Chimeric antigen receptor targeting CD19 and BCMA double targets and application of chimeric antigen receptor

Abstract

The invention relates to the technical field of biological medicine, and provides a chimeric antigen receptor targeting CD19 and BCMA double targets and application thereof. The chimeric antigen receptor comprises a targeted CD19 antibody light chain, a Linker, a targeted BCMA humanized nano antibody and a targeted CD19 antibody heavy chain which are sequentially connected in series. According to the invention, the CAR structure of the dual-targeting CD19 / BCMA is optimized, the humanized nano antibody of the targeting BCMA is arranged between an antibody light chain of the targeting CD19 and an antibody heavy chain of the targeting CD19, and respective expression of IL7 and CCL9 in T cells is realized by utilizing a self-cleavage P / T 2A site. The chimeric antigen receptor with the structure shows relatively strong tumor killing ability and can be used for preparing a medicine for treating hematologic 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 hematological malignancy, mostly occurring in the elderly population. MM is a plasma cell disease characterized by terminal differentiation of plasma cells, plasma cell infiltration of the bone marrow, and the presence of monoclonal immunoglobulins (or immunoglobulin fragments) in serum and / or urine, usually accompanied by osteolytic bone disease, anemia, and renal failure. In recent years, thanks to the continuous emergence of new drugs and the improvement of detection methods, the diagnosis and treatment of MM have been continuously improved and perfected, and the overall survival (OS) of MM patients has been significantly prolonged, but it is still regarded as an incurable disease, and almost all patients will experience a relapsed and refractory stage. The effective and tolerable treatment options for patients with relapsed and refractory (R / R) MM are still unsatisfactory, with the median overall survival (OS) of patients with triple-refractory disease being 9.3 months and that of patients with penta-refractory disease being 5.6 months. Therefore, finding an effective treatment option for patients with relapsed and refractory (R / R) MM is a scientific problem that needs to be solved urgently.

[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). At present, 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 a total 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 obtaining a response. After CAR-T treatment, BCMA expression downregulation or loss was observed in 4%-33% of progressive patients.

[0004] To solve this problem, researchers began to try 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 of T cell amplification and tumor killing ability in the CAR structure to enhance T cell amplification 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 nanoantibody targeting BCMA, and a CD19-targeting antibody heavy chain connected in series in sequence.

[0007] On the basis of the above technical scheme, preferably, the amino acid sequence of the light chain of the antibody targeting CD19 is shown as SEQ ID NO: 2, the amino acid sequence of Linker is shown as SEQ ID NO: 3, the amino acid sequence of the nanoantibody targeting BCMA is shown as SEQ ID NO: 4 or SEQ ID NO: 5, and the amino acid sequence of the heavy chain of the antibody targeting CD19 is shown as 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 scheme, 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] On the basis of the above technical scheme, 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 also includes cytokines IL7 and CCL19, and the cytokines IL7 and CCL19 utilize self-cleavage P2A and T2A sites to insert into the 4-1BB-CD3ζ intracellular activation domain.

[0012] Based on the above technical scheme, 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 cytokine IL7 is shown as SEQ ID NO: 11, and the amino acid sequence of cytokine CCL19 is shown as SEQ ID NO: 12.

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

[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 above-mentioned 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 blood tumors.

[0017] The chimeric antigen receptor targeting CD19 and BCMA dual targets 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 the dual-targeted CD19 / BCMA CAR, places the humanized nanoantibody targeting BCMA between the light chain of the antibody targeting CD19 and the heavy chain of the antibody targeting CD19, and uses the self-cleaving 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 blood tumor therapeutic drug. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0020] Figure 1 This is a structural diagram of the chimeric antigen receptor targeting CD19 and BCMA dual targets of the present invention; 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 sequence.

[0021] Figure 2 Figure 1 is a diagram of the lentiviral transduction efficiency carrying 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 This is a comparison of the in vitro killing ability of the two dual-target CAR-T cells. Figure A is a specific killing experiment of the two dual-target CAR-T cells, and Figure B is a graph showing the changes in the degranulation values ​​of CD4+T and CD8+T cells 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 anti-multiple myeloma experiment of dual-target CAR-T. 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 effect 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 described embodiments 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 creative work 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 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 nanoantibody targeting BCMA 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 Listing

[0031]

[0032] 1. Construction of Lentiviral Vector

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

[0034] like Figure 1 As shown, this embodiment constructs two chimeric antigen receptors that simultaneously target CD19 and BCMA: 19CAR-38AL and 19CAR-38BL. The amino acid sequence of the nanobody targeting BCMA in 19CAR-38AL is shown in SEQ ID NO: 4, and the amino acid sequence of the nanobody targeting BCMA 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 molecules (4-1BB),

[0042] Signaling domain (CD3ζ),

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

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

[0045] 2. Transfection of Lentivirus Packaging Cells

[0046] HEK293T cells were inoculated in a T175 culture flask (cultured to 70%-80% confluence). After mixing the CAR plasmid with the auxiliary plasmid, transfection was performed using Lipofectamine 3000. Fresh culture medium was replaced after 6-8 hours. 48-72 hours after transfection, the supernatant was collected. The supernatant was filtered with a 0.45μm filter membrane 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 (Ficoll gradient centrifugation). PBMCs were stimulated with anti-CD3 / CD28 antibody magnetic beads to activate T cells and guide their expansion. Activated T cells were cultured in RPMI 1640 medium containing 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 cells / mL). Incubate the lentiviral particles (MOI = 5-10) with T cells for 48 hours. Polybrene (such as Polybrene, 8μg / mL) can be added to improve the transduction efficiency. After transduction, replace the culture medium and continue to culture and expand T cells.

[0050] 4. Transduction efficiency detection

[0051] Flow cytometry antibody labeling: Collect transduced T cells and use antibodies against specific markers on the dual-target CAR for fluorescent labeling (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] Tumor target cells MM.1S and JeKo-1 were labeled with luciferase, and then 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, and the Mock group and the single target cell group (background control group) were set up. Co-culture for 4 hours at 37°C and 5% CO2. The cells in each well were treated with luciferase substrate, the luciferase signal intensity was measured, the target cell killing rate was calculated, and the killing curve was drawn (see Figure 3 A).

[0059] Figure 3A shows that both CAR-T cells showed good killing ability against MM.1S (BCMA+CD19-) and JeKo-1 (CD19+BCMA-) target cells, and the killing ability gradually weakened as the E:T ratio decreased. In terms of target cell killing ability, there was no significant difference between 19CAR-38AL and 19CAR-38BL, but both were significantly better than the Mock control.

[0060] 2. Degranulation Detection Experiment

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

[0062] Consistent with the specific killing experiment, the target cells (MM.1S or JeKo-1) were co-cultured with the 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 respectively. The changes in degranulation values ​​of 19CAR-38AL, 19CAR-38BL and Mock groups were compared (see Figure 3 B).

[0063] Figure 3 B shows that in 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 and antigen-specific binding ability

[0065] In order 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), and different lentiviral vectors (MOI = 0.5-1) were added respectively: 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, and then detected and analyzed by flow cytometry. The results are shown in Figure 4 A.

[0067] Figure 4 A The results showed that 19CAR-38AL and 19CAR-38BL had similar efficiency in binding to 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 The cells were co-incubated with CD19-Fc proteins at different concentrations (1:9, 1:27, 1:81, 1:243, 1:729, 1:2187) at 4°C for 30 minutes, washed twice with PBS, and then added with fluorescently labeled anti-Fc secondary antibody. The cells were incubated at 4°C in the dark for 30 minutes, washed twice with PBS, and resuspended. The cells were 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 A. Under conditions of higher antigen concentration, the binding abilities of the two CARs are similar, but under conditions of low antigen concentration (such as 1:243), 19CAR-38AL is significantly stronger than 19CAR-38BL. Therefore, 19CAR-38AL was selected as a CAR-T cell therapy with dual targeting of CD19 and BCMA to further evaluate its anti-tumor effect in vivo.

[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 detection.

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

[0073] Use 8-week-old male NSG immunodeficient mice. 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 to 6 mice per group):

[0074] Experimental group: 19CAR-38AL CAR-T cells were transfused, 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 intravenous model for anti-tumor effect 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 transfused, 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 protection scope of the present invention.

Claims

1. A chimeric antigen receptor targeting CD19 and BCMA, characterized in that: The chimeric antigen receptor includes a CD19-targeting antibody light chain, a linker, a humanized nanobody targeting BCMA, 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 CD19 and BCMA dual targets as claimed in claim 1, characterized in that: The chimeric antigen receptor also includes 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 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; 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 CD19 and BCMA dual targets as claimed in claim 2, characterized in that: The chimeric antigen receptor also includes cytokines IL7 and CCL19, and the cytokines IL7 and CCL19 utilize self-cleavage P2A and T2A sites to insert into the 4-1BB-CD3ζ intracellular activation domain; The amino acid sequence of the P / T2A site is shown in SEQ ID NO: 9, the amino acid sequence of the T2A site is shown in SEQ ID NO: 10, the amino acid sequence of cytokine IL7 is shown in SEQ ID NO: 11, and the amino acid sequence of cytokine CCL19 is shown in SEQ ID NO:

12.

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

5. A lentiviral vector, characterized in that: The lentiviral vector comprises the nucleic acid of claim 4.

6. 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 mammalian blood tumors.

Images