BCMA-targeted nano antibody, chimeric antigen receptor and application of BCMA-targeted nano antibody and chimeric antigen receptor

Abstract

The invention relates to the technical field of biological medicine, and provides a BCMA-targeted nano antibody, a chimeric antigen receptor and application thereof. The nano antibody comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2 and a heavy chain variable region CDR3, the amino acid sequence of the CDR1 is shown as SEQ ID No. 1, the amino acid sequence of the CDR2 is shown as SEQ ID No. 2, and the amino acid sequence of the CDR3 is shown as SEQ ID No. 3. The BCMA nano antibody sequence provided by the invention is specifically combined with a tumor cell BCMA target spot, and can be used for subsequently preparing a hematologic tumor treatment medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a nanobody targeting BCMA, a chimeric antigen receptor, and their applications. Background Art

[0002] Multiple myeloma (MM) is the second most common hematological malignancy after non-Hodgkin lymphoma. In recent years, despite great progress in chemotherapy, proteasome inhibitors, immunomodulatory thalidomide derivatives, and CD38-targeting antibodies, almost all patients will eventually relapse. Therefore, there is an urgent need for new treatment regimens.

[0003] B cell maturation antigen (BCMA) is an extremely important B cell biomarker that is widely present on the surface of multiple myeloma (MM) cells and has become a very popular immunotherapeutic target for MM and other hematological malignancies in recent years. BCMA CAR-T has shown encouraging effects in the treatment of severe refractory MM and has potential important application value. Therefore, developing antibody recognition sequences targeting BCMA is helpful for MM immunotherapy. Summary of the Invention

[0004] In view of this, the present invention provides a nanobody targeting BCMA, a chimeric antigen receptor, and their applications. The BCMA nanobody sequence is used for the construction of CAR and the subsequent preparation of CAR-T cells, thereby mediating the specific binding of CAR-T cells to the BCMA target of tumor cells and promoting the clinical treatment of MM.

[0005] The technical solution of the present invention is realized as follows: In the first aspect, the present invention provides a nanobody targeting the BCMA target. The nanobody comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3 of the heavy chain variable region. The amino acid sequence of CDR1 is as shown in SEQ ID No.1, the amino acid sequence of CDR2 is as shown in SEQ ID No.2, and the amino acid sequence of CDR3 is as shown in SEQ ID No.3.

[0006] Based on the above technical solution, preferably, the nanobody further comprises framework regions FR1, FR2, FR3, and FR4. The amino acid sequence of FR1 is as shown in SEQ ID No.4, the amino acid sequence of FR2 is as shown in SEQ ID No.5, the amino acid sequence of FR3 is as shown in SEQ ID No.6, and the amino acid sequence of FR4 is as shown in SEQ ID No.7.

[0007] Based on the above technical solution, preferably, the amino acid sequence of the nanobody is as shown in SEQ ID No.8.

[0008] In a second aspect, the present invention provides a chimeric antigen receptor targeting BCMA based on a nanobody, and the chimeric antigen receptor comprises the above-mentioned nanobody.

[0009] On the basis of the above technical solutions, preferably, the chimeric antigen receptor further comprises a signal peptide, a transmembrane region CD8TM, and an intracellular activation domain of 4-1BB-CD3ζ;

[0010] The chimeric antigen receptor comprises a signal peptide, a nanobody targeting BCMA, a transmembrane region CD8TM, and an intracellular activation domain of 4-1BB-CD3ζ connected in series in sequence.

[0011] On the basis of the above technical solutions, preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 9, the amino acid sequence of the transmembrane region CD8TM is as shown in SEQ ID NO: 10, and the amino acid sequence of the intracellular activation domain of 4-1BB-CD3ζ is as shown in SEQ ID NO: 11.

[0012] In a third aspect, the present invention provides an isolated nucleic acid, and the isolated nucleic acid encodes the above-mentioned nanobody or chimeric antigen receptor.

[0013] In a fourth aspect, the present invention provides an expression vector, and the expression vector comprises the above-mentioned isolated nucleic acid.

[0014] In a fifth aspect, the present invention provides the application of the above-mentioned nanobody, chimeric antigen receptor, isolated nucleic acid or expression vector in the preparation of a drug for treating multiple myeloma.

[0015] The nanobody targeting BCMA, chimeric antigen receptor and their application of the present invention have the following beneficial effects compared with the prior art:

[0016] The nanobody targeting the BCMA target provided by the present invention can better bind to the BCMA protein and has good application prospects in the treatment of MM. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is a flow chart for antibody sequence screening;

[0019] Figure 2It is a figure of ELISA test results for BCMA antibody sequences. Figure A shows BCMA in the experimental group, and Figure B shows the control, Milk;

[0020] Figure 3 It is a flow cytometry detection figure of the binding of BCMA CAR-T cells to BCMA protein;

[0021] Figure 4 It is a structural diagram of BCMA CAR-T sequence. Detailed implementation manners

[0022] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0023] The present invention provides a nanobody targeting the BCMA target. The nanobody includes complementarity-determining region 1 (CDR1), CDR2, and CDR3 of the heavy chain variable region and framework regions FR1, FR2, FR3, and FR4.

[0024] Among them, the amino acid sequence of CDR1 is shown in SEQ ID No.1, the amino acid sequence of CDR2 is shown in SEQ ID No.2, and the amino acid sequence of CDR3 is shown in SEQ ID No.3. The nanobody also includes framework regions FR1, FR2, FR3, and FR4. The amino acid sequence of FR1 is shown in SEQ ID No.4, the amino acid sequence of FR2 is shown in SEQ ID No.5, the amino acid sequence of FR3 is shown in SEQ ID No.6, and the amino acid sequence of FR4 is shown in SEQ ID No.7. The amino acid sequence of the nanobody is shown in SEQ ID No.8.

[0025]

[0026] The present invention also provides a chimeric antigen receptor targeting BCMA based on the nanobody. The chimeric antigen receptor includes a signal peptide, a nanobody targeting BCMA, a transmembrane region CD8TM, and an intracellular activation domain of 4-1BB-CD3ζ connected in series.

[0027] The amino acid sequence of the signal peptide is shown in SEQ ID NO:9, the amino acid sequence of the transmembrane region CD8TM is shown in SEQ ID NO:10, and the amino acid sequence of the intracellular activation domain of 4-1BB-CD3ζ is shown in SEQ ID NO:11.

[0028] The present invention also provides a separated nucleic acid, which encodes the above-mentioned nanobody or chimeric antigen receptor.

[0029] The present invention provides an expression vector, which contains the above-mentioned separated nucleic acid.

[0030] The vector can be a DNA vector, an RNA vector, a plasmid and / or a vector derived from a virus. The vector derived from a virus can be a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector and / or a herpesviral vector.

[0031] The present invention provides the application of the above-mentioned nanobody, chimeric antigen receptor, separated nucleic acid or expression vector in the preparation of a drug for treating multiple myeloma.

[0032] The following verifies the nanobody and chimeric antigen receptor of the present invention and their applications through examples.

[0033] Example 1 Phage library panning of alpaca nanobody library

[0034] 1) Select recombinant human BCMA protein (KaiKa Biotech, product number BCM-HM217) as the antigen for immunizing and activating VHH antibodies.

[0035] 2) Select 1 healthy alpaca for immunization. After mixing and emulsifying the complete Freund's adjuvant and the antigen at a ratio of 1:1, inject subcutaneously at multiple points, with a dosage of 0.2 mg per camel. A total of 5 immunizations are carried out, with an interval of 2 weeks.

[0036] 3) After the 5th immunization, collect 200 mL of alpaca peripheral blood, and isolate mononuclear cells by density centrifugation. Extract RNA by the TRIZOL lysis method and reverse transcribe it into cDNA, and then amplify the VHH gene fragment by nested PCR.

[0037] 4) Connect the VHH gene fragment to the pCANTAB-5E phage display plasmid, and then electrotransform it into TG1 competent cells to establish a phage library.

[0038] 5) Coat an immunization tube with BCMA antigen and block it with 5% skim milk powder. Add 1 mL of the phage library and incubate with rotation at room temperature for 1 hour. After washing the immunization tube with 2 mL of PBST (1x PBS plus 0.1% Tween20), add 500 μL of Gly-HCl neutralization buffer for neutralization, and transfer the solution in the immunization tube to a new 1.5 ml centrifuge tube, which is the phage eluate for the first round of screening.

[0039] 6) Take the phage eluate from the first round to infect TG1 strains, and perform 10 5 and 10 6Two gradient dilutions were performed for plate counting, and the results are shown in Figure 1 and Table 1.

[0040] Figure 1 The number of single colonies at dilutions where single colonies can be clearly distinguished on the statistical plate of Figure 1 was used to calculate the panning efficiency by clone counting.

[0041] Table 1 Results of the first round of ELISA

[0042] 1 2 3 4 5 6 7 8 9 10 A 5.034 5.013 6.896 35.335 4.058 26.162 1.805 3.475 0.906 27.116 B 23.206 0.996 10.008 33.331 36.762 35.866 24.893 28.585 0.862 13.463 C 5.738 25.227 19.387 17.730 32.304 1.025 38.276 35.579 7.756 0.786 D 9.998 17.762 13.457 33.774 34.398 35.487 30.133 1.025 0.960 4.383 E 1.154 22.006 1.006 21.711 23.936 28.454 34.517 1.019 1.000 20.096 F 23.397 4.687 31.398 14.025 36.683 26.044 43.925 31.965 37.051 5.917 G 8.523 25.859 28.355 29.242 15.953 30.124 5.890 1.049 0.948 5.686 H 5.313 2.160 32.538 23.501 1.018 1.002 1.071 20.901 4.547 0.927

[0043] Example 2 Phage ELISA

[0044] 1) Single colonies from the second-round panning clone plate were picked for culture. When the OD 600 reached 0.5, M13KO7 helper phage was added, and after overnight culture at 25 °C, the supernatant was collected by centrifugation.

[0045] 2) The BCMA antigen (KaiKa Biotech, catalog number BCM-HM217) was coated on an ELISA plate (1 ng / μl, 100 μl / well). After blocking and washing, 100 μl of the phage culture supernatant was added to each well. After incubation and washing, horseradish peroxidase-labeled anti-M13 antibody (diluted 10,000-fold with PBS) was added. After incubation and washing, TMB chromogenic solution was added for color development, and finally, the stop solution was added to terminate the reaction. The optical density was measured at 450 nm, and the results are shown in Table 2.

[0046] Table 2 Results of the second round of ELISA

[0047] 1 2 3 4 5 6 7 8 9 10 11 12 A 17.120 7.873 10.804 7.004 1.026 13.898 16.310 0.919 8.807 8.875 0.984 1.040 B 7.375 6.688 0.941 8.805 0.987 13.098 1.002 8.715 8.489 11.282 11.643 9.091 C 11.675 10.640 10.813 9.846 10.182 11.454 15.696 12.540 11.657 11.534 12.002 4.197 D 7.954 9.218 0.899 8.819 7.449 9.926 8.052 1.031 10.041 1.135 8.395 4.830 E 7.173 12.061 12.225 0.946 10.574 11.862 9.173 7.324 10.522 11.343 11.404 7.173 F 10.829 1.041 10.592 9.683 9.290 8.946 10.112 1.047 11.572 0.774 12.068 9.256 G 11.071 9.139 1.122 10.378 11.802 14.481 9.498 10.837 14.564 10.917 9.064 8.488 H 14.098 15.367 7.396 9.141 10.568 10.940 13.089 12.147 11.898 10.947 5.501 10.150

[0048] 3) After removing redundant sequences by sequencing, 20 unique antibody sequences were obtained.

[0049] Example 3 Flow cytometry detection of the binding ability of CAR-T cells derived from anti-BCMA nanobodies to BCMA protein

[0050] 1) The selected sequences were constructed into a CAR lentiviral vector, and membrane-expressed NGFR was used as a tag to identify the expression of CAR (Gene ID: 4804).

[0051] CAR structural design targeting BCMA:

[0052] As Figure 2 shown, the chimeric antigen receptor targeting BCMA constructed in this example includes a signal peptide, a nanobody targeting BCMA, a transmembrane region CD8TM, and a 4-1BB-CD3ζ intracellular activation domain in series.

[0053] The amino acid sequence of the signal peptide is shown in SEQ ID NO: 9, the amino acid sequence of the transmembrane region CD8TM is shown in SEQ ID NO: 10, and the amino acid sequence of the 4-1BB-CD3z intracellular activation domain is shown in SEQ ID NO: 11.

[0054]

[0055] 2) Prepare a lentiviral vector for infecting T cells to express the CAR molecule and detect the functional titer.

[0056] 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.

[0057] 3) The BCMA-CAR lentivirus was used to infect T cells from the peripheral blood of healthy donors to prepare CAR-T cells.

[0058] 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.

[0059] 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.

[0060] 4) Flow cytometry antibody labeling: collect transduced T cells, and take 1×10 6 After washing, the cells were added with a mixture of antibodies: APC-BCMA (Acro, Catalog No. BCA-HA2H9) and PE-NGFR (Biolegend, Catalog No. 345106). After incubation, the cells were washed twice and flow cytometry was performed. The results are shown in Figure 3 .

[0061] Figure 3 The flow cytometry results showed that sequence 8B (SEQ ID No. 8) had the highest ability to bind to BCMA protein.

[0062] Sequence of 8B antibody: EVQLQASGGGFVQPGGSLRLSCAASGYTSYTEIMGWFRQAPGKEREFVSAISYSPNYKTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCALWDWEAGPKFFDYWGQGTQVTVSS.

[0063] Sequence optimization of BCMA antibody in Example 4

[0064] To reduce the potential immunogenicity in subsequent CAR-T applications, the nanobody sequence of BCMA from alpaca obtained by screening and identification was modified, mainly involving local amino acid changes: Q5V, A6E, F11L, Y27F, S29F, Y30S, F37V, E44G, R45L, F47W, T89V, L94R, Q108L. The optimized BCMA nanobody sequence is:

[0065] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTEIMGWVRQAPGKGLEWVSAISYSPNYKTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARWDWEAGPKFFDYWGQGTLVTVSS (SEQ ID NO: 12).

[0066] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanobody targeting BCMA, characterized in that: The nanobody comprises heavy chain variable region CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID No.1, the amino acid sequence of CDR2 is shown as SEQ ID No.2, and the amino acid sequence of CDR3 is shown as SEQ ID No.

3.

2. The nanobody targeting BCMA according to claim 1, wherein: The nanobody further comprises framework regions FR1, FR2, FR3, FR4, the amino acid sequence of FR1 is shown as SEQ ID No.4, the amino acid sequence of FR2 is shown as SEQ ID No.5, the amino acid sequence of FR3 is shown as SEQ ID No.6, and the amino acid sequence of FR4 is shown as SEQ ID No.

7.

3. The nanobody targeting BCMA according to claim 1, characterized in that: The amino acid sequence of the nanobody is shown as SEQ ID No.

8.

4. A chimeric antigen receptor targeting BCMA based on nanobody, characterized in that: The chimeric antigen receptor comprises the nanobody according to any one of claims 1 to 3.

5. The chimeric antigen receptor according to claim 4, wherein: The chimeric antigen receptor further comprises a signal peptide, a transmembrane region CD8TM, and a 4-1BB-CD3ζ intracellular activation domain; The chimeric antigen receptor comprises a signal peptide, a nanobody targeting BCMA, a transmembrane region CD8TM, and a 4-1BB-CD3ζ intracellular activation domain in series.

6. The chimeric antigen receptor according to claim 5, wherein: The amino acid sequence of the signal peptide is shown as SEQ ID NO:9, the amino acid sequence of the transmembrane region CD8TM is shown as SEQ ID NO:10, and the amino acid sequence of the 4-1BB-CD3ζ intracellular activation domain is shown as SEQ ID NO:

11.

7. An isolated nucleic acid, characterized in that: The isolated nucleic acid encodes the nanobody according to any one of claims 1 to 3, or the chimeric antigen receptor according to any one of claims 4 to 6.

8. An expression vector, characterized in that: The expression vector comprises the isolated nucleic acid according to claim 7.

9. Use of the nanobody according to any one of claims 1 to 3, or the chimeric antigen receptor according to any one of claims 4 to 6, or the isolated nucleic acid according to claim 7, or the expression vector according to claim 8 in the preparation of a medicament for treating multiple myeloma.

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