, the expression of bispecific target CAR on NK cells was detected by flow cytometry; 0.5 M of NK or CAR-NK cells were used, and antibodies against CD19 CAR and BCMA CAR were detected using Miltenyi CD19 CAR and BCMA CAR at a concentration of 1:50-100, at room temperature, incubated in the dark for 30 minutes; and then the cells were analyzed. Based on the different tandem sequences of CD19 and BCMA, the cells were divided into: dual CAR-NK-1 cells (BCMA-CD19 tandem) and dual CAR-NK-2 cells (CD19-BCMA tandem).

[0080] 2. Experimental Results

[0081] Experimental results are as follows Figure 2 and Figure 3 As shown, it illustrates the expression of the bispecific target CAR on NK cells at different time points, as detailed below:

[0082] (1) The cells were divided into 5 groups. The first group, CTRL (no virus added), consisted of unmodified NK cells, serving as the negative control group. No CAR expression was detected in these cells. The second group, CTRL (CD19 CAR lentivirus added only), consisted of positive control cells expressing only CD19 CAR. CD19 CAR expression was detected in these cells, but BCMA CAR expression was absent. The third group, CTRL (BCMA CAR lentivirus added only), consisted of positive control cells expressing only BCMA CAR. BCMA CAR expression was detected in these cells, but CD19 CAR expression was absent. The fourth group consisted of dual CAR-NK-1 expression tandem BCMA-CD19 dual CAR (i.e., BCMA scFv first, CD19 scFv second). The fifth group consisted of dual CAR-NK-2 expression tandem CD19-BCMA dual CAR (i.e., CD19 scFv first, BCMA scFv second).

[0083] (2) Flow cytometry was used to detect the expression of CAR on NK cells in each group at 4, 8, 12, and 14 days after viral infection. In the first group (CTRL, no virus added), no CAR was expressed at any time point. The second and third groups expressed only one specific target CAR, and CAR expression was observed at all time points, with the expression increasing over time. The fourth and fifth groups showed bispecific target CAR expression at all time points, with the expression increasing over time.

[0084] (3) Based on the expression of bispecific target CAR at each time point, the expression level of CAR in the fourth group of bispecific CAR-NK-1 was always significantly higher than that in the fifth group of CAR-NK-2 (D4: 15.03% vs 11.92%; D8: 14.62% vs 11.49%; D12: 28.94% vs 26.85%; D14: 47.19% vs 44.31%).

[0085] The above results indicate that, in terms of CAR expression level, the cascaded arrangement of BCMA scFv first and CD19 scFv last in the CAR structure is significantly better than the cascaded arrangement of CD19 scFv first and BCMA scFv last. This result is a technical effect that would not have been anticipated by those skilled in the art based on the prior art.

[0086] Example 2: In vitro target cell killing experiment of CAR-NK cells with dual specific targets

[0087] 1. Experimental Methods

[0088] NK cells, CD19 CAR NK cells constructed in Example 1 above, BCMA-CD19 dual CAR NK cells (i.e., dual CAR-NK-1 cells), and CD19-BCMA dual CAR NK cells (i.e., dual CAR-NK-2 cells) were co-cultured with two target cell types (CD19-positive Nalm-6 cells and BCMA-positive MM.1S cells) at an effector-to-target ratio (all effector-to-target ratios were 1:4). The killing effect of CAR-NK cells on these two cell types was detected at different time points (0 h, 24 h, and 48 h of co-culture), and the effect of different tandem sequences on killing was compared. The specific experimental methods are as follows:

[0089] Nalm-6 tumor cells were used as target cells expressing CD19 antigen, and MM.1S tumor cells were used as target cells expressing BCMA antigen. CAR-NK cells with bispecific targets were co-cultured with the above two types of cells at an effector-target ratio of 1:4, and the killing effect of CAR-NK cells on these two types of cells was detected at different time points.

[0090] Control NK cells, CD19 CAR-NK cells, BCMA CAR-NK cells, and bispecific target CAR-NK-1 and bispecific target CAR-NK-2 cells were counted separately, and 0.125 M was used as effector cells. Nalm-6 cells and MM.1S cells were counted separately, and 0.5 M was used as target cells. Each group of NK and CAR-NK cells was co-cultured with the two target cell types at an effector-to-target ratio of 1:4. At 0, 24, and 48 hours after co-culture, changes in the proportion of effector cells and target cells in the co-culture system were detected by flow cytometry to evaluate the cytotoxic effect of CAR-NK cells.

[0091] 2. Experimental Results

[0092] The experimental results of the bispecific target CAR-NK cell in vitro killing assay (target cells: CD19-positive Nalm-6 cells, i.e., CD19-positive B-type acute lymphoblastic leukemia cells) are as follows: Figure 4 As shown, the results are displayed below:

[0093] (1) The target cells were CD19-positive Nalm-6 cells, and the ratio of effector cells to target cells was 1:4;

[0094] (2) Effector cells were divided into four groups. The first group consisted of NK cells without any gene modification. The second group consisted of NK cells expressing CD19CAR, which served as a positive control. The third and fourth groups consisted of CAR-NK cells expressing different tandem sequences of bispecific target CARs.

[0095] (3) Flow cytometry results after 0 hours of co-culture showed that the effect-to-target ratio was 1:4 for each group;

[0096] (4) Flow cytometry results after 24 hours of co-culture showed that the first group of NK cells did not have a clear killing effect on CD19-positive Nalm-6 cells. The second, third, and fourth groups of CAR-NK cells all had significant killing effects on target cells, and the proportion of target cells was significantly reduced to 14.06%, 12.02%, and 19.14%, respectively.

[0097] (5) The killing ability of CAR-NK cells with different tandem sequences in the third and fourth groups is significantly different. The CAR-NK killing effect of the third group with BCMA-CD19 scFv tandem sequence (i.e., BCMA scFv first and CD19 scFv last) is the best and significantly better than that of the fourth group of CAR-NK cells. This result is also a technical effect that cannot be expected by those skilled in the art based on the existing technology.

[0098] (6) Flow cytometry results after 48 hours of co-culture showed that the first group of NK cells still did not have a clear killing effect on CD19-positive Nalm-6 cells. The second, third and fourth groups of CAR-NK cells had cleared the target cells, with the proportion of target cells remaining at only 0.61%, 0.16% and 0.58%, respectively, and the CAR-NK cells of the third group with BCMA-CD19 scFv tandem sequence had a significantly better killing effect.

[0099] The experimental results of the bispecific target CAR-NK cell in vitro killing assay (target cells: BCMA-positive MM.1S cells, i.e., BCMA-positive multiple myeloma cells) are as follows: Figure 5 As shown, the results are displayed below:

[0100] (1) The target cells were BCMA-positive MM.1S cells, and the ratio of effector cells to target cells was 1:4;

[0101] (2) Effector cells were divided into four groups. The first group consisted of NK cells without any gene modification. The second group consisted of NK cells expressing BCMACAR, which served as a positive control. The third and fourth groups consisted of dual CAR-NK cells expressing different tandem sequences.

[0102] (3) Flow cytometry results after 0 hours of co-culture showed that the effect-to-target ratio was 1:4 for each group;

[0103] (4) Flow cytometry results after 24 hours of co-culture showed that the first group of NK cells did not have a clear killing effect on BCMA-positive MM.1S cells. The second, third and fourth groups of CAR-NK cells could clear the target cells within 24 hours, with only 1.93%, 0.79% and 1.85% of the target cells remaining, respectively. Among them, the third group of CAR-NK cells with the BCMA-CD19 scFv tandem sequence (i.e., BCMA scFv first and CD19 scFv second) had the best killing effect and was significantly better than the fourth group of CAR-NK cells. This result is also a technical effect that could not have been expected by those skilled in the art based on the existing technology.

[0104] (5) Flow cytometry results after 48 hours of co-culture showed that the first group of NK cells did not significantly kill BCMA-positive MM.1S cells. The second, third and fourth groups of CAR-NK cells completely cleared the target cells and maintained them at very low levels: 0.05%, 0.08% and 0.08%, respectively.

[0105] The above results show that, in terms of lethality, the cascaded configuration of BCMA with scFv first and CD19 with scFv last in the CAR structure is significantly better than the cascaded configuration of CD19 with scFv first and BCMA with scFv last. This result is also a technical effect that would not have been expected by those skilled in the art based on the prior art.

Claims

1. A bispecific chimeric antigen receptor targeting CD 19 and BCMA, characterized in that, The chimeric antigen receptor comprises an antibody targeting CD19 and an antibody targeting BCMA, the antibody targeting BCMA in front, the antibody targeting CD19 in back; The amino acid sequences of HCDR1-3 in the heavy chain variable region of the antibody targeting CD19 are respectively shown as SEQ ID NOs: 17-19; The amino acid sequences of LCDR1-3 in the light chain variable region of the antibody targeting CD19 are respectively shown as SEQ ID NOs: 20-22; The amino acid sequences of HCDR1-3 in the heavy chain variable region of the antibody targeting BCMA are respectively shown as SEQ ID NOs: 25-27; The amino acid sequences of LCDR1-3 in the light chain variable region of the antibody targeting BCMA are respectively shown as SEQ ID NOs: 28-30.

2. The chimeric antigen receptor of claim 1, wherein, The amino acid sequence of the heavy chain variable region of the antibody targeting CD19 is shown as SEQ ID NO: 15; The amino acid sequence of the light chain variable region of the antibody targeting CD19 is shown as SEQ ID NO: 16; The amino acid sequence of the heavy chain variable region of the antibody targeting BCMA is shown as SEQ ID NO: 23; The amino acid sequence of the light chain variable region of the antibody targeting BCMA is shown as SEQ ID NO:

24.

3. The chimeric antigen receptor of claim 2, wherein, The chimeric antigen receptor comprises, from N-terminal to C-terminal: a signal peptide, the antibody targeting BCMA, a linker 2, the antibody targeting CD19, a hinge region and transmembrane domain, a costimulatory signal domain, an intracellular signaling domain.

4. The chimeric antigen receptor of claim 3, wherein, The antibody targeting BCMA is obtained by concatenation of the light chain variable region of the antibody targeting BCMA, a linker 1, the heavy chain variable region of the antibody targeting BCMA, in order; The antibody targeting CD19 is obtained by concatenation of the light chain variable region of the antibody targeting CD19, a linker 1, the heavy chain variable region of the antibody targeting CD19, in order; The hinge region and transmembrane domain are CD8a hinge region and transmembrane domain; The costimulatory signal domain is 4-1BB costimulatory signal domain; The intracellular signaling domain is CD3zeta intracellular signaling domain.

5. The chimeric antigen receptor of claim 4, wherein, The amino acid sequence of the antibody targeting BCMA is shown as SEQ ID NO: 3; The amino acid sequence of the antibody targeting CD19 is shown as SEQ ID NO: 1; The amino acid sequence of the linker 1 is shown as SEQ ID NO: 5; The amino acid sequence of the linker 2 is shown as SEQ ID NO: 7; The amino acid sequence of the CD8a hinge region and transmembrane domain is shown as SEQ ID NO: 9; The amino acid sequence of the 4-1BB costimulatory signal domain is shown as SEQ ID NO: 11; The amino acid sequence of the CD3zeta intracellular signaling domain is shown as SEQ ID NO:

13.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor of any one of claims 1-5.

7. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule of claim 6.

8. An engineered immune cell, characterized in that, The engineered immune cell expresses the chimeric antigen receptor of any one of claims 1-5.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the engineered immune cell of claim 8.

10. A biological agent, characterized in that, The biological agent comprises the pharmaceutical composition of claim 9.

11. A bispecific antibody, characterized in that, The bispecific antibody is a tandem combination of the antibody targeting BCMA and the antibody targeting CD19 as described in claim 1, the antibody targeting BCMA being in front, the antibody targeting CD19 being in back.

12. Use of any of the following aspects, characterized in that The application comprises: (1) use of the chimeric antigen receptor of any one of claims 1-5, the nucleic acid molecule of claim 6 or the expression vector of claim 7 in the preparation of an engineered immune cell targeting CD19 and BCMA; (2) use of the engineered immune cell of claim 8 in the preparation of a medicament for treating a CD19 and / or BCMA related disease.

13. Use according to claim 12, characterized in that, The CD19 and / or BCMA related disease comprises a B cell malignancy or a B cell related autoimmune disease; The B cell malignancy is one or more of B cell leukemia, B cell lymphoma, multiple myeloma; The B cell related autoimmune disease is one or more of systemic lupus erythematosus, rheumatoid arthritis, membranous nephropathy, nephritis, multiple sclerosis.

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