CART cell targeting BCMA and application thereof

Abstract

The invention relates to the technical field of biology, in particular to BCMA CART as well as preparation and application thereof. The invention firstly provides a single-chain antibody aiming at BCMA, the single-chain antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2) and a heavy chain variable region complementarity determining region 3 (HCDR3); the light chain comprises a light chain variable region, and the light chain comprises a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2) and a light chain variable region complementarity determining region 3 (LCDR3). The single-chain antibody can be used for preparing BCMA-targeted chimeric antigen receptors, chimeric antigen receptor T cells and tumor treatment drugs, and has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a BCMA CART cell and a preparation and application thereof. Background Art

[0002] Multiple myeloma (MM) is a disease characterized by the malignant proliferation of clonal plasma cells. The main feature is that malignantly proliferating plasma cells (i.e., myeloma cells) infiltrate bones and soft tissues and produce abnormal immunoglobulins (M proteins) and their polypeptide chains. The main clinical manifestations are bone pain, pathological fractures, hypercalcemia, hyperviscosity syndrome and other abnormalities. MM accounts for about 1% of all tumors and about 10% of hematological tumors, making it the second most common hematological malignancy. The main treatments currently include chemotherapy, immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, and immunotherapy. However, it still cannot be completely cured, and most patients still face the risk of recurrence.

[0003] Chimeric antigen receptor T cell (CART) therapy is an emerging immunotherapy that specifically recognizes the target antigen through the extracellular ligand recognition region (generally single-chain antibody or single-chain receptor), and activates the intracellular signal region, causing the cell to undergo directional changes due to the signal. T cells are modified with specific chimeric antigen receptors, and co-stimulatory signals are added intracellularly to construct chimeric antigen receptor T lymphocytes (CART). Its characteristic is that the activation of CART cells does not depend on TCR, but only on the recognition between CAR and antigen, which greatly reduces the cellular immune escape caused by the low level of MHC molecules expressed on the surface of tumor cells. Chimeric antigen receptor T cells can specifically recognize tumor-associated antigens, providing a new option for some patients with relapsed and refractory tumors, especially in the treatment of hematological malignancies, and have achieved remarkable success.

[0004] B-cell maturation antigen (BCMA) is also known as CD269 and TNF receptor superfamily 17 (TNFRSF17). BCMA ligands include B cell activating factor (BAFF, TNFSF13B) and proliferation-inducing ligand (APRIL, TNFSF13). BCMA is a member of the tumor necrosis factor receptor superfamily that is normally expressed on the surface of plasma cells and mature B cells. It is not expressed on the surface of hematopoietic stem cells and non-hematopoietic cells. The maintenance of B cell survival is through proliferation-inducing ligands and cell activation factors, and the expression of this molecule can be significantly increased when plasma cells undergo abnormal clonal changes. At the same time, studies have confirmed that the killing effect of BCMA CAR-T cells is limited to the expression of BCMA in myeloma cells and patient-derived MM cells, and BCMA is not expressed on CD34. + BCMA is expressed on the surface of hematopoietic stem cells or most normal tissue cells, so it has become an ideal target for MM immunotherapy strategies.

[0005] However, CART still faces many problems in tumor treatment, such as CART is insensitive to tumor cells and has low killing activity. Since the targeted antibody part has an important influence on the activity of immunotherapy, a person skilled in the art would provide a new BCMA targeting antibody and its preparation BCMA CART to enhance the killing ability of CART. Summary of the invention

[0006] In view of the prior art, the object of the present invention is to provide a BCMACART and its preparation and application.

[0007] In order to achieve the above-mentioned purpose and other related purposes, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a single-chain antibody against BCMA is provided, comprising a heavy chain variable region (V H ) and light chain variable region (V L ), wherein the heavy chain\light chain variable region respectively includes three complementarity determining 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 as 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 as 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 of is shown in SEQ ID NO.7, wherein 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 connected by a connecting peptide, and the amino acid sequence of the connecting peptide 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] The second aspect of the present invention provides use of the aforementioned single-chain antibody against BCMA in the preparation of a chimeric antigen receptor targeting BCMA.

[0015] In a third aspect of the present invention, a chimeric antigen receptor targeting BCMA (BCMACAR) is provided, wherein the chimeric antigen receptor comprises a signal peptide, the aforementioned single-chain antibody against BCMA, a hinge region, a transmembrane domain, a co-stimulatory region and a cytoplasmic signaling domain connected in sequence.

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

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

[0018] In some embodiments, the hinge region comprises the hinge region of IgG1, IgG4, CD8, CD28, CD34, CD137, CD3ε, or a variant thereof.

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

[0020] In some embodiments, the intracellular signaling domain is the intracellular signaling 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 domain 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 a variant thereof.

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

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

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

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

[0026] In some embodiments, the chimeric antigen receptor comprises 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 signaling domain, which are sequentially connected.

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

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

[0029] The 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 adenoviral vector, a herpes virus 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, wherein the cell comprises 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 cell comprises an immune cell. In some embodiments, the immune cell comprises a T cell, a B cell, a NK cell, a NKT cell, a monocyte, a macrophage, a dendritic cell, a myeloid cell, or any combination thereof. In some embodiments, the immune cell is a T cell, more preferably a primary T cell or a Jurkat cell.

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

[0034] The seventh aspect of the present invention provides a method for preparing the cell described in the sixth aspect of the present invention, the method comprising introducing 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 into the cell.

[0035] In an eighth aspect, 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 anti-tumor drugs.

[0036] In some embodiments, the tumor is a hematological tumor. In some embodiments, the tumor is selected from multiple myeloma (MM) or plasma cell leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figures 1A-1C The results shown are that BCMA CART cells were expressed in different tumor cells (MM.1S ( Figure 1A )、RPMI-8266( Figure 1B )、K562( Figure 1C )) in cytotoxicity.

[0038] Figure 2 The results shown are the activity of BCMA CART cells in inducing interferon-γ release in different tumor cells.

[0039] Figure 3 The results shown are that in MM.1S model mice, BCMA CART cells prolonged the survival of mice. DETAILED DESCRIPTION

[0040] In order to promote understanding of the present invention, some embodiments will be referred to below, and specific language will be used to describe the present invention. However, it should be understood that these specific embodiments are not intended to limit the scope of the present invention. Any changes and further modifications in the described embodiments, as well as any further applications of the present invention, are generally expected 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 by subcloning. The BCMA antigen was obtained after purification. The BCMA antigen was mixed with an equal volume of adjuvant and injected intraperitoneally to immunize BALB / C mice, 200 μL per mouse. After several rounds of immunization, the spleen of the immunized mice was taken, RNA was extracted, and cDNA was obtained by reverse transcription. V H and V K Specific primers amplify V H and V K The fragment was purified by gel recovery and ligated to V H and V K , construct scFv and clone it into the phage display plasmid pDAN5. Then, the plasmid was electroporated into E. coli TG1, and the phage was used to infect E. coli TG1 to obtain the scFv antibody phage display library. Then, the BCMA antigen was coated on the ELISA plate. The constructed phage display library was added to the ELISA plate at an input of 4×10 11 pfu / well, incubated at 37°C for 2 hours. After that, the positive phages binding to BCMA antigen were screened using anti-M13 antibody (SinoBiological, 11973-MM05T-H), and the positive phages were eluted with the eluent. After multiple rounds of screening, the positive phages were enriched. Through further ELISA detection, the anti-BCMA single-chain antibody G18 was obtained. The positive clones were sequenced to obtain the amino acid sequence corresponding to the G18 antibody.

[0043] To obtain the mouse antibody G18 V H / V L Based on the typical structure of CDR, the heavy and light chain variable region sequences were compared with the sequences in the antibody germline database to obtain a human germline template with high homology. The CDR region of the mouse antibody was transplanted to the selected human germline template to produce a humanized variable region. The 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 the present invention includes 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 signaling domain connected in sequence. The complete BCMACAR gene sequence is synthesized by Heyuan Biotechnology (Shanghai) Co., Ltd. in the above order. During synthesis, the head and tail ends of the CAR complete gene fragment are respectively connected to the recognition sequence fragments of the restriction endonucleases BamHI and EcoRI. Then, the CAR complete gene fragment is inserted between the BamHI and EcoRI restriction sites of the pLenti lentiviral expression plasmid produced by the company to construct the BCMACAR lentiviral expression plasmid.

[0046] Example 3. BCMACAR lentiviral packaging

[0047] 293T cells were used for virus packaging. First, 293T cells were passaged and cultured until the cell density reached about 90%, and the solution was changed for standby use. Take 3μg pLenti lentiviral expression plasmid (Example 2 Preparation of BCMA CAR lentiviral expression plasmid or control empty pLenti lentiviral expression plasmid) and pLenti lentiviral packaging plasmid (3μg each), add to a centrifuge tube, add 500μL serum-free high-glucose DMEM medium, and mix. Take 36μLPolyJet transfection reagent and add it to a new centrifuge tube, add 500μL serum-free high-glucose DMEM medium, and mix. Add the transfection reagent mixture to a 1.5mL centrifuge tube containing the plasmid solution, invert and mix, and let stand at room temperature for 10 minutes to form a PolyJet-DNA transfection complex. The PolyJet-DNA transfection complex was added dropwise and evenly to the cell culture dish, mixed evenly, and cultured at 37°C and 5% CO2. Continue culturing after changing the solution the next day. 72 hours after transfection, use a sterile syringe to remove the cell culture supernatant. Use a 0.45 μm syringe filter to filter the cell culture supernatant. The filtered liquid is the lentiviral liquid, which is stored in a -86°C refrigerator after aliquoting.

[0048] Example 4. BCMA CART cell preparation

[0049] Use an anticoagulant tube to extract 20mL of peripheral blood from healthy volunteers, add 20mL of PBS and mix to form a cell suspension. Take another 50mL centrifuge tube and add 15mL of human lymphocyte separation solution (purchased from Dakoway Bioengineering Co., Ltd.), take 30mL of the above cell suspension and slowly add it to the centrifuge tube. After centrifugation at 2000rpm for 20min, obvious stratification can be seen, from top to bottom, there are plasma layer, mononuclear cell layer, separation liquid layer, and red blood cell layer. Remove the top plasma layer, carefully pipette the mononuclear cell layer liquid into a 50mL centrifuge tube, add 3-5 times the volume of PBS and gently blow to mix, centrifuge at 1200rpm for 5min to wash, and repeat twice. Resuspend the above human peripheral blood mononuclear cells with BDbuffer (0.5% BSA, 2mM EDTAPH7.2 PBS solution) and adjust the cell suspension density to 4×10 8 / mL. Add 50μL of cell suspension and 50μl of CD3 magnetic beads (purchased from BD, USA) to each sterile flow tube, mix well and incubate for 1h. After incubation, add 1mL of ice BD buffer to each tube to terminate the reaction, place the flow tube in a BD magnetic stand, and separate the cells for 8-10min. After removing the supernatant, add 1mL of ice BD buffer to resuspend the cells adsorbed on the tube wall, and place the flow tube in the magnetic stand again to separate for 5min. After discarding the supernatant, resuspend the cells and wash them for later use. Add 5μg / mL of CD3mAb (OKT3, Miltenyi Biotec, 1mL / well) to the 24-well plate and coat at 37℃ for 2h. Aspirate the antibody solution, wash 3 times with PBS, add the above-sorted CD3-positive T lymphocytes to the above-mentioned 24-well plate, and add CD28mAb (purchased from BD, USA) at a final concentration of 0.5μg / ml, rhIL-2 (purchased from Peprotech, USA) and rhIL-7 (purchased from Peprotech, USA) at 10ng / ml, and place in a 37°C, 5% CO2 cell culture incubator for activation for 48 hours. Centrifuge at 24°C, 1000rpm for 5min, and discard the supernatant. Add 2% FBS 1640 medium (containing rhIL-2 and rhIL-7) to resuspend the cells and adjust the cell density to

[0050] 2×10 6 / ml, and inoculated in 24-well plates (100 μL / well). Take 100 μL concentrated virus solution and infect overnight. The next day, add 1 mL of fresh culture medium. After 6 days of 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 the present invention are named BCMA-hG18CART.

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

[0052] 5.1 LDH kit to detect CAR-T cell toxicity

[0053] The 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 a ratio of 5:1 and 1:1 for 16 h (wherein the target cells were 3×10 4 / well, and the number of BCM A CART cells added was adjusted according to the effector-target ratio (E:T). The killing effect of CAR-T was determined using an LDH detection kit (Shanghai Biotech, C0016).

[0054] According to the instruction manual, set up the culture medium background well, effector cell spontaneous release well, target cell spontaneous release well, and target cell maximum release well. 1 hour before collecting the supernatant, add 15 μL of LDH lysate to the target cell maximum release well, mix well, and place in the incubator for 1 hour. Take the above cell culture plate, centrifuge at 12000rpm for 3 minutes, take 120 μL of the supernatant from each well, add 60 μL of LDH working solution (prepare LDH working solution according to 20 μL enzyme solution, 20 μL lactic acid solution, 20 μL INT solution, and 18 μL INT diluent for each reaction), and place on a horizontal shaker and incubate at room temperature in the dark for 30 minutes. Detect the OD490 value of each well.

[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 natural release group - OD value of the corresponding effector cell natural release group) / (OD value of the corresponding target cell maximum release group - OD value of the corresponding effector cell natural release group) × 100%.

[0056] The results are as follows Figures 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 )) has significant cytotoxicity, and the cytotoxicity of BCMA-hG18 CART is better than that of C-CAR088, while for BCMA-negative cells (K562( Figure 1C )), BCMA-hG18 CART, C-CAR088 and NT cells showed no cytotoxicity.

[0057] 5.2 ELISA to detect cytokine release

[0058] The effector cells (BCMA-hG18 CART, C-CAR088, NT cells, 3×10 4 / well) and target cells (MM.1S, RPMI-8266, K562, 3×10 4 After mixing, the cells were co-cultured at 37°C and 5% CO2 for 16 hours. The cell culture plate was centrifuged at 24°C and 1000 rpm for 5 minutes, and 50 μL of supernatant was taken to detect the amount of interferon γ released using an interferon γ detection kit (BD Biosciences, 560111).

[0059] Dilute the standard according to the instructions. Prepare a 100 μL reaction system with 1 μL interferon gamma capture microspheres, 1 μL PE detector, and 98 μL PBS for each reaction. Add 50 μL of standard or sample to be tested to each reaction system and incubate at room temperature for 2 hours. Add 1 mL PBS to each reaction, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 150 μL PBS to each tube and resuspend, collect 3000 effective microspheres on the flow cytometer and analyze with 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 better than that of C-CAR088, while in BCMA-negative cells (K562), there is no difference in interferon γ release between BCMA-hG18 CART or C-CAR088 and NT cells, that is, in BCMA-hG18 CART and C-CAR088, there is no activity in inducing interferon γ.

[0061] Example 6. In vivo toxicity detection of BCMA CART cells

[0062] Twelve-week-old B-NDG mice were randomly divided into three groups, with five mice in each group. Each mouse was injected with 3×10 6 NT cells, BCMA-hG18 CART or C-CAR088 (the date of T cell / CART cell injection was marked as D0), and 1×10 6 The survival status of each mouse was observed from day 0 to day 45, the death time was recorded, and the survival curve was drawn 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 can significantly prolong the survival of MM.1S mice. And the effect of BCMA-hG18 CART in prolonging the survival of mice is also better than C-CAR088.

Claims

1. A single-chain antibody against BCMA, characterized in that: include: V H , which comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; and V L , which comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

2. The single-chain antibody against BCMA according to claim 1, characterized in that: include: V H , the V H The amino acid sequence of is shown in SEQ ID NO.7; V L , the V L The amino acid sequence is shown in SEQ ID NO.

8.

3. The single-chain antibody against BCMA according to claim 2, characterized in that: The amino acid sequence of the single-chain antibody against BCMA is shown in SEQ ID NO.

10.

4. A chimeric antigen receptor targeting BCMA, characterized in that: The chimeric antigen receptor comprises a CD8 signal peptide, a single-chain antibody against BCMA as described in any one of claims 1 to 3, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory region and a CD3ζ cytoplasmic signaling domain, which are sequentially connected.

5. The chimeric antigen receptor targeting BCMA according to claim 4, wherein the amino acid sequence of the chimeric antigen receptor targeting BCMA (BCMACAR) is shown in SEQ ID NO.

16.

6. A polynucleotide, characterized in that: It encodes the single-chain antibody against BCMA as described in any one of claims 1 to 3 or the chimeric antigen receptor targeting BCMA as described in any one of claims 4 to 5.

7. A carrier, characterized in that: It contains the polynucleotide as claimed in claim 6.

8. A host cell, characterized in that: It comprises or expresses the single-chain antibody against BCMA as described in any one of claims 1 to 3, the chimeric antigen receptor targeting BCMA as described in any one of claims 4 to 5, the polynucleotide as described in claim 6, or the vector as described in claim 7.

9. A chimeric antigen receptor T cell, characterized in that: It expresses a chimeric antigen receptor targeting BCMA as described in any one of claims 4-5.

10. A tumor treatment drug, characterized in that: It contains the chimeric antigen receptor T cell as claimed in claim 9.

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