A chimeric antigen receptor targeting CD19 and BCMA and its application

Abstract

The present invention relates to the field of biomedicine technology, and proposes a chimeric antigen receptor targeting CD19 and BCMA dual targets and its application. The chimeric antigen receptor comprises a CD19-targeting antibody light chain, a linker, a humanized nano antibody targeting BCMA, and a CD19-targeting antibody heavy chain connected in series. The present invention optimizes the structure of the dual-targeting CD19 / BCMA CAR, places the humanized nano antibody targeting BCMA between the antibody light chain targeting CD19 and the antibody heavy chain targeting CD19, and utilizes the self-cleavage P / T 2A site to achieve the separate expression of IL7 and CCL9 in T cells. The chimeric antigen receptor with this structure exhibits a strong tumor-killing ability and can be used to prepare a drug for treating blood tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor targeting CD19 and BCMA dual targets and applications thereof. Background Art

[0002] Multiple myeloma (MM) is the second most common hematologic malignancy, often occurring in the elderly. MM is a plasma cell disorder characterized by terminally differentiated plasma cells, plasma cell infiltration of the bone marrow, and the presence of monoclonal immunoglobulins (or immunoglobulin fragments) in the serum and / or urine. It is often accompanied by osteolytic bone disease, anemia, and renal failure. In recent years, thanks to the continuous emergence of new drugs and improved testing methods, the diagnosis and treatment of MM have been continuously improved and refined, and the overall survival (OS) of MM patients has been significantly prolonged. However, MM is still considered an incurable disease, and nearly all patients will experience a relapsed or refractory phase. Effective and tolerable treatment options for patients with relapsed and refractory (R / R) MM remain unsatisfactory, with a median overall survival (OS) of 9.3 months for triple-refractory patients and 5.6 months for penta-refractory patients. Therefore, finding effective treatment options for patients with relapsed and refractory (R / R) MM is an urgent scientific challenge.

[0003] The emergence of a new generation of immunotherapy has brought new hope for the treatment of patients with relapsed and refractory (R / R) MM. B-cell maturation antigen (BCMA) is a transmembrane glycoprotein receptor that is overexpressed in the bone marrow plasma cells of MM patients and not expressed in important tissues. It has become the main therapeutic target for multiple myeloma (MM). Currently, in clinical trials at home and abroad, CAR-T cells targeting BCMA have achieved significant therapeutic effects in the treatment of relapsed / refractory MM, with an overall remission rate of 48%-100% and a complete remission rate of 6%-76%. However, some patients do not respond to BCMA-targeted CAR T cells or relapse shortly after achieving a response. After CAR-T treatment, downregulation or loss of BCMA expression has been observed in 4%-33% of progressive patients.

[0004] To solve this problem, researchers have begun to attempt dual-targeted CAR T cells, such as using CAR T cells targeting CD19 / CD22 to treat leukemia and lymphoma, CAR T cells targeting CD19 / CD20 to treat lymphoma, and CART cells targeting BCMA / CD38 to treat MM. Studies have shown that less differentiated MM cells or myeloma-like stem cells are CD19 positive, and in most patients, CD19 is expressed at low density in some myeloma cells (10.3%-80%), so it can be eliminated using target CD19CAR T cells. Based on the above conclusions, it is feasible to use humanized anti-CD19 and anti-BCMACAR T cells for the treatment of relapsed / relapsed MM patients. In the present invention, the inventors further designed CAR T cells targeting CD19 and BCMA, and enhanced the IL7 / CCL19 expression elements that enhance T cell expansion and tumor killing ability in the CAR structure to enhance T cell expansion and tumor killing ability. Summary of the Invention

[0005] In view of this, the present invention proposes a chimeric antigen receptor targeting CD19 and BCMA dual targets and its application for enhancing T cell expansion and tumor killing ability.

[0006] The technical solution of the present invention is implemented as follows: In the first aspect, the present invention provides a chimeric antigen receptor targeting CD19 and BCMA dual targets, including a CD19-targeting antibody light chain, a linker, a humanized nanobody targeting BCMA, and a CD19-targeting antibody heavy chain connected in series.

[0007] Based on the above technical solution, preferably, the amino acid sequence of the light chain of the CD19-targeting antibody is shown in SEQ ID NO: 2, the amino acid sequence of the Linker is shown in SEQ ID NO: 3, the amino acid sequence of the BCMA-targeting nanobody is shown in SEQ ID NO: 4 or SEQ ID NO: 5, and the amino acid sequence of the heavy chain of the CD19-targeting antibody is shown in SEQ ID NO: 6.

[0008] On the basis of the above technical solution, preferably, the chimeric antigen receptor further includes a signal peptide, a hinge region, a transmembrane region CD8, and a 4-1BB-CD3z intracellular activation domain.

[0009] On the basis of the above technical solution, preferably, the chimeric antigen receptor includes a signal peptide, a CD19-targeting antibody light chain, a linker, a humanized nanobody targeting BCMA, a hinge region, a CD19-targeting antibody heavy chain, a transmembrane region CD8 and a 4-1BB-CD3ζ intracellular activation domain connected in series.

[0010] Based on the above technical solution, preferably, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 7, and the amino acid sequence of the 4-1BB-CD3ζ intracellular activation domain is shown in SEQ ID NO: 8.

[0011] On the basis of the above technical solution, preferably, the chimeric antigen receptor further comprises cytokines IL7 and CCL19, and the cytokines IL7 and CCL19 utilize the self-cleavage P2A and T2A sites to insert into the 4-1BB-CD3ζ intracellular activation domain.

[0012] Based on the above technical solution, preferably, the amino acid sequence of the P / T2A site is shown as SEQ ID NO: 9, the amino acid sequence of the T2A site is shown as SEQ ID NO: 10, the amino acid sequence of the cytokine IL7 is shown as SEQ ID NO: 11, and the amino acid sequence of the cytokine CCL19 is shown as SEQ ID NO: 12.

[0013] In a second aspect, the present invention provides a nucleic acid encoding the above-mentioned chimeric antigen receptor.

[0014] In a third aspect, the present invention provides a lentiviral vector comprising the above-mentioned nucleic acid.

[0015] In a fourth aspect, the present invention provides a host cell containing the aforementioned lentiviral vector.

[0016] In a fifth aspect, the present invention provides the use of the above-mentioned chimeric antigen receptor, nucleic acid, lentiviral vector or host cell in the preparation of a drug for treating mammalian hematological tumors.

[0017] The chimeric antigen receptor targeting CD19 and BCMA and its application in the present invention have the following beneficial effects compared with the prior art:

[0018] (1) The present invention optimizes the structure of a dual-targeting CD19 / BCMA CAR, placing a humanized nanobody targeting BCMA between the light chain and heavy chain of an antibody targeting CD19, and utilizing a self-cleaving P / T 2A site to achieve separate expression of IL7 and CCL9 in T cells. The chimeric antigen receptor with this structure exhibits strong tumor-killing ability and can be used to prepare drugs for treating hematologic malignancies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is a structural diagram of the chimeric antigen receptor of the present invention targeting CD19 and BCMA dual targets; VL: CD19-targeting antibody light chain; 38A / 38B: BCMA-targeting nanoantibody; VH: CD19-targeting antibody heavy chain; CD8TM: CD8 transmembrane region; 4-1BB CD3ζ: intracellular activation domain; 2A: P / T 2A site; 7×19: cytokine IL7 and CCL19 expression sequences.

[0021] Figure 2 Figure 1 is a diagram of the lentiviral transduction efficiency of the dual-target chimeric antigen receptor sequence of the present invention. Figure A is a diagram of the lentiviral transduction results, and Figure B is a diagram of the expression of the dual-target CAR on T cells.

[0022] Figure 3 Figure 3 is a comparison of the in vitro killing ability of two dual-target CAR-T cells. Figure A is a graph showing the specific killing experiment of the two dual-target CAR-T cells, and Figure B is a graph showing the changes in CD4+T and CD8+T cell degranulation values during the killing process of the two dual-target CAR-T cells.

[0023] Figure 4 These are verification diagrams of the specific binding ability of different CAR vectors with antigens. Figure A shows the effect of two CAR lentiviral vectors specifically binding to BCMA and CD19 proteins, and Figure B shows the effect of co-incubation of three CAR cells with CD19-Fc proteins at different concentrations.

[0024] Figure 5 This is an in vivo experiment of dual-target CAR-T against multiple myeloma. Figure A shows the establishment of a multiple myeloma and lymphoma xenograft model, Figure B shows the establishment of a multiple myeloma and lymphoma xenograft model, Figures C and D show the imaging results, Figure E shows the establishment of an intravenous model of anti-tumor effects targeting CD19, and Figures F and G show the imaging results. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] The present invention provides a chimeric antigen receptor targeting CD19 and BCMA dual targets, such as Figure 1 As shown, the chimeric antigen receptor includes a signal peptide, a CD19-targeting antibody light chain (VL), a linker, a humanized nanobody targeting BCMA (38A or 38B), a hinge region, a CD19-targeting antibody heavy chain (VH), a transmembrane region CD8 (CD8TM) and a 4-1BB-CD3ζ intracellular activation domain, which are connected in series.

[0027] Among them, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the light chain of the CD19-targeting antibody is shown in SEQ ID NO: 2, the amino acid sequence of the Linker is shown in SEQ ID NO: 3, the amino acid sequence of the BCMA-targeting nanoantibody is shown in SEQ ID NO: 4 or SEQ ID NO: 5, the amino acid sequence of the heavy chain of the CD19-targeting antibody is shown in SEQ ID NO: 6, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 7, and the amino acid sequence of the 4-1BB-CD3z intracellular activation domain is shown in SEQ ID NO: 8.

[0028] In addition, if Figure 1 As shown, the chimeric antigen receptor also includes cytokines IL7 and CCL19, which utilize the self-cleavage P / T 2A site to insert the 4-1BB-CD3z intracellular activation domain.

[0029] The amino acid sequence of the P / T2A site is shown in SEQ ID NO: 9, the amino acid sequence of the cytokine IL7 is shown in SEQ ID NO: 10, and the amino acid sequence of the cytokine CCL19 is shown in SEQ ID NO: 11.

[0030] Table 1 Amino acid sequence table

[0031]

[0032] 1. Construction of Lentiviral Vectors

[0033] 1. Dual-target CAR structure design:

[0034] like Figure 1 As shown, in this example, two chimeric antigen receptors targeting both CD19 and BCMA were constructed: 19CAR-38AL and 19CAR-38BL. The amino acid sequence of the BCMA-targeting nanobody in 19CAR-38AL is shown in SEQ ID NO: 4, and the amino acid sequence of the BCMA-targeting nanobody in 9CAR-38BL is shown in SEQ ID NO: 5.

[0035] The dual-target CAR sequence targeting BCMA and CD19 includes the following components arranged in sequence (such as Figure 1 shown):

[0036] VL (CD19 antibody light chain),

[0037] 38A / 38B (BCMA-targeted nanoantibodies),

[0038] VH (CD19 antibody heavy chain),

[0039] Hinge area,

[0040] transmembrane domain (CD8TM),

[0041] Co-stimulatory molecule (4-1BB),

[0042] Signaling domain (CD3ζ),

[0043] P2A site (2A, used for separate expression of multiple genes),

[0044] Cytokine expression sequence (7x19, promotes the secretion of IL-7 and CCL19).

[0045] 2. Transfection of Lentiviral Packaging Cells

[0046] HEK293T cells were seeded in a T175 flask (cultivated to 70%-80% confluency). The CAR plasmid and helper plasmid were mixed and transfected using Lipofectamine 3000. Fresh medium was replaced 6-8 hours later. Supernatant was collected 48-72 hours after transfection. The supernatant was filtered through a 0.45 μm filter and concentrated to obtain high-titer lentivirus.

[0047] 3. BT Cell Activation and Transduction

[0048] PBMC Isolation and T Cell Activation: PBMCs were isolated from the peripheral blood of healthy donors by Ficoll gradient centrifugation. PBMCs were stimulated with anti-CD3 / CD28 antibody magnetic beads to activate T cells and induce their expansion. Activated T cells were cultured in RPMI 1640 medium supplemented with IL-2 (50-100 IU / mL) for 3 days.

[0049] Lentiviral transduction: 3 days after activation, T cells were adjusted to an appropriate concentration (1×10 6 Incubate lentiviral particles (MOI = 5-10) with T cells for 48 hours. Polybrene (e.g., 8 μg / mL) may be added to improve transduction efficiency. After transduction, replace the culture medium and continue culturing and expanding the T cells.

[0050] 4. Transduction Efficiency Detection

[0051] Flow cytometry antibody labeling: Collect transduced T cells and fluorescently label them with antibodies targeting specific markers on the dual-target CAR (such as fluorescent Fc secondary antibodies that bind to the CAR antibody structure). After labeling, wash twice with PBS. Use flow cytometry to detect transduction efficiency. The results are shown in Figure 2 .

[0052] Figure 2 As shown, dual-target CAR (19CAR-38AL and 19CAR-38BL) are expressed on T cells ( Figure 2 A), the transduction efficiency can reach more than 72% by calculating the proportion of positive cells ( Figure 2 B).

[0053] 2. Verification of the ability to kill target cells in vitro

[0054] 1. Specific Killing Experiment

[0055] Tumor target cells: MM.1S (BCMA+CD19-, multiple myeloma cells) and JeKo-1 (CD19+BCMA-, lymphoma cells).

[0056] CAR-T cells: 19CAR-38AL, 19CAR-38BL, and Mock (empty vector control).

[0057] Gradient effect-target ratio: 8:1, 4:1, 2:1, 1:1, 1:2.

[0058] Luciferase was used to label tumor target cells MM.1S and JeKo-1, and then the target cells were co-cultured with CAR-T cells under different effector-target ratios (E:T, i.e. effector cells: target cells): each group was set up in a 96-well plate in a gradient recursive manner, with 1×10 4 Target cells (MM.1S or JeKo-1) were added with the calculated number of CAR-T cells according to the E:T ratio. Mock and target cell groups (background control groups) were set up. Co-culture was continued for 4 hours at 37°C and 5% CO2. Luciferase substrate was used to treat cells in each well, and the luciferase signal intensity was measured. The target cell killing rate was calculated and the killing curve was plotted (see Figure 3 A).

[0059] Figure 3As shown in Figure A, both CAR-T cells demonstrated good cytotoxicity against MM.1S (BCMA+CD19-) and JeKo-1 (CD19+BCMA-) target cells, with cytotoxicity gradually weakening as the E:T ratio decreased. There was no significant difference in target cell cytotoxicity between 19CAR-38AL and 19CAR-38BL, but both were significantly superior to the mock control.

[0060] 2. Degranulation Detection Experiment

[0061] Given that the specific killing of tumors by CAR-T cells depends on cytotoxicity mediated by degranulation, flow cytometry was used to detect changes in the degranulation values of CD4+ T cells and CD8+ T cells during the killing process. The specific method is as follows:

[0062] Consistent with the specific killing experiment, target cells (MM.1S or JeKo-1) were co-cultured with two CAR-T cells at a ratio of E:T=10:1. BrefeldinA and Monensin were added (to prevent the granules produced by killing from being discharged from the cells, thereby detecting the degranulation marker CD107a). After the co-culture, the co-cultured cells were collected. Anti-CD107a antibody was used for surface labeling. Anti-CD4 and CD8 antibodies were used for staining at the same time to distinguish CD4+T and CD8+T cell subsets. The proportion of CD107a-positive cells in each group was detected by flow cytometry, and the degranulation ability of CD4+T and CD8+T cell subsets was analyzed separately. The changes in degranulation values of 19CAR-38AL, 19CAR-38BL and Mock groups were compared (see Figure 3 B).

[0063] Figure 3 Figure B shows that during the process of killing tumor target cells, the CD107a degranulation values of 19CAR-38AL and 19CAR-38BL were significantly higher than those of the Mock group. There was no significant difference in the degranulation ability of the two CAR-T cells, but both showed good degranulation behavior.

[0064] 3. CAR vector-antigen specific binding ability

[0065] To compare the ability of the two CAR lentiviral vectors to specifically bind to BCMA and CD19 proteins, the dual-targeting 19CAR-38AL, 19CAR-38BL, and single-target 19CAR lentiviruses were used to infect Jurkat cell lines, respectively, and the specific binding ability of the CAR vectors to the two proteins was detected. The specific method is as follows:

[0066] Jurkat cells were seeded in 24-well plates (2 × 10 5Cells / well) were added with different lentiviral vectors (MOI = 0.5-1): 19CAR-38AL, 19CAR-38BL, 1915N (19CAR single target control), Mock (empty vector control), and cells were collected 48 hours later. Three low-copy CAR cells were selected and co-incubated with CD19-Fc and BCMA-Fc proteins (4°C, incubation for 30 min), and then washed twice with PBS (1200 rpm, 5 min). Fluorescently labeled anti-Fc secondary antibody was added, incubated at 4°C in the dark for 30 min, washed twice with PBS, and resuspended in 200 μL PBS. The cells were then detected and analyzed by flow cytometry. The results are shown in the figure. Figure 4 A.

[0067] Figure 4 Results A showed that 19CAR-38AL and 19CAR-38BL had similar binding efficiencies for BCMA protein, but 19CAR-38AL bound to CD19-Fc protein better than 19CAR-38BL.

[0068] Concentration gradient binding assay: Three CAR cells (1×10 6 Cells / tube) were co-incubated with CD19-Fc protein at different ratios (1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187) at 4°C for 30 minutes, washed twice with PBS, and then washed twice with PBS. Fluorescently labeled anti-Fc secondary antibody was added and incubated in the dark at 4°C for 30 minutes. The cells were washed twice with PBS, resuspended, and then detected and analyzed by flow cytometry. The results are shown in the figure. Figure 4 B.

[0069] Figure 4 B results are similar to the above Figure 4 Consistent with A, at higher antigen concentrations, the two CARs had similar binding abilities. However, at lower antigen concentrations (e.g., 1:243), 19CAR-38AL was significantly more potent than 19CAR-38BL. Therefore, 19CAR-38AL was selected as a CD19 and BCMA dual-targeting CAR-T cell therapy to further evaluate its in vivo anti-tumor efficacy.

[0070] IV. Establishment of Multiple Myeloma Xenograft Tumor Model

[0071] In order to further explore the specific anti-tumor effect of dual-target CAR-T (19CAR-38AL) in vivo, the present invention established multiple myeloma and lymphoma xenograft models for testing.

[0072] 1. Establishment of a peritoneal model for the anti-tumor effect of targeting BMCA

[0073] Use 8-week-old male NSG immunodeficient mice, as Figure 5 As shown in A, each NSG mouse was intraperitoneally injected with 0.5M MM.1S (BCMA+) cells and then divided into groups (5-6 mice per group):

[0074] Experimental group: 19CAR-38AL CAR-T cells were infused, with an E:T ratio of 10:1 (i.e., 0.5M CAR-T cells were injected into each mouse).

[0075] Control group: Mock CAR-T cells were reinfused (same E:T ratio).

[0076] Tumor growth was monitored after injection and imaged regularly. Figure 5 B and 5C.

[0077] Imaging results showed that CAR-T cells significantly inhibited tumor growth and significantly prolonged the survival time of mice compared with the Mock group.

[0078] 2. Establishment of an intravenous model for the anti-tumor effect of targeting CD19

[0079] like Figure 5 As shown in E, each NSG mouse was injected with 0.01M JeKo-1 (CD19+) cells via the tail vein and then divided into groups (5 to 6 mice per group):

[0080] Experimental group: 19CAR-38AL CAR-T cells were infused with an E:T ratio of 10:1 (i.e., 0.1M CAR-T cells were injected into each mouse).

[0081] Control group: Mock CAR-T cells were reinfused (same E:T ratio).

[0082] Tumor growth was monitored after injection and imaged regularly. Figure 5 FH.

[0083] Imaging results showed that in this model, CAR-T cells significantly inhibited tumor growth compared with the Mock group ( Figure 5 F, Figure 5 G) and significantly prolonged the survival time of mice ( Figure 5 H).

[0084] The above animal experimental results show that BCMA and CD19 dual-targeting CAR-T can effectively fight tumors in multiple myeloma and lymphoma xenograft models and prolong the survival time of mice.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor targeting both CD19 and BCMA, characterized by: The chimeric antigen receptor includes a CD19-targeting antibody light chain, a linker, a BCMA-targeting humanized nanobody, and a CD19-targeting antibody heavy chain connected in series; The amino acid sequence of the light chain of the CD19-targeting antibody is shown in SEQ ID NO: 2, the amino acid sequence of the Linker is shown in SEQ ID NO: 3, the amino acid sequence of the BCMA-targeting nanobody is shown in SEQ ID NO: 4 or SEQ ID NO: 5, and the amino acid sequence of the heavy chain of the CD19-targeting antibody is shown in SEQ ID NO:

6.

2. A chimeric antigen receptor targeting both CD19 and BCMA according to claim 1, characterized in that: The chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane region CD8, and a 4-1BB-CD3ζ intracellular activation domain. The chimeric antigen receptor includes a signal peptide, a light chain of an antibody targeting CD19, a linker, a humanized nanobody targeting BCMA, a hinge region, a heavy chain of an antibody targeting CD19, a transmembrane region CD8, and a 4-1BB-CD3ζ intracellular activation domain connected in series; The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 7, and the amino acid sequence of the 4-1BB-CD3z intracellular activation domain is shown in SEQ ID NO:

8.

3. A chimeric antigen receptor targeting both CD19 and BCMA according to claim 2, characterized in that: The chimeric antigen receptor also includes cytokines IL7 and CCL19, which utilize self-cleavage P2A and T2A to insert into the 4-1BB-CD3ζ intracellular activation domain; The amino acid sequence of the P2A is shown in SEQ ID NO: 9, the amino acid sequence of the T2A site is shown in SEQ ID NO: 11, the amino acid sequence of the cytokine IL7 is shown in SEQ ID NO: 10, and the amino acid sequence of the cytokine CCL19 is shown in SEQ ID NO:

12.

4. A nucleic acid encoding the chimeric antigen receptor according to any one of claims 1 to 3.

5. A lentiviral vector, characterized in that: The lentiviral vector comprises the nucleic acid of claim 4. A host cell containing the lentiviral vector according to claim 5 .

7. Use of the chimeric antigen receptor according to any one of claims 1 to 3, the nucleic acid according to claim 4, the lentiviral vector according to claim 5, or the host cell according to claim 6 in the preparation of a drug for treating multiple myeloma and lymphoma in a mammal.

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