CART cells targeting bcma and uses thereof

Abstract

This invention relates to the field of biotechnology, specifically to a BCMA CAR-T and its preparation and application. The invention first provides a single-chain antibody targeting BCMA, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain variable region complementarity-determining region 1 (HCDR1), heavy chain variable region complementarity-determining region 2 (HCDR2), and heavy chain variable region complementarity-determining region 3 (HCDR3). The light chain includes a light chain variable region, specifically light chain variable region complementarity-determining region 1 (LCDR1), light chain variable region complementarity-determining region 2 (LCDR2), and light chain variable region complementarity-determining region 3 (LCDR3). This single-chain antibody can be used to prepare chimeric antigen receptors, chimeric antigen receptor T cells, and tumor therapeutic drugs targeting BCMA, and has significant clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a BCMACART cell and its preparation and application. Background Technology

[0002] Multiple myeloma (MM) is a clonal malignant proliferation of plasma cells. Its main characteristic is the infiltration of bone and soft tissue by malignantly proliferating plasma cells (myeloma cells) and the production of abnormal immunoglobulins (M proteins) and their polypeptide chains. Clinically, it mainly manifests as bone pain, pathological fractures, hypercalcemia, and hyperviscosity syndrome. MM accounts for approximately 1% of all cancers and about 10% of hematologic malignancies, making it the second most common hematologic malignancy. Current treatment options include chemotherapy, immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, and immunotherapy. However, it is currently not completely curable, and most patients still face the risk of relapse.

[0003] Chimeric antigen receptor T cell (CART) therapy is an emerging immunotherapy that specifically recognizes target antigens through extracellular ligand recognition regions (usually single-chain antibodies or single-chain receptors) and activates intracellular signaling regions, causing cellular changes in response to this signal. By modifying T cells with specific chimeric antigen receptors and increasing intracellular co-stimulatory signals, chimeric antigen receptor T lymphocytes (CARTs) are constructed. A key characteristic of CARTs is that their activation is independent of the TCR (tumor retrieval system) and relies solely on the recognition between the CAR and the antigen. This significantly reduces cellular immune escape caused by low-level expression of MHC molecules on the surface of tumor cells. CARTs can specifically recognize tumor-associated antigens, providing a new option for patients with relapsed or refractory cancers, and has achieved significant success, particularly in the treatment of hematologic malignancies.

[0004] B-cell maturation antigen (BCMA), also known as CD269 and TNF receptor superfamily 17 (TNFRSF17), contains B-cell activating factor (BAFF, TNFSF13B) and proliferation-inducing ligand (APRIL, TNFSF13B). BCMA is a member of the tumor necrosis factor receptor superfamily, normally expressed on the surface of plasma cells and mature B cells, but not on the surface of hematopoietic stem cells or non-hematopoietic cells. B-cell survival is maintained through proliferation-inducing ligand and cell activating factor, and its expression is significantly increased during abnormal clonal changes in plasma cells. Studies have also confirmed that the cytotoxic effect of BCMA-T cells is limited to expression in BCMA-containing myeloma cells and patient-derived MM cells; BCMA is not expressed on CD34. + BCMA is located on the surface of hematopoietic stem cells or most normal tissue cells, thus making it an ideal target for MM immunotherapy strategies.

[0005] However, CAR-T therapy still faces many challenges in cancer treatment, such as its insensitivity to tumor cells and low killing activity. Since the targeting antibody portion significantly influences the activity of immunotherapy, those skilled in the art provide a novel BCMA-targeting antibody and its preparation, BCMA-ART, to enhance the killing ability of CAR-T. Summary of the Invention

[0006] In view of the current state of the technology, the purpose of this invention is to provide a BCMACART and its preparation and application.

[0007] To achieve the above and other related objectives, the present invention adopts the following technical solution:

[0008] A first aspect of the present invention provides a single-chain antibody against BCMA, comprising a heavy chain variable region (V0). H ) and light chain variable region (V L The heavy chain and light chain variable regions each include three complementary determinant regions (CDRs), namely HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0009] In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0010] In some embodiments, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0011] In some embodiments, the V H The amino acid sequence is shown in SEQ ID NO.7, and the V L The amino acid sequence is shown in SEQ ID NO.8.

[0012] In some embodiments, the V H and V L They are linked by a linker peptide, the amino acid sequence of which is shown in SEQ ID NO.9.

[0013] In some embodiments, the amino acid sequence of the single-chain antibody against BCMA is shown in SEQ ID NO.10.

[0014] A second aspect of the invention provides the use of the aforementioned single-chain antibody against BCMA in the preparation of a chimeric antigen receptor targeting BCMA.

[0015] A third aspect of the present invention provides a chimeric antigen receptor (BCMACAR) targeting BCMA, the chimeric antigen receptor comprising, in sequence, a signal peptide, the aforementioned single-chain antibody against BCMA, a hinge region, a transmembrane domain, a co-stimulatory region, and a cytoplasmic signal transduction domain.

[0016] In some embodiments, the transmembrane domain comprises the transmembrane domains of the following molecules: CD8, CD28, CD45, CD4, CD80, CD86, PD-1, CD34, OX40, CD3ε, or variants thereof.

[0017] In some embodiments, the transmembrane domain is a CD8 transmembrane domain, the amino acid sequence of which is shown in SEQ ID NO. 11.

[0018] In some embodiments, the hinge region comprises the hinge region of the following molecules: IgG1, IgG4, CD8, CD28, CD34, CD137, CD3ε, or variants thereof.

[0019] In some embodiments, the hinge region is a CD8 hinge region, the amino acid sequence of which is shown in SEQ ID NO.12.

[0020] In some embodiments, the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ, whose amino acid sequence is shown in SEQ ID NO.13.

[0021] In some embodiments, the co-stimulatory signaling domain comprises the co-stimulatory signaling domains of the following molecules: CD19, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, ICAM, LFA-1, ICOS, CD2, CD4, CD5, CD7, CD226, LIGHIT, B7-H3, or variants thereof.

[0022] In some embodiments, the co-stimulation signaling domain includes a 4-1BB co-stimulation signaling domain, a CD28 co-stimulation signaling domain, or a combination of 4-1BB and CD28 co-stimulation signaling domains.

[0023] In some embodiments, the amino acid sequence of the 4-1BB co-stimulatory signaling domain is shown in SEQ ID NO.14.

[0024] In some embodiments, the signal peptide comprises signal peptides of the following molecules: CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD64, CD80, CD86, CD134, CD137, CD154, GITR, ICOS, or variants thereof.

[0025] In some embodiments, the signal peptide is a CD8 signal peptide, the amino acid sequence of which is shown in SEQ ID NO.15.

[0026] In some embodiments, the chimeric antigen receptor comprises, in sequence, a CD8 signal peptide, the aforementioned single-chain antibody against BCMA, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory region, and a CD3ζ cytoplasmic signal transduction domain.

[0027] In some embodiments, the amino acid sequence of the chimeric antigen receptor (BCMACAR) targeting BCMA is shown in SEQ ID NO.16.

[0028] A fourth aspect of the present invention provides a polynucleotide encoding the chimeric antigen receptor targeting BCMA.

[0029] A fifth aspect of the present invention provides an expression vector containing the polynucleotide.

[0030] In some embodiments, the vector comprises a DNA vector, an RNA vector, a plasmid, a transposon vector, or a viral vector. In some embodiments, the viral vector comprises a lentiviral vector, an adenovirus vector, a herpesvirus vector, or a retroviral vector. In some embodiments, the viral vector is a lentiviral vector.

[0031] The sixth aspect of the present invention provides an engineered cell that contains or expresses the chimeric antigen receptor described in the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, or the vector described in the fifth aspect of the present invention.

[0032] In some embodiments, the cells comprise immune cells. In some embodiments, the immune cells comprise T cells, B cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, myeloid cells, or any combination thereof. In some embodiments, the immune cells are T cells, more preferably primary T cells or Jurkat cells.

[0033] In some embodiments, the engineered cells are chimeric antigen receptor T cells (BCMACART) targeting BCMA.

[0034] The seventh aspect of the present invention provides a method for preparing the cells described in the sixth aspect of the present invention, the method comprising introducing a nucleic acid molecule described in the fourth aspect of the present invention or a vector described in the fifth aspect of the present invention into the cells.

[0035] The eighth aspect of the present invention provides the use of the single-chain antibody targeting BCMA as described above, or the nucleic acid molecule as described above, or the chimeric antigen receptor targeting BCMA as described above, or the vector as described above, or the engineered cells as described above (such as chimeric antigen receptor T cells targeting BCMA (BCMA CART)) in the preparation of antitumor drugs.

[0036] In some embodiments, the tumor is a hematologic malignancy. In some embodiments, the tumor is selected as multiple myeloma (MM) or plasma cell leukemia. Attached Figure Description

[0037] Figure 1A-1C The results show that BCMACART cells are present in different tumor cell types (MM.1S). Figure 1A ), RPMI-8266 ( Figure 1B K562 Figure 1C Cytotoxicity in ))

[0038] Figure 2 The results show the activity of BCMACART cells in inducing interferon-γ release in different tumor cells.

[0039] Figure 3 The results show that BCMACART cells prolong the survival of MM.1S model mice. Detailed Implementation

[0040] To facilitate understanding of the invention, reference will be made to certain embodiments below, and specific language will be used to describe the invention. However, it should be understood that these specific embodiments are not intended to limit the scope of the invention. Any changes and further modifications to the described embodiments, as well as any further applications of the invention, are those commonly conceived by those skilled in the art.

[0041] Example 1. Preparation and screening of anti-BCMA single-chain antibodies

[0042] The cDNA encoding the extracellular domain of human BCMA was constructed into a prokaryotic or eukaryotic expression vector via subcloning. The BCMA antigen was obtained after purification. The BCMA antigen was mixed with an equal volume of adjuvant and injected intraperitoneally into BALB / c mice (200 μL per mouse). After several rounds of immunization, the spleens of the immunized mice were harvested, RNA was extracted, and cDNA was obtained through reverse transcription. V... H and V K Specific primer amplification V H and V K The fragment, after gel recovery and purification, was ligated with V... H and V K The scFv antibody was constructed and cloned into the phage display plasmid pDAN5. Subsequently, the plasmid was electroporated into *E. coli* TG1, and *E. coli* TG1 was infected with phage to obtain the scFv antibody phage display library. Then, BCMA antigen was coated onto an ELISA plate. The constructed phage display library was added to the ELISA plate at a volume of 4 × 10⁻⁶. 11 PFU / well, incubated at 37°C for 2 hours. Subsequently, BCMA antigen-binding positive phages were screened using anti-M13 antibody (SinoBiological, 11973-MM05T-H), and the positive phages were eluted with elution buffer. Positive phages were enriched after multiple rounds of screening. Further ELISA detection yielded the anti-BCMA single-chain antibody G18. The positive clones were sequenced to obtain the amino acid sequence corresponding to the G18 antibody.

[0043] V of the obtained mouse antibody G18 H / V L Based on the typical CDR structure, the heavy and light chain variable region sequences were compared with sequences in the antibody germline database to obtain a human germline template with high homology. The CDR region of the mouse antibody was transplanted onto the selected human germline template to generate a humanized variable region. This humanized antibody was named hG18. The heavy chain variable region sequence of hG18 is shown in SEQ ID NO: 7, and the light chain variable region sequence is shown in SEQ ID NO: 8.

[0044] Example 2. Construction of BCMACAR lentiviral expression vector

[0045] The CAR structure used in this invention comprises, in sequence, a CD8 signal peptide, a single-chain antibody hG18 targeting BCMA, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory region, and a CD3ζ cytoplasmic signal transduction domain. The complete BCMACAR gene sequence was synthesized by Heyuan Biotechnology (Shanghai) Co., Ltd. in the above order. During synthesis, recognition sequences of the restriction endonucleases BamHI and EcoRI were attached to the first and last ends of the complete CAR gene fragment, respectively. Then, the complete CAR gene fragment was inserted between the BamHI and EcoRI restriction sites of the pLenti lentiviral expression plasmid produced by Heyuan Biotechnology (Shanghai) Co., Ltd., to construct the BCMACAR lentiviral expression plasmid.

[0046] Example 3. BCMACAR Lentiviral Packaging

[0047] Virus packaging was performed using 293T cells. First, 293T cells were passaged and cultured until the cell density reached approximately 90%, then the medium was changed. 3 μg of pLenti lentiviral expression plasmid (BCMA CAR lentiviral expression plasmid prepared in Example 2 or control empty pLenti lentiviral expression plasmid) and 3 μg of pLenti lentiviral packaging plasmid were added to a centrifuge tube, followed by 500 μL of serum-free high-glucose DMEM medium, and mixed well. 36 μL of PolyJet transfection reagent was added to a new centrifuge tube, followed by 500 μL of serum-free high-glucose DMEM medium, and mixed well. The transfection reagent mixture was added to a 1.5 mL centrifuge tube containing the plasmid solution, inverted to mix, and incubated at room temperature for 10 minutes to allow the PolyJet-DNA transfection complex to form. The PolyJet-DNA transfection complex was slowly and evenly added dropwise to a cell culture dish, mixed thoroughly, and then incubated at 37°C with 5% CO2. The medium was changed the next day, and the cells were cultured further. 72 hours after transfection, the cell culture supernatant was removed using a sterile syringe. The cell culture supernatant was filtered using a 0.45μm needle filter, and the filtered liquid was the lentivirus solution, which was aliquoted and stored at -86℃.

[0048] Example 4. Preparation of BCMACART cells

[0049] 20 mL of peripheral blood was drawn from a healthy volunteer using an anticoagulant tube. 20 mL of PBS was added and mixed thoroughly to form a cell suspension. Separately, 15 mL of human lymphocyte separation medium (purchased from Dakowei Biotechnology Co., Ltd.) was added to a 50 mL centrifuge tube. 30 mL of the cell suspension was then slowly added to the centrifuge tube, adhering to the wall of the tube. After centrifugation at 2000 rpm for 20 min, distinct layers were observed, from top to bottom: plasma layer, mononuclear cell layer, separation medium layer, and erythrocyte layer. The top plasma layer was removed. The mononuclear cell layer was carefully aspirated into a 50 mL centrifuge tube, and 3-5 times the volume of PBS was added and gently mixed by pipetting. The tube was centrifuged at 1200 rpm for 5 min and washed. This process was repeated twice. The human peripheral blood mononuclear cells were resuspended in BD buffer (0.5% BSA, 2 mM EDTAPH 7.2 PBS solution), and the cell suspension density was adjusted to 4 × 10⁻⁶ cells / mL. 8 / mL. Add 50μL of cell suspension and 50μL of CD3 magnetic beads (purchased from BD Biosciences, USA) to each sterile flow cytometry tube, mix well, and incubate for 1 h. After incubation, add 1mL of ice-cold BD buffer to each tube to stop the reaction, place the flow cytometry tube in a BD magnetic rack, and separate the cells for 8-10 min. After removing the supernatant, add 1mL of ice-cold BD buffer to resuspend the cells adsorbed on the tube wall, and place the flow cytometry tube in the magnetic rack again for separation for 5 min. After discarding the supernatant, resuspend the cells and wash them for later use. Add 5μg / mL of CD3 mAb (OKT3, Miltenyi, 1mL / well) to a 24-well plate and coat at 37℃ for 2 h. Discard the antibody solution, wash three times with PBS, and add the sorted CD3-positive T lymphocytes to the 24-well plate. Add CD28 mAb (purchased from BD Biosciences, USA) to a final concentration of 0.5 μg / ml, rhIL-2 (purchased from Peprotech, USA) to 10 ng / ml, and rhIL-7 (purchased from Peprotech, USA). Incubate at 37°C and 5% CO2 for 48 hours to activate the cells. Centrifuge at 24°C and 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells in 1640 medium (containing rhIL-2 and rhIL-7) with 2% FBS and adjust the cell density.

[0050] 2×10 6 / ml, and seeded into 24-well plates (100μL / well). Take 100μL of concentrated virus solution and infect overnight. On the second day, add 1mL of fresh culture medium. Six days after infection, the positive rate of BCMACAR expression on the surface of T cells was determined by FACS method. The results showed that the BCMACAR positive rate was 82.1%. The BCMACART cells prepared in this invention were named BCMA-hG18CART.

[0051] Example 5. In vitro antitumor activity of BCMACART cells

[0052] 5.1 LDH kit for detecting CAR-T cell cytotoxicity

[0053] Effector cells (BCMA-hG18 CART, C-CAR088 (positive control, sequence information see CN116997345A), NT cells (untransfected T cells, negative control)) cultured on day 7 in Example 4 were co-cultured with target cells (BCMA-positive multiple myeloma cell line MM.1S (BFN60808555, purchased from ATCC-Shanghai Cell Center), RPMI-8266 (BFN60806107, purchased from ATCC-Shanghai Cell Center), BCMA-negative K562 tumor cells (GDC037, purchased from China Center for Type Culture Collection)) at ratios of 5:1 and 1:1 for 16 h (with target cells at 3 × 10⁶ cells per cell line). 4 / well, adjust the number of BCMACART cells added according to the effector-to-target ratio (E:T). The killing effect of CAR-T cells was determined using an LDH detection kit (Shanghai Beyotime, C0016).

[0054] According to the instruction manual, separate wells were set up for background culture, spontaneous release of effector cells, spontaneous release of target cells, and maximum release of target cells. One hour before collecting the supernatant, 15 μL of LDH lysis buffer was added to the maximum release well of target cells, mixed well, and incubated for 1 hour. The cell culture plate was then centrifuged at 12,000 rpm for 3 minutes. 120 μL of supernatant from each well was collected, and 60 μL of LDH working solution was added (LDH working solution was prepared by using 20 μL enzyme solution, 20 μL lactate solution, 20 μL INT solution, and 18 μL INT dilution buffer per reaction). The plate was then incubated on a horizontal shaker at room temperature in the dark for 30 minutes. The OD490 value of each well was measured.

[0055] After subtracting the absorbance of the blank control group from the absorbance of each well, the cytotoxicity was calculated according to the following formula: (OD value of each experimental well - OD value of the corresponding target cell spontaneous release group - OD value of the corresponding effector cell spontaneous release group) / (OD value of the corresponding target cell maximum release group - OD value of the corresponding effector cell spontaneous release group) × 100%.

[0056] The results are as follows Figure 1A-1C As shown, in vitro, BCMA-hG18 CART and C-CAR088 have an effect on BCMA-positive cells (MM.1S). Figure 1A ), RPMI-8266 ( Figure 1B It exhibits significant cytotoxicity, and BCMA-hG18 CART is superior to C-CAR088 in cytotoxicity, while it is less cytotoxic to BCMA-negative cells (K562). Figure 1C BCMA-hG18 CART, C-CAR088 and NT cells did not exhibit cytotoxicity.

[0057] 5.2 ELISA method for detecting cytokine release

[0058] The effector cells (BCMA-hG18 CART, C-CAR088, NT cells) cultured on day 7 in Example 4, 3 × 10⁻⁶ 4 / well) were respectively used with target cells (MM.1S, RPMI-8266, K562, 3×10⁶) 4 After mixing with the culture medium (in each well), the cells were co-cultured at 37°C and 5% CO2 for 16 h. The cell culture plates were then centrifuged at 24°C and 1000 rpm for 5 min. 50 μL of the supernatant was collected, and the amount of interferon-γ released was detected using an interferon-γ detection kit (BDBiosciences, 560111).

[0059] Dilute the standard according to the instructions. Prepare a 100 μL reaction system by adding 1 μL of interferon-γ capture microspheres, 1 μL of PE detection reagent, and 98 μL of PBS to each reaction system. Add 50 μL of standard or test sample to each reaction system and incubate at room temperature for 2 hours. Add 1 mL of PBS to each reaction system, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend in 150 μL of PBS in each tube. Collect 3000 effective microspheres by flow cytometry and analyze using FCAP Array V3 software.

[0060] The results are as follows Figure 2 As shown, both BCMA-hG18 CART and C-CAR088 can induce BCMA-positive cells (MM.1S, RPMI-8266) to release higher levels of interferon-γ; and the activity of BCMA-hG18 CART in inducing interferon-γ release is superior to that of C-CAR088. However, in BCMA-negative cells (K562), there is no difference in interferon-γ release between BCMA-hG18 CART and C-CAR088 and NT cells. That is, in BCMA-negative cells, BCMA-hG18 CART and C-CAR088 have no activity in inducing interferon-γ.

[0061] Example 6. BCMACART in vivo toxicity assay

[0062] Twelve-week-old B-NDG mice were randomly divided into three groups of five each. Each mouse was injected with 3 × 10⁻⁶ mg / L via the tail vein. 6 One NT cell, BCMA-hG18 CART or C-CAR088 (the date of T cell / CART cell injection is recorded as D0), is injected via tail vein at a dose of 1×10⁻⁶ on day D1. 6 MM.1s cells. The survival status of each mouse was observed from day 0 to day 45, and the time of death was recorded. Survival curves were plotted using Graph Pad.

[0063] The results are as follows Figure 3 As shown, compared with the control group mice injected with NT cells, injection of BCMA-hG18CART or C-CAR088 significantly prolonged the survival of MM.1S mice. Furthermore, BCMA-hG18CART was more effective than C-CAR088 in prolonging mouse survival.

Claims

1. A chimeric antigen receptor T cell, characterized in that: It expresses a chimeric antigen receptor that targets BCMA, the amino acid sequence of which is shown in SEQ ID NO.

16.

2. A drug for treating multiple myeloma, characterized in that: It contains the chimeric antigen receptor T cells as described in claim 1.

Images