Nanobodies targeting bcma, chimeric antigen receptors and uses thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a nano antibody targeting BCMA, a chimeric antigen receptor and application thereof. The nano antibody comprises a heavy chain variable region CDR1, CDR2 and 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 application is specifically combined with a tumor cell BCMA target point, and can be used for subsequent preparation of a blood tumor treatment drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanobody targeting BCMA, a chimeric antigen receptor, and their applications. Background Technology

[0002] Multiple myeloma (MM) is the second most common hematologic malignancy after non-Hodgkin's lymphoma. Despite significant advancements in chemotherapy, proteasome inhibitors, immunomodulatory agents such as thalidomide derivatives, and CD38-targeting antibodies in recent years, almost all patients eventually relapse. Therefore, there is an urgent need for new treatment options.

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

[0004] In view of this, the present invention proposes a BCMA-targeting nanobody, a chimeric antigen receptor and its application. 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 implemented as follows: In a first aspect, the present invention provides a nanobody targeting BCMA, the nanobody comprising heavy chain variable regions CDR1, CDR2 and CDR3, wherein 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.

[0006] Based on the above technical solutions, preferably, the nanobody further comprises backbone regions FR1, FR2, FR3, and FR4, wherein 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.

[0007] Based on the above technical solutions, preferably, the amino acid sequence of the nanobody is as shown in SEQ ID No. 18.

[0008] Secondly, the present invention provides a chimeric antigen receptor based on nanobodies that targets BCMA, wherein the chimeric antigen receptor comprises the aforementioned nanobodies.

[0009] Based on the above technical solutions, preferably, the chimeric antigen receptor further includes a signal peptide, a transmembrane region CD8TM, and a 4-1BB-CD3ζ intracellular activation domain;

[0010] The chimeric antigen receptor comprises a signal peptide, a nanobody targeting BCMA, a transmembrane region CD8TM, and a 4-1BB-CD3ζ intracellular activation domain, which are sequentially linked.

[0011] Based on 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 4-1BB-CD3z intracellular activation domain is as shown in SEQ ID NO: 11.

[0012] Thirdly, the present invention provides an isolated nucleic acid that encodes the aforementioned nanobody or chimeric antigen receptor.

[0013] Fourthly, the present invention provides an expression vector comprising the isolated nucleic acid described above.

[0014] Fifthly, the present invention provides the application of the above-mentioned nanobodies, chimeric antigen receptors, isolated nucleic acids or expression vectors in the preparation of drugs for treating multiple myeloma.

[0015] The BCMA-targeting nanobody, chimeric antigen receptor, and their applications of the present invention have the following advantages over existing technologies:

[0016] The nanobody targeting BCMA provided by this invention can bind to BCMA protein better and has a promising application prospect in the treatment of MM. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Flowchart for antibody sequence screening;

[0019] Figure 2The images show the ELISA results of BCMA antibody sequence detection. Image A shows the experimental group BCMA, and image B shows the control group Milk.

[0020] Figure 3 Flow cytometry data showing the binding of BCMACAR-T cells to BCMA protein;

[0021] Figure 4 This is a diagram of the BCMACAR-T sequence structure. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention provides a nanobody targeting BCMA, wherein the nanobody comprises heavy chain variable regions CDR1, CDR2 and CDR3 and backbone regions FR1, FR2, FR3 and FR4.

[0024] 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 further comprises backbone 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] This invention also provides a chimeric antigen receptor based on nanobodies that targets BCMA. The chimeric antigen receptor comprises, in tandem, a signal peptide, a BCMA-targeting nanobody, a transmembrane region CD8TM, and a 4-1BB-CD3ζ intracellular activation domain.

[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 4-1BB-CD3z intracellular activation domain is shown in SEQ ID NO: 11.

[0028] The present invention also provides an isolated nucleic acid that encodes the aforementioned nanobody or chimeric antigen receptor.

[0029] The present invention provides an expression vector comprising the isolated nucleic acid described above.

[0030] The vector can be a DNA vector, an RNA vector, a plasmid, and / or a viral vector. Viral vectors can be lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, and / or herpesvirus vectors.

[0031] This invention provides the application of the above-mentioned nanobodies, chimeric antigen receptors, isolated nucleic acids, or expression vectors in the preparation of drugs for treating multiple myeloma.

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

[0033] Example 1: Screening of phage libraries for alpaca nanobody libraries

[0034] 1) Recombinant human BCMA protein (Kaikai Biotechnology, catalog number BCM-HM217) was selected as the antigen for immune activation of VHH antibody.

[0035] 2) Select one healthy alpaca for immunization. Use a 1:1 mixture of complete Freund's adjuvant and antigen, emulsify, and inject subcutaneously at multiple sites. The dosage is 0.2 mg per alpaca. Administer a total of 5 immunizations, with an interval of 2 weeks between each immunization.

[0036] 3) After the 5th immunization, 200 mL of peripheral blood was collected from alpacas, and mononuclear cells were obtained by density centrifugation. RNA was extracted using the TRIZOL lysis method and reverse transcribed into cDNA, which was then amplified by nested PCR to obtain the VHH gene fragment.

[0037] 4) The VHH gene fragment was ligated into the pCANTAB-5E phage display plasmid and then electroporated into TG1 competent cells to establish a phage library.

[0038] 5) Coat the immunotubes with BCMA antigen and block with 5% skim milk powder. Add 1 mL of phage library and incubate at room temperature for 1 hour by rotation. Wash the immunotubes with 2 mL of PBST (1x PBS with 0.1% Tween 20), then add 500 μL of Gly-HCl neutralization buffer to neutralize. Transfer the solution from the immunotubes to a new 1.5 mL centrifuge tube; this is the phage elution buffer for the first round of screening.

[0039] 6) Infect TG1 strain with the first round of phage elution buffer, and centrifuge in a 1.5 mL tube for 10... 5 and 10 6Two serial dilutions were performed, and plate counts were conducted. The results are shown in [the table]. Figure 1 See Table 1.

[0040] Figure 1 The statistical panel clearly distinguishes the dilution of single colonies and the number of single colonies, and the screening efficiency is calculated by cloning.

[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) Select single colonies from the second round of screening and culturing, OD 600 When the concentration reaches 0.5, add M13KO7 helper phage, incubate overnight at 25 degrees Celsius, and then centrifuge to collect the supernatant.

[0045] 2) Coat the ELISA plate with BCMA antigen (KaiKa Biotechnology, catalog number BCM-HM217) (1 ng / ul, 100ul / well). After blocking and washing, add 100ul of phage culture supernatant to each well. After incubation and washing, add horseradish peroxidase-labeled anti-M13 antibody (diluted 10,000 times with PBS). After incubation and washing, add TMB chromogenic solution for color development. Finally, add stop solution to terminate the reaction. Measure the optical density at 450nm. 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 through sequencing, 20 unique antibody sequences were obtained.

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

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

[0051] CAR structure design targeting BCMA:

[0052] like Figure 2 As shown, the chimeric antigen receptor targeting BCMA constructed in this embodiment includes a signal peptide, a BCMA-targeting nanobody, a transmembrane region CD8TM, and a 4-1BB-CD3ζ intracellular activation domain, which are connected 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 lentiviral vectors for infecting T cells to express CAR molecules and detect functional titers.

[0056] HEK293T cells were seeded in T175 culture flasks (cultured to 70%-80% confluence). The CAR plasmid and helper plasmid were mixed and transfected using Lipofectamine 3000. The culture medium was replaced with fresh medium 6-8 hours later. The supernatant was collected 48-72 hours post-transfection. The supernatant was filtered through a 0.45 μm filter and concentrated to obtain high-titer lentivirus.

[0057] 3) Infect T cells derived from peripheral blood of healthy donors with BCMA-CAR lentivirus to prepare CAR-T cells.

[0058] PBMC isolation and T cell activation: PBMCs were isolated from 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 for 3 days in RPMI 1640 medium containing IL-2 (50-100 IU / mL).

[0059] Lentiviral transduction: 3 days after activation, T cells were adjusted to an appropriate concentration (1×10⁻⁶). 6 Lentiviral particles (MOI = 5–10) were co-incubated with T cells for 48 hours. Polybrene (e.g., 8 μg / mL) can be added to improve transduction efficiency. After transduction, the culture medium was changed, and T cells were cultured and expanded again.

[0060] 4) Flow cytometry antibody labeling: Collect transduced T cells, taking 1×10⁻⁶ cells per cell sample. 6 Cells were washed and then incubated with an antibody mixture of APC-BCMA (Acro, catalog number BCA-HA2H9) and PE-NGFR (Biolegend, catalog number 345106). After washing twice, flow cytometry analysis was performed. Results are shown below. Figure 3 .

[0061] Figure 3 Flow cytometry results showed that sequence 8B (SEQ ID No. 8) had the highest binding capacity to BCMA protein.

[0062] Sequence 8B Antibody sequence: EVQLQASGGGFVQPGGSLRLSCAASGYTSYTEIMGWFRQAPGKEREFVSAISYSPNYKTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCALWDWEAGPKFFDYWGQGTQVTVSS.

[0063] Example 4: Sequence optimization of BCMA antibody

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

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

[0066] 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 within the protection scope of the present invention.

Claims

1. A nanobody targeting BCMA, characterized in that: The nanobody comprises heavy chain variable regions CDR1, CDR2 and CDR3, 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.

2. The BCMA-targeting nanobody as described in claim 1, characterized in that: The nanobody further comprises backbone regions FR1, FR2, FR3, and FR4, wherein 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.

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

8.

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

5. The chimeric antigen receptor as described in claim 4, characterized in that: The chimeric antigen receptor also includes 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, which are sequentially linked.

6. The chimeric antigen receptor as described in claim 5, characterized in that: 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.

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

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

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