WO2022033057A1 - Single-domain antibody-based bcma chimeric antigen receptor, and application thereof - Google Patents

Single-domain antibody-based bcma chimeric antigen receptor, and application thereof Download PDF

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WO2022033057A1
WO2022033057A1 PCT/CN2021/086417 CN2021086417W WO2022033057A1 WO 2022033057 A1 WO2022033057 A1 WO 2022033057A1 CN 2021086417 W CN2021086417 W CN 2021086417W WO 2022033057 A1 WO2022033057 A1 WO 2022033057A1
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seq
bcma
chimeric antigen
antigen receptor
domain antibody
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French (fr)
Chinese (zh)
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姜舒
王冰
彭方理
张芸
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深圳市茵冠生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of immune cell therapy, in particular to a single domain antibody-based BCMA chimeric antigen receptor and application thereof.
  • MM Multiple Myeloma
  • M protein monoclonal immunoglobulin or its fragment
  • ROTI organ or tissue damage
  • Chimeric Antigen Receptor T cells Receptor modified T cells is a synthetic CAR molecule (Chimeric Antigen Receptor) is expressed on the T cell membrane, enabling T cells to recognize and kill tumor cells in the manner of antigen-antibody binding.
  • the CAR molecule includes an extracellular binding region, a hinge region, a transmembrane region and an intracellular signal segment, wherein the extracellular binding region is a single-chain antibody (scFv) derived from a monoclonal antibody that can specifically recognize the target antigen.
  • scFv single-chain antibody
  • CAR-T cells can overcome tumor cells to escape immune attack by downregulating MHC molecules, and CAR recognizes tumor antigens without MHC restriction, the same CAR can be applied to different patients.
  • CAR molecules can recognize any type of antigen on the cell surface, including proteins, carbohydrates, and glycolipids.
  • TCR T cell receptor
  • CAR enables T cells to recognize the tumor surface. The range of markers is greatly increased.
  • the binding of the scFv segment of CAR-T to the antigen is also affected by factors such as the affinity of the binding domain, the structure of the antigenic epitope, the number of tumor cell surface antigens, pH, temperature, and ionic strength.
  • Chinese patent 201580050638.9 discloses a chimeric antigen receptor comprising: an extracellular domain; a transmembrane domain; one or more intracellular costimulatory signal transduction domains; and a primary signal transduction domain,
  • the extracellular domain comprises a humanized anti-BCMA antibody or antigen-binding fragment thereof capable of binding one or more epitopes of a human BCMA (B cell maturation antigen) polypeptide.
  • the coding gene is obtained by genetic engineering technology, and the gene fragment is inserted into a lentivirus expression vector, packaged into a lentivirus, and infected with human T cells, so that the T cells express the chimeric antigen receptor.
  • Such chimeric antigen receptor T cells can be used for the treatment of B cell-related malignancies.
  • Single-domain antibodies also known as nanobodies (Nbs)
  • Nbs nanobodies
  • HcAbs heavy chain-only antibodies
  • the antigen-binding fragment in each arm of a camelid-only heavy chain antibody has a single heavy chain variable domain (VHH), and this antibody contains only one heavy chain variable region VHH and two conventional CH2 and CH3 constant domains
  • VHH single heavy chain variable domain
  • the molecular weight is only half that of traditional antibodies.
  • VHH can have high affinity for antigen without the help of light chain and is one of the smallest functional antigen-binding fragments with a molecular weight of about 15kD.
  • VHH consists of 3 antigenic complementarity determining regions (complementarity determining regions, CDRs) and 4 framework regions (frame regions, FR), generally arranged from the N-terminal to the C-terminal structure: FR1-CDR1-FR2-CDR2-FR3 -CDR3-FR4.
  • the 3 CDRs are the binding regions of the sdAb to the antigen, while traditional antibodies require 6 CDRs to maintain binding to the antigen.
  • the amino acid sequences of CDR1 and CDR3 of the sdAb were longer, which to some extent compensated for the loss of antigen-binding ability due to the deletion of the light chain.
  • sdAbs After antigenic stimulation, the production of sdAbs mainly depends on somatic hypermutation, so longer CDR sequences can also generate more antibody diversity. Antibody crystallography studies have shown that the longer CDR3 region endows sdAbs with greater antigen-binding capacity, enabling them to bind epitopes that traditional antibodies cannot. Therefore, Nb exhibits comparable or even stronger antigen-binding ability compared to monoclonal antibodies.
  • Nb Another advantage of Nb is that it is found by sequence alignment that Nb is highly homologous to the VH domain of human immunoglobulin IgG, and only the FR2 and CDR3 regions have significant differences. Studies have shown that repeated administration of Nb does not cause humoral and cellular immune responses, but whether long-term repeated use of Nb drugs can cause its immunogenicity to the body remains to be studied.
  • Cibia chimeric antigen receptor the CAR comprises: a BCMA antigen binding domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; wherein the The BCMA antigen binding domain comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3).
  • Immune cells such as CAR-T cells
  • the CAR expression efficiency is low and needs to be further improved.
  • the present invention constructs a chimeric antigen receptor against BCMA antigen by screening the specific single domain antibody against BCMA antigen and recombining its VHH through the method of gene recombination. Insert into the genome of human T lymphocytes to express specific chimeric antigen receptors (BCMA antigens) on their cell membranes CAR-T), by expanding BCMA CAR-T in vitro and returning it to the patient, to achieve specific immune cell therapy against tumor cells (myeloma cells) expressing BCMA antigens, and at the same time, avoid scFv derived from mouse antibodies
  • the disadvantage of mouse-derived BCMA CAR-T cells is that they are prone to produce anti-mouse antibodies and lead to treatment failure.
  • BCMA (antigen): B cell maturation antigen
  • CAR Chimeric Antigen Receptor
  • CAR-T cells Chimeric antigen receptor T lymphocytes.
  • the present invention provides an anti-BCMA single domain antibody.
  • the anti-BCMA single domain antibody comprises CDR1, CDR2 and CDR3.
  • the CDR1 is one of SEQ ID NOs: 15-17 or a sequence with 80% identity to SEQ ID NOs: 15-17;
  • the CDR2 is one of SEQ ID NOs: 18-20 or A sequence with 80% identity to SEQ ID NO: 18-20;
  • the CDR3 is one of SEQ ID NO: 21-23 or a sequence with 80% identity to SEQ ID NO: 21-23.
  • the amino acid sequence of the anti-BCMA single domain antibody is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: The amino acid sequence shown in NO:7.
  • the present invention provides a chimeric antigen receptor.
  • the chimeric antigen receptor comprises the aforementioned anti-BCMA single domain antibody.
  • the chimeric antigen receptor also comprises a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain.
  • the structural gene of the chimeric antigen receptor comprises the coding gene of the anti-BCMA single domain antibody, and the nucleotide sequence of the coding gene of the anti-BCMA single domain antibody is SEQ ID NO: 8, SEQ ID NO : 9 or the sequence shown in SEQ ID NO: 10.
  • the structural gene of the chimeric antigen receptor comprises the nucleotide sequence shown in SEQ ID NO: 11.
  • nucleotide sequence of the structural gene of the chimeric antigen receptor is the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
  • the present invention provides a biological material.
  • the biological material is a recombinant vector, a recombinant cell or a biological product for the purpose of treatment or prevention.
  • the biological material comprises the aforementioned anti-BCMA single-domain antibody or a gene encoding the anti-BCMA single-domain antibody.
  • the biological material comprises the aforementioned chimeric antigen receptor or a gene encoding the chimeric antigen receptor.
  • the biological material is a recombinant vector, and the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector.
  • the viral vector is a lentiviral vector.
  • the viral vector is pCDH-EF1a-BCMA CAR viral vector; the viral vector is the lentiviral vector pCDH comprising any of the nucleotide sequences shown in SEQ ID NOs: 12-14 -EF1a.
  • the biological material is immune cells
  • the immune cells include but are not limited to: T cells, NK cells, peripheral blood mononuclear cells (PBMC), hematopoietic stem cells, pluripotent stem cells or embryonic stem cells, preferably human peripheral cells blood T cells.
  • PBMC peripheral blood mononuclear cells
  • hematopoietic stem cells pluripotent stem cells or embryonic stem cells, preferably human peripheral cells blood T cells.
  • each single cell in the cells carries 3-4 chimeric antigen receptor molecules.
  • the present invention provides a medical formulation for the treatment of myeloma.
  • the medical preparation comprises the aforementioned anti-BCMA single-domain antibody or a gene encoding the anti-BCMA single-domain antibody.
  • the medical preparation comprises the aforementioned chimeric antigen receptor or a gene encoding the chimeric antigen receptor.
  • the medical formulation includes the aforementioned biological material.
  • the medical formulation comprises T cells expressing the BCMA chimeric antigen receptor.
  • the cell concentration of the BCMA chimeric antigen receptor-expressing T cells in the medical preparation is: 1 ⁇ 108 positive BCMA CAR-T/100mL.
  • the administration amount of the medical formulation was 2 ⁇ 10 6 positive BCMA CAR-T/kg body weight.
  • the dosage forms of the medical preparations include but are not limited to infusions and injections.
  • the administration modes of the medical formulation include but are not limited to: intravenous injection, intraperitoneal injection.
  • the present invention provides the use of the aforementioned anti-BCMA single domain antibody, chimeric antigen receptor or biological material in the preparation of a medicament for treating myeloma.
  • the myeloma is multiple myeloma.
  • the invention aims to solve the problem that patients with relapsed/drug-resistant multiple myeloma currently have no effective clinical treatment means, and apply BCMA CART technology specifically kills myeloma cells in the bone marrow, thereby achieving the purpose of treating patients with relapsed/drug-resistant multiple myeloma.
  • the present invention adopts the tandem composition of the BCMA single-domain antibody gene and the second-generation CAR structural gene sequence, which is different from the prior art.
  • the present invention uses different sequences from known BCMA antibodies, and the used antibody is a single domain antibody, which has stronger binding force to target cells, better killing effect, and longer in vivo duration than traditional antibody-derived CARs.
  • Fig. 1 is a graph showing the result of flow cytometry to detect the binding ability of BCMA VHH recombinant antibody to the recombinant cell line CHO-BCMA highly expressing BCMA.
  • Figure 2 is a partial detection result of flow cytometry to detect the binding ability of humanized antibody to BCMA.
  • Figure 3 shows the results of antibody affinity detection.
  • A represents the screened anti-BCMA single-domain antibody B-6-14;
  • B and C represent the humanized antibodies NW2-1222-6-14-1 and NW2-1222-6-14-2, respectively.
  • Figure 4 shows the results of flow cytometry detection of CAR-T cell membrane surface markers, where A is the positive rate of CD8, B is the positive rate of CD4, C is the BCMA negative control, and D is the B-6-14 BCMA CAR positive rate of CAR-T, E is the CAR positive rate of NW2-1222-6-14-1 BCMA CAR-T, F is NW2-1222-6-14-2 CAR positive rate of BCMA CAR-T.
  • Figure 5 shows the results of RT-PCR detection of the copy number of CAR on BCMA CAR-T cells.
  • A is the standard curve and B is the copy number of CAR.
  • Figure 6 shows the positive rate of BCMA detected by flow cytometry in the recombinant Nalm6-BCMA-GFP-LUC cell line, where A is the FSC-SSC scatter plot, B is the Nalm6 negative control, and C is the recombinant Nalm6-BCMA-GFP-LUC cell BCMA Positive rate (BCMA-GFP double positive).
  • A is the FSC-SSC scatter plot
  • B is the Nalm6 negative control
  • C is the recombinant Nalm6-BCMA-GFP-LUC cell BCMA Positive rate (BCMA-GFP double positive).
  • FIG 7 shows the experimental results of in vitro functional evaluation of BCMA CAR-T cells.
  • A is for Nalm6-BCMA- Killing effect of GFP-LUC cells
  • B is the detection result of IFN- ⁇ release.
  • Figure 8 shows the in vivo imaging results of mice in the in vivo functional evaluation of BCMA CAR-T cells. The stronger the color signal (the darker the color) in the in vivo image of the mouse, the more tumor cells in the mouse.
  • Figure 9 shows the results of changes in the body weight of mice in the in vivo functional evaluation of BCMA CAR-T cells.
  • Figure 10 is the mouse survival curve in the in vivo functional evaluation of BCMA CAR-T cells.
  • VHH of alpaca heavy chain antibody was amplified by two rounds of PCR. After the VHH fragment and the antibody display vector were digested by SfiI respectively, the VHH fragment was cloned into the display vector using T4 DNA ligase, and then the ligated product was electro-transformed into Pichia cells by electro-transformation, and screened. , to obtain a single-domain antibody yeast display library.
  • VHH yeast display library medium add methanol with a final concentration of 0.5% to induce yeast to display single-domain antibodies on the yeast cell wall, and add biotin-labeled BCMA recombinant protein to detect positive yeast clones in the yeast library. Labeling, using streptividin-labeled magnetic beads to isolate the clones in the yeast display library that can bind to the target protein BCMA, repeat the above enrichment screening process 2-3 times, and coat the clones that can bind to the BCMA recombinant protein on a solid plate. Clonal identification was performed after picking single clones.
  • VHH antibody sequences were separately synthesized and cloned into the expression vector Lenti-hlgG1-Fc. After the vector was verified by sequencing, the plasmid was extracted.
  • This application has screened a variety of anti-BCMA single-domain antibodies.
  • the amino acid sequence of the CDR1 region is selected from SEQ ID NO: 15-17
  • the amino acid sequence of the CDR2 region is selected from SEQ ID NO: 18-20
  • the CDR3 region is selected from SEQ ID NO: 18-20.
  • the amino acid sequences of the regions are selected from SEQ ID NOs: 21-23.
  • This example takes the screened anti-BCMA single-domain antibody B-6-14 as an example, and its amino acid sequence is shown in SEQ ID NO: 1.
  • SEQ ID NO: 1 the design of surface amino acid substitution is used to carry out humanization design, and the humanized antibody sequences are as shown in SEQ ID NO: 2-7.
  • the above six humanized antibody fragments were synthesized and cloned into the expression vector Lenti-hlgG1-Fc. After the vector was verified by sequencing, the plasmid was extracted.
  • Humanized antibody affinity detection The BCMA recombinant protein was immobilized on the CM5 chip using 10 mM Acetate buffer, and the humanized antibodies NW2-1222-6-14-1 and NW2-1222-6-14-2 were used for the above-mentioned humanized antibodies respectively. and the original sequence antibody as the mobile phase to detect the binding ability of the antibody to the target protein BCMA before and after humanization.
  • SEQ ID NOs: 12-14 were obtained by PCR, enzyme digestion and enzyme ligation.
  • the lentiviral vector pCDH-EF1a and SEQ ID NOs: 12-14 were double digested and ligated with T4 DNA ligase, respectively, and then transformed into competent cells and single clones were picked. After the bacterial liquid was sequenced correctly, the plasmids were extracted and sequences were obtained. Correct three lentiviral vectors pCDH-EF1a-BCMA 1#, 2#, 3#, of which 1# is the original sequence of anti-BCMA single-domain antibody, and 2# and 3# are the sequences of two humanized anti-BCMA single-domain antibodies.
  • the lentiviral packaging plasmid mixture (including PSPAX2 and VSVG) was mixed with pCDH-EF1a-BCMA 1#, 2#, and 3# according to the pre-optimized ratio, and then the co-transfection reagent was added, and incubated at room temperature for 15 minutes. The transfection mixture was then added dropwise to the 293T cells. After 1-3 days, the medium supernatant was collected to obtain the crude lentivirus solution. The supernatant was collected after centrifugation at 500 g for 10 min at 4°C. Ultracentrifugation was then performed for lentiviral concentration.
  • lentiviral particles were carefully resuspended in pre-chilled DPBS.
  • the virus was aliquoted and stored at -80°C. Physical and infectious titers of lentivirus were determined based on RT-PCR method and flow cytometry.
  • PBMC Preparation of PBMC: Centrifuge 30 mL of human venous blood at 800 g for 30 minutes at 20°C. After the centrifugation, transfer the upper plasma into another centrifuge tube. Add 15mL of human lymphocyte separation solution (Tianjin Haoyang Biotechnology) into a 50mL centrifuge tube, and slowly add the centrifuged blood above the lymphocyte separation solution with an electric pipette, centrifuge at 250g, 20°C for 10 minutes. Discard the supernatant and resuspend in 10 ml of PBS for T cell purification.
  • human lymphocyte separation solution Trianjin Haoyang Biotechnology
  • T cell purification Miltenyi was used according to the manufacturer's protocol as described below Pan T Cell Isolation Kit (Cat. No. 130-096-535) purifies human T cells from PBMC. After PBMC counting, centrifuge at 300g for 10 minutes at 20°C. Pour off the supernatant, resuspend the cell pellet in buffer per 107/40 ⁇ L buffer, add 10 ⁇ L/107 Pan T cell biotin-antibody mixture, mix well and incubate at 4°C for 5 minutes. Then add 30 ⁇ L of buffer, and 20 ⁇ L of Pan T cell bead mix. Mix well and incubate at 4°C for 10 minutes.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • the cell suspension flowed out with gravity.
  • the effluent ie, the T cell fraction
  • T cells were then enriched by centrifugation and resuspended in lymphocyte medium + 1000 IU/mL IL-2.
  • CAR-T preparation After pre-activating the prepared T cells with a human T cell activation/expansion kit (Miltenyi #130-091-441) for 24-96 hours, lentivirus infection was performed.
  • a human T cell activation/expansion kit Miltenyi #130-091-441
  • B-6-14 BCMA CAR-T cells pCDH-EF1a-BCMA 1# lentivirus infection;
  • B-6-14-2 BCMA CAR-T cells pCDH-EF1a-BCMA 2# lentivirus infection;
  • B-6-14-3 BCMA CAR-T cells pCDH-EF1a-BCMA 3# lentivirus infection.
  • the test results are shown in Figure 4.
  • the results in Figure 4 show that in the BCMA CAR-T cells prepared in Example 4, the CAR expression efficiencies of the three sequences are all above 90%.
  • the DNA of the three BCMA CAR-T cells was extracted by RT-PCR to detect the copy number of CAR on the BCMA CAR-T cells.
  • the results are shown in Figure 5.
  • the relevant data are as follows:
  • Recombinant Nalm6-BCMA-GFP-LUC cell line (gifted from Elken Biomedical Technology (Suzhou) Co., Ltd.) was used to detect the positive rate of BCMA by flow cytometry.
  • the results are shown in Figure 6.
  • the results show that BCMA molecules are highly expressed in Nalm6-BCMA-LUC recombinant cell line, but not expressed in Nalm6 cells (purchased from Guangzhou Saiku Biotechnology Co., Ltd.), which can be used as target cells and control cells for CAR-T killing experiments respectively. .
  • Tumor cell Nalm6-BCMA-Luciferase cells 1 ⁇ 106/200 ⁇ L were infused through the tail vein of mice to establish a mouse tumor model.
  • the mice were randomly divided into 4 groups, 5 days, 11 days, 17 days and In vivo imaging observation was performed on 25 days, and control T cells (isolated and purified from the same peripheral blood PBMC sample) and two humanized single-domain antibody sequence BCMA CAR-T cells (B) were infused through the tail vein on the 5th day after modeling.
  • mouse-derived BCMA CAR-T was used as a positive control, and the injection dose of each cell was as follows:
  • B-6-14-1 BCMA CAR-T cells 1 ⁇ 107;
  • mice in the control group had dead mice from about 25 days after modeling, while the mice in the CAR-T treatment group were in a normal state and continued to be observed for 60+ days.
  • the mouse-derived BCMA All the mice injected with CAR-T died, while most of the mice injected with the humanized single-domain antibody sequence BCMA CAR-T survived, indicating that the single-domain antibody sequence BCMA CAR-T lasted longer in vivo and had better effects ( Figure 10). .

Abstract

Provided is a single-domain antibody-based BCMA chimeric antigen receptor (CAR). The BCMA antigen-binding domain of the BCMA CAR comprises an anti-BCMA single-domain antibody, and the anti-BCMA single-domain antibody comprises CDR1, CDR2, and CDR3. CDR1 is one of SEQ ID NOs: 15-17 or a sequence having 80% identity with SEQ ID NOs: 15-17, CDR2 is one of SEQ ID NOs: 18-20 or a sequence having 80% identity with SEQ ID NOs: 18-20, and CDR3 is one of SEQ ID NOs: 21-23 or a sequence having 80% identity with SEQ ID NOs: 21-23. Compared with conventional antibody-derived BCMA CARs, the present single-domain antibody-based BCMA CAR has a stronger binding force to target cells, better killing effect, and a longer duration of persistence in the body.

Description

一种基于单域抗体的BCMA嵌合抗原受体及其应用A kind of BCMA chimeric antigen receptor based on single domain antibody and its application 技术领域technical field
本发明属于免疫细胞治疗领域，具体涉及一种基于单域抗体的BCMA嵌合抗原受体及其应用。The invention belongs to the field of immune cell therapy, in particular to a single domain antibody-based BCMA chimeric antigen receptor and application thereof.
背景技术Background technique
多发性骨髓瘤（Multiple Myeloma，MM）是浆细胞恶性增殖性疾病，骨髓中克隆性浆细胞异常增生，并分泌单克隆免疫球蛋白或其片段（M蛋白），并导致相关器官或组织损伤（ROTI）。常见临床表现为骨痛、贫血、肾功能不全、感染等。统计显示，每年将有近86000名患者被诊断为骨髓瘤，而每年有约63000名患者死于疾病相关的并发症。近年新药蛋白酶体抑制剂硼替佐米、免疫调节药沙利度胺和来那度胺等的应用尽管改善了MM患者的缓解率和无病生存时间，但总生存时间与传统治疗无明显差异。并且MM应用化疗取得缓解后，大多数病人终将复发，且对原来敏感的药物产生耐药，加大剂量并不能使患者再次获得缓解，反而容易产生骨髓抑制、继发感染、肝功能损害等副作用。解决复发/耐药性多发性骨髓瘤患者目前临床上无有效治疗手段。Multiple Myeloma (MM) is a malignant proliferative disease of plasma cells, in which clonal plasma cells in the bone marrow proliferate abnormally and secrete monoclonal immunoglobulin or its fragment (M protein), and cause associated organ or tissue damage ( ROTI). Common clinical manifestations include bone pain, anemia, renal insufficiency, and infection. Statistics show that nearly 86,000 patients will be diagnosed with myeloma every year, and about 63,000 patients will die of disease-related complications every year. In recent years, the application of new drugs such as proteasome inhibitor bortezomib, immunomodulatory drugs thalidomide and lenalidomide has improved the remission rate and disease-free survival time of MM patients, but the overall survival time is not significantly different from traditional treatment. And after MM achieves remission with chemotherapy, most patients will eventually relapse and become resistant to the original sensitive drugs. Increasing the dose will not make the patient remission again, but is prone to bone marrow suppression, secondary infection, liver function damage, etc. side effect. There is currently no effective clinical treatment for patients with relapsed/drug-resistant multiple myeloma.
嵌合抗原受体T细胞（Chimeric Antigen Receptor modified T cells，CAR-T）是通过基因修饰的手段，将一个人工合成的CAR分子（Chimeric Antigen Receptor）表达在T细胞膜上，使T细胞以抗原抗体结合的方式识别并杀伤肿瘤细胞。CAR分子包括胞外结合区域、铰链区、跨膜区和胞内的信号段，其中胞外结合区域为能特异性识别靶抗原的单克隆抗体来源的单链抗体（scFv）。这一技术有几个优势，例如，CAR分子对肿瘤表面的TAA（肿瘤相关抗原，tumor associated antigen）的识别是以一种MHC(major histocompatibility complex)-非依赖性的方式进行的，因而CAR-T细胞可以克服肿瘤细胞通过下调MHC分子逃脱免疫攻击，并且CAR识别肿瘤抗原没有MHC限制性，同一种CAR可以应用于不同的患者。另外，CAR分子可以识别细胞表面的任何类型的抗原，包括蛋白、糖类、糖脂，这样，相对于TCR（T cell receptor）只能识别MHC-肽段，CAR使T细胞能识别的肿瘤表面标志物的范围大大增加。与抗原-抗体反应一样，CAR-T的scFv段与抗原结合也受结合域的亲和力、抗原表位的结构、肿瘤细胞表面抗原的数量、pH值、温度、离子强度等因素的影响。Chimeric Antigen Receptor T cells Receptor modified T cells, CAR-T) is a synthetic CAR molecule (Chimeric Antigen Receptor) is expressed on the T cell membrane, enabling T cells to recognize and kill tumor cells in the manner of antigen-antibody binding. The CAR molecule includes an extracellular binding region, a hinge region, a transmembrane region and an intracellular signal segment, wherein the extracellular binding region is a single-chain antibody (scFv) derived from a monoclonal antibody that can specifically recognize the target antigen. This technology has several advantages. For example, the recognition of TAA (tumor associated antigen) on the tumor surface by CAR molecules is an MHC (major associated antigen). Histocompatibility complex)-independent manner, so CAR-T cells can overcome tumor cells to escape immune attack by downregulating MHC molecules, and CAR recognizes tumor antigens without MHC restriction, the same CAR can be applied to different patients. In addition, CAR molecules can recognize any type of antigen on the cell surface, including proteins, carbohydrates, and glycolipids. In this way, compared with TCR (T cell receptor), which can only recognize MHC-peptides, CAR enables T cells to recognize the tumor surface. The range of markers is greatly increased. Like the antigen-antibody reaction, the binding of the scFv segment of CAR-T to the antigen is also affected by factors such as the affinity of the binding domain, the structure of the antigenic epitope, the number of tumor cell surface antigens, pH, temperature, and ionic strength.
中国专利201580050638 .9公开了一种嵌合抗原受体，其包含：胞外结构域；跨膜结构域；一个或多个胞内共刺激信号转导结构域；以及初级信号转导结构域，所述胞外结构域包含能结合人BCMA(B细胞成熟抗原)多肽的一个或多个表位的人源化的抗BCMA抗体或其抗原结合片段。利用基因工程技术获得编码基因，将该基因片段***慢病毒表达载体，包装成慢病毒，感染人T细胞，使T细胞表达该嵌合抗原受体。这种嵌合抗原受体T细胞能够用于B细胞相关的恶性肿瘤的治疗。Chinese patent 201580050638.9 discloses a chimeric antigen receptor comprising: an extracellular domain; a transmembrane domain; one or more intracellular costimulatory signal transduction domains; and a primary signal transduction domain, The extracellular domain comprises a humanized anti-BCMA antibody or antigen-binding fragment thereof capable of binding one or more epitopes of a human BCMA (B cell maturation antigen) polypeptide. The coding gene is obtained by genetic engineering technology, and the gene fragment is inserted into a lentivirus expression vector, packaged into a lentivirus, and infected with human T cells, so that the T cells express the chimeric antigen receptor. Such chimeric antigen receptor T cells can be used for the treatment of B cell-related malignancies.
单域抗体(sdAb)也称为纳米抗体（Nb），由于具有单个单体抗体可变域而不同于传统的4链抗体。骆驼科动物和鲨鱼产生天然缺乏轻链的sdAb，其被称为仅重链抗体(HcAb)。骆驼科动物仅重链抗体的每个臂中的抗原结合片段具有单个重链可变域(VHH)，这种抗体只包含一个重链可变区VHH和两个常规的CH2和CH3恒定结构域区，分子量只有传统抗体的一半。VHH可在无需轻链的帮助下，对抗原具有高亲和力，是最小的功能性抗原结合片段之一，分子量为大约15kD。VHH由3个抗原互补决定区（complementarity determining region，CDR）和4个框架区域（frame region，FR）组成，一般从N-末端至C-末端排列结构为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。3个CDR是sdAb与抗原的结合区域，而传统抗体需要6个CDR来维持与抗原的结合。此外，sdAb的CDR1和CDR3的氨基酸序列更长，在一定程度上弥补了由于轻链缺失而导致抗原结合能力的损失。在接受抗原刺激后，sdAb的产生主要依赖于体细胞超突变，因此更长的CDR序列也可产生更多的抗体多样性。抗体晶体学研究表明，更长的CDR3区域赋予sdAb更强的抗原结合能力，从而能够结合传统抗体无法到达的抗原表位。因此，相比于单克隆抗体，Nb展示出相当甚至更强的抗原结合能力。Single-domain antibodies (sdAbs), also known as nanobodies (Nbs), differ from traditional 4-chain antibodies by having a single monomeric antibody variable domain. Camelids and sharks produce sdAbs that naturally lack light chains, which are referred to as heavy chain-only antibodies (HcAbs). The antigen-binding fragment in each arm of a camelid-only heavy chain antibody has a single heavy chain variable domain (VHH), and this antibody contains only one heavy chain variable region VHH and two conventional CH2 and CH3 constant domains The molecular weight is only half that of traditional antibodies. VHH can have high affinity for antigen without the help of light chain and is one of the smallest functional antigen-binding fragments with a molecular weight of about 15kD. VHH consists of 3 antigenic complementarity determining regions (complementarity determining regions, CDRs) and 4 framework regions (frame regions, FR), generally arranged from the N-terminal to the C-terminal structure: FR1-CDR1-FR2-CDR2-FR3 -CDR3-FR4. The 3 CDRs are the binding regions of the sdAb to the antigen, while traditional antibodies require 6 CDRs to maintain binding to the antigen. In addition, the amino acid sequences of CDR1 and CDR3 of the sdAb were longer, which to some extent compensated for the loss of antigen-binding ability due to the deletion of the light chain. After antigenic stimulation, the production of sdAbs mainly depends on somatic hypermutation, so longer CDR sequences can also generate more antibody diversity. Antibody crystallography studies have shown that the longer CDR3 region endows sdAbs with greater antigen-binding capacity, enabling them to bind epitopes that traditional antibodies cannot. Therefore, Nb exhibits comparable or even stronger antigen-binding ability compared to monoclonal antibodies.
Nb另外一个优点是，通过序列比对发现，Nb与人源免疫球蛋白IgG的VH结构域高度同源，仅FR2和CDR3区域存在显著差异。有研究表明，Nb反复给药并不会引起体液和细胞免疫反应，但长期重复使用Nb药物是否引起其产生对机体的免疫原性仍有待研究。Another advantage of Nb is that it is found by sequence alignment that Nb is highly homologous to the VH domain of human immunoglobulin IgG, and only the FR2 and CDR3 regions have significant differences. Studies have shown that repeated administration of Nb does not cause humoral and cellular immune responses, but whether long-term repeated use of Nb drugs can cause its immunogenicity to the body remains to be studied.
中国专利201810972053 .8公开了一种嵌合抗原受体，所述CAR包含：BCMA抗原结合结构域、跨膜结构域、一个或多个共刺激结构域、以及胞内信号传导结构域；其中所述BCMA抗原结合结构域包含重链互补决定区1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)。包含所述的嵌合抗原受体的免疫细胞(如CAR-T细胞)对相关肿瘤杀伤力强、特异性强。但是，根据所述嵌合抗原受体制备的BCMA CAR-T细胞中，CAR表达效率较低，有待进一步提升。Chinese Patent 201810972053.8 discloses a chimeric antigen receptor, the CAR comprises: a BCMA antigen binding domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; wherein the The BCMA antigen binding domain comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3). Immune cells (such as CAR-T cells) containing the chimeric antigen receptor have strong killing power and specificity to related tumors. However, in the BCMA CAR-T cells prepared according to the chimeric antigen receptor, the CAR expression efficiency is low and needs to be further improved.
技术问题technical problem
本发明通过筛选特异性的针对BCMA抗原的单域抗体，并将其VHH通过基因重组的方法，构建一个针对BCMA抗原的嵌合抗原受体，使用慢病毒通过基因转导的方式将该重组基因***人T淋巴细胞的基因组，使其细胞膜表面表达特异性的针对BCMA抗原的嵌合抗原受体（BCMA CAR-T），通过体外扩增BCMA CAR-T后回输患者体内，达到特异性的针对表达BCMA抗原的肿瘤细胞（骨髓瘤细胞）的免疫细胞疗法，同时，避免来源于小鼠抗体的scFv的小鼠源性BCMA CAR-T细胞的容易产生抗小鼠抗体导致治疗失败的缺点。The present invention constructs a chimeric antigen receptor against BCMA antigen by screening the specific single domain antibody against BCMA antigen and recombining its VHH through the method of gene recombination. Insert into the genome of human T lymphocytes to express specific chimeric antigen receptors (BCMA antigens) on their cell membranes CAR-T), by expanding BCMA CAR-T in vitro and returning it to the patient, to achieve specific immune cell therapy against tumor cells (myeloma cells) expressing BCMA antigens, and at the same time, avoid scFv derived from mouse antibodies The disadvantage of mouse-derived BCMA CAR-T cells is that they are prone to produce anti-mouse antibodies and lead to treatment failure.
技术解决方案technical solutions
术语：the term:
BCMA（抗原）：B细胞成熟抗原；BCMA (antigen): B cell maturation antigen;
CAR：嵌合抗原受体；CAR: Chimeric Antigen Receptor;
CAR-T细胞：嵌合抗原受体T淋巴细胞。CAR-T cells: Chimeric antigen receptor T lymphocytes.
一方面，本发明提供了一种抗BCMA单域抗体。In one aspect, the present invention provides an anti-BCMA single domain antibody.
所述的抗BCMA单域抗体包含CDR1、CDR2和CDR3。The anti-BCMA single domain antibody comprises CDR1, CDR2 and CDR3.
所述的CDR1为SEQ ID NO：15-17中的一种或与SEQ ID NO：15-17有80%同一性的序列；所述的CDR2为SEQ ID NO：18-20中的一种或与SEQ ID NO：18-20有80%同一性的序列；所述的CDR3为SEQ ID NO：21-23中的一种或与SEQ ID NO：21-23有80%同一性的序列。The CDR1 is one of SEQ ID NOs: 15-17 or a sequence with 80% identity to SEQ ID NOs: 15-17; the CDR2 is one of SEQ ID NOs: 18-20 or A sequence with 80% identity to SEQ ID NO: 18-20; the CDR3 is one of SEQ ID NO: 21-23 or a sequence with 80% identity to SEQ ID NO: 21-23.
所述的抗BCMA单域抗体的氨基酸序列为SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的氨基酸序列。The amino acid sequence of the anti-BCMA single domain antibody is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID The amino acid sequence shown in NO:7.
另一方面，本发明提供了一种嵌合抗原受体。In another aspect, the present invention provides a chimeric antigen receptor.
所述的嵌合抗原受体包含前述抗BCMA单域抗体。The chimeric antigen receptor comprises the aforementioned anti-BCMA single domain antibody.
所述的嵌合抗原受体还包含跨膜结构域、一个或多个共刺激结构域、以及胞内信号传导结构域。The chimeric antigen receptor also comprises a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain.
所述的嵌合抗原受体的结构基因包含所述的抗BCMA单域抗体的编码基因，所述的抗BCMA单域抗体的编码基因的核苷酸序列为SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10所示的序列。The structural gene of the chimeric antigen receptor comprises the coding gene of the anti-BCMA single domain antibody, and the nucleotide sequence of the coding gene of the anti-BCMA single domain antibody is SEQ ID NO: 8, SEQ ID NO : 9 or the sequence shown in SEQ ID NO: 10.
所述的嵌合抗原受体的结构基因包含SEQ ID NO：11所示的核苷酸序列。The structural gene of the chimeric antigen receptor comprises the nucleotide sequence shown in SEQ ID NO: 11.
所述的嵌合抗原受体的结构基因的核苷酸序列为SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14所示的序列。The nucleotide sequence of the structural gene of the chimeric antigen receptor is the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
再一方面，本发明提供了一种生物材料。In yet another aspect, the present invention provides a biological material.
所述的生物材料为重组载体、重组细胞或以治疗或预防为目的生物制品。The biological material is a recombinant vector, a recombinant cell or a biological product for the purpose of treatment or prevention.
所述的生物材料包含前述抗BCMA单域抗体或编码所述抗BCMA单域抗体的基因。The biological material comprises the aforementioned anti-BCMA single-domain antibody or a gene encoding the anti-BCMA single-domain antibody.
所述的生物材料包含前述嵌合抗原受体或编码所述嵌合抗原受体的基因。The biological material comprises the aforementioned chimeric antigen receptor or a gene encoding the chimeric antigen receptor.
进一步地，所述的生物材料为重组载体，所述的载体选自DNA载体，RNA载体，质粒，慢病毒载体，腺病毒载体和逆转录病毒载体。Further, the biological material is a recombinant vector, and the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector.
优选地，所述的病毒载体为慢病毒载体。Preferably, the viral vector is a lentiviral vector.
在一些实施例中，所述的病毒载体为pCDH-EF1a-BCMA CAR病毒载体；所述的病毒载体为包括SEQ ID NO：12-14所示的任一种核苷酸序列的慢病毒载体pCDH-EF1a。In some embodiments, the viral vector is pCDH-EF1a-BCMA CAR viral vector; the viral vector is the lentiviral vector pCDH comprising any of the nucleotide sequences shown in SEQ ID NOs: 12-14 -EF1a.
进一步地，所述的生物材料为免疫细胞，所述的免疫细胞包括但不限于：T细胞、NK细胞、外周血单核细胞(PBMC)、造血干细胞、多能干细胞或胚胎干细胞，优选为人外周血T细胞。Further, the biological material is immune cells, and the immune cells include but are not limited to: T cells, NK cells, peripheral blood mononuclear cells (PBMC), hematopoietic stem cells, pluripotent stem cells or embryonic stem cells, preferably human peripheral cells blood T cells.
优选地，所述的细胞中每单个细胞所携带的嵌合抗原受体分子为3-4个。Preferably, each single cell in the cells carries 3-4 chimeric antigen receptor molecules.
又一方面，本发明提供了一种用于治疗骨髓瘤的医用配制品。In yet another aspect, the present invention provides a medical formulation for the treatment of myeloma.
所述的医用配制品包含前述抗BCMA单域抗体或编码所述抗BCMA单域抗体的基因。The medical preparation comprises the aforementioned anti-BCMA single-domain antibody or a gene encoding the anti-BCMA single-domain antibody.
所述的医用配制品包含前述嵌合抗原受体或编码所述嵌合抗原受体的基因。The medical preparation comprises the aforementioned chimeric antigen receptor or a gene encoding the chimeric antigen receptor.
所述的医用配制品包含前述生物材料。The medical formulation includes the aforementioned biological material.
优选地，所述的医用配制品包括表达BCMA嵌合抗原受体的T细胞。Preferably, the medical formulation comprises T cells expressing the BCMA chimeric antigen receptor.
进一步地，所述的表达BCMA嵌合抗原受体的T细胞在医用配制品中的细胞浓度为：1×108个阳性BCMA CAR-T/100mL。Further, the cell concentration of the BCMA chimeric antigen receptor-expressing T cells in the medical preparation is: 1×108 positive BCMA CAR-T/100mL.
所述的医用配制品的施用量为2×106个阳性BCMA CAR-T/kg体重。The administration amount of the medical formulation was 2×10 6 positive BCMA CAR-T/kg body weight.
所述的医用配制品剂型包括但不限于输液剂、注射剂。The dosage forms of the medical preparations include but are not limited to infusions and injections.
所述的医用配制品的施用方式包括但不限于：静脉注射，腹腔注射。The administration modes of the medical formulation include but are not limited to: intravenous injection, intraperitoneal injection.
又一方面，本发明提供了前述抗BCMA单域抗体、嵌合抗原受体或生物材料在制备具有治疗骨髓瘤的药物中的用途。In yet another aspect, the present invention provides the use of the aforementioned anti-BCMA single domain antibody, chimeric antigen receptor or biological material in the preparation of a medicament for treating myeloma.
优选地，所述的骨髓瘤为多发性骨髓瘤。Preferably, the myeloma is multiple myeloma.
有益效果beneficial effect
本发明旨在解决复发/耐药性多发性骨髓瘤患者目前临床上无有效治疗手段，应用BCMA CART技术特异性杀死骨髓中的骨髓瘤细胞，从而达到治疗复发/耐药性多发性骨髓瘤患者的目的。The invention aims to solve the problem that patients with relapsed/drug-resistant multiple myeloma currently have no effective clinical treatment means, and apply BCMA CART technology specifically kills myeloma cells in the bone marrow, thereby achieving the purpose of treating patients with relapsed/drug-resistant multiple myeloma.
本发明采用了与现有技术不同的，由BCMA单域抗体基因和2代CAR结构基因序列串联组成。本发明使用和已知BCMA抗体不同的序列，使用的抗体为单域抗体，较传统抗体来源的CAR与靶细胞结合力更强、杀伤效果更好、体内持续时间更久。The present invention adopts the tandem composition of the BCMA single-domain antibody gene and the second-generation CAR structural gene sequence, which is different from the prior art. The present invention uses different sequences from known BCMA antibodies, and the used antibody is a single domain antibody, which has stronger binding force to target cells, better killing effect, and longer in vivo duration than traditional antibody-derived CARs.
附图说明Description of drawings
图1为流式细胞术检测BCMA VHH重组抗体与高表达BCMA的重组细胞株CHO-BCMA的结合能力结果图。Fig. 1 is a graph showing the result of flow cytometry to detect the binding ability of BCMA VHH recombinant antibody to the recombinant cell line CHO-BCMA highly expressing BCMA.
图2为流式细胞术检测人源化抗体与BCMA的结合力的部分检测结果。Figure 2 is a partial detection result of flow cytometry to detect the binding ability of humanized antibody to BCMA.
图3为抗体亲和力检测结果。其中A代表筛选的抗BCMA单域抗体B-6-14；B、C分别代表人源化后的抗体NW2-1222-6-14-1、NW2-1222-6-14-2。Figure 3 shows the results of antibody affinity detection. A represents the screened anti-BCMA single-domain antibody B-6-14; B and C represent the humanized antibodies NW2-1222-6-14-1 and NW2-1222-6-14-2, respectively.
图4为CAR-T细胞膜表面标志物的流式细胞术检测结果，其中A为CD8 阳性率，B为CD4阳性率，C为BCMA 阴性对照，D为B-6-14 BCMA CAR-T 的CAR阳性率，E为NW2-1222-6-14-1 BCMA CAR-T 的CAR阳性率，F为NW2-1222-6-14-2 BCMA CAR-T 的CAR阳性率。Figure 4 shows the results of flow cytometry detection of CAR-T cell membrane surface markers, where A is the positive rate of CD8, B is the positive rate of CD4, C is the BCMA negative control, and D is the B-6-14 BCMA CAR positive rate of CAR-T, E is the CAR positive rate of NW2-1222-6-14-1 BCMA CAR-T, F is NW2-1222-6-14-2 CAR positive rate of BCMA CAR-T.
图5为RT-PCR的方法检测BCMA CAR-T细胞上CAR的拷贝数结果。其中A为标准曲线，B为CAR的拷贝数。Figure 5 shows the results of RT-PCR detection of the copy number of CAR on BCMA CAR-T cells. where A is the standard curve and B is the copy number of CAR.
图6为重组Nalm6-BCMA-GFP-LUC细胞株流式细胞术检测BCMA阳性率，其中A为FSC-SSC散点图，B为Nalm6阴性对照，C为重组Nalm6-BCMA- GFP-LUC细胞BCMA 阳性率(BCMA-GFP双阳)。该图中数据可以对结果进行判定。Figure 6 shows the positive rate of BCMA detected by flow cytometry in the recombinant Nalm6-BCMA-GFP-LUC cell line, where A is the FSC-SSC scatter plot, B is the Nalm6 negative control, and C is the recombinant Nalm6-BCMA-GFP-LUC cell BCMA Positive rate (BCMA-GFP double positive). The data in this figure can be used to judge the results.
图7为BCMA CAR-T细胞体外功能评价实验结果。其中A为对Nalm6-BCMA- GFP-LUC细胞的杀伤作用，B为IFN-γ释放检测结果。Figure 7 shows the experimental results of in vitro functional evaluation of BCMA CAR-T cells. where A is for Nalm6-BCMA- Killing effect of GFP-LUC cells, B is the detection result of IFN-γ release.
图8为BCMA CAR-T细胞体内功能评价中小鼠活体成像结果，小鼠活体图像中彩色信号越强（颜色越暗）表示小鼠体内肿瘤细胞数量较多。Figure 8 shows the in vivo imaging results of mice in the in vivo functional evaluation of BCMA CAR-T cells. The stronger the color signal (the darker the color) in the in vivo image of the mouse, the more tumor cells in the mouse.
图9为BCMA CAR-T细胞体内功能评价中小鼠体重变化结果。Figure 9 shows the results of changes in the body weight of mice in the in vivo functional evaluation of BCMA CAR-T cells.
图10为BCMA CAR-T细胞体内功能评价中小鼠生存曲线。Figure 10 is the mouse survival curve in the in vivo functional evaluation of BCMA CAR-T cells.
本发明的实施方式Embodiments of the present invention
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。The present invention will be described in further detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but are only used to illustrate the present invention. The experimental methods used in the following examples, unless otherwise specified, the experimental methods that do not specify specific conditions in the examples are usually in accordance with conventional conditions, and the materials, reagents, etc. used in the following examples, unless otherwise specified, are all Commercially available.
实施例1 抗BCMA单域抗体VHH基因获取Example 1 Acquisition of VHH gene of anti-BCMA single domain antibody
1、抗BCMA单域抗体文库构建：利用自制的BCMA抗原蛋白对成年健康的羊驼在颈背部皮下多点注射免疫。免疫时，将抗原和等体积Gerbu佐剂充分混匀后，多点注射羊驼皮下，共进行5次免疫，每次免疫时间间隔为14天；第三次免疫结束，通过颈静脉采集部分外周血分离血清后测定抗原免疫效价，结果见下表：1. Construction of anti-BCMA single-domain antibody library: The self-made BCMA antigen protein was used to immunize adult healthy alpacas subcutaneously on the back of the neck at multiple points. During immunization, the antigen and an equal volume of Gerbu adjuvant were thoroughly mixed, and the alpaca was injected subcutaneously at multiple points for a total of 5 immunizations, with an interval of 14 days for each immunization; after the third immunization, some peripheral tissues were collected through the jugular vein. The antigen immune titer was determined after blood separation of serum, and the results are shown in the following table:
Figure 577694dest_path_image001
Figure 577694dest_path_image001
抗体效价>1：160000。Antibody titer > 1:160000.
2、第5次免疫结束后采外周血150mL，分离PBMC后，提取总RNA，并反转录成cDNA，通过两轮PCR扩增羊驼重链抗体的可变区片段VHH。将VHH片段及抗体展示载体分别通过SfiI进行酶切后，采用T4 DNA连接酶将VHH片段克隆至展示载体中，然后通过电转化的方法将连接产物电转入毕赤酵母细胞中，并进行筛选，获得单域抗体酵母展示库。2. After the fifth immunization, 150 mL of peripheral blood was collected. After separating PBMC, total RNA was extracted and reverse transcribed into cDNA. The variable region fragment VHH of alpaca heavy chain antibody was amplified by two rounds of PCR. After the VHH fragment and the antibody display vector were digested by SfiI respectively, the VHH fragment was cloned into the display vector using T4 DNA ligase, and then the ligated product was electro-transformed into Pichia cells by electro-transformation, and screened. , to obtain a single-domain antibody yeast display library.
3、向获得的VHH酵母展示库培养基中，加入终浓度为0.5%的甲醇，诱导酵母展示单域抗体至酵母细胞壁上，加入生物素标记的BCMA重组蛋白，对酵母库中阳性的酵母克隆进行标记，采用Streptividin标记的磁珠分离酵母展示库中能够与目标蛋白BCMA结合的克隆，重复上述富集筛选流程2-3次，将能够与BCMA重组蛋白结合的克隆涂布到固体平板上，挑选单克隆后进行克隆鉴定。3. To the obtained VHH yeast display library medium, add methanol with a final concentration of 0.5% to induce yeast to display single-domain antibodies on the yeast cell wall, and add biotin-labeled BCMA recombinant protein to detect positive yeast clones in the yeast library. Labeling, using streptividin-labeled magnetic beads to isolate the clones in the yeast display library that can bind to the target protein BCMA, repeat the above enrichment screening process 2-3 times, and coat the clones that can bind to the BCMA recombinant protein on a solid plate. Clonal identification was performed after picking single clones.
4、将上述挑选的单克隆酵母菌扩增后，培养基中加入终浓度为0.5%的甲醇，进行诱导VHH表达，采用流式细胞术检测展示的VHH片段能否与BCMA蛋白结合，从流式细胞术验证为阳性的克隆中抽提基因组DNA、PCR扩增VHH片段、并进行Sanger测序，获得VHH抗体序列。4. After amplifying the above-selected monoclonal yeast, add methanol with a final concentration of 0.5% to the medium to induce VHH expression, and use flow cytometry to detect whether the displayed VHH fragments can bind to BCMA protein. Genomic DNA was extracted from the clones that were positive by cytometry, the VHH fragment was amplified by PCR, and Sanger sequencing was performed to obtain the VHH antibody sequence.
5、将VHH抗体序列分别进行基因合成，克隆至表达载体Lenti-hIgG1-Fc中。载体经测序验证无误后，大提质粒。5. The VHH antibody sequences were separately synthesized and cloned into the expression vector Lenti-hlgG1-Fc. After the vector was verified by sequencing, the plasmid was extracted.
6、取一个6孔板接种293T后，用Lenti-VHH-hIgG1-Fc表达载体瞬时转染，3天后收集培养基上清，离心后将上清通过0.45μm的滤膜，收集得到BCMA VHH 重组抗体。6. After inoculating a 6-well plate with 293T, transiently transfected with the Lenti-VHH-hIgG1-Fc expression vector, collect the supernatant of the medium after 3 days, and pass the supernatant through a 0.45 μm filter after centrifugation to collect the BCMA VHH recombinant antibody.
7、使用流式细胞术检测 BCMA VHH 重组抗体与高表达BCMA的重组细胞株CHO-BCMA的结合能力：将对照细胞CHO和CHO-BCMA细胞各分为若干份，每份细胞的数量为5×105个细胞；将100μL BCMA VHH 重组抗体分别与靶细胞和对照细胞混匀后，4度孵育1小时；PBS洗涤细胞3次后加入100μL PBS 重悬细胞后加入1 μL PE标记的抗人IgG抗体，充分混匀后，4度避光孵育30分钟；PBS洗涤细胞3次后上机检测，结果见图1，其中A-D为CHO-BCMA细胞，E-H为CHO细胞。7. Use flow cytometry to detect the binding ability of BCMA VHH recombinant antibody to the recombinant cell line CHO-BCMA with high expression of BCMA: divide the control cells CHO and CHO-BCMA cells into several parts, and the number of cells in each part is 5× 105 cells; 100 μL BCMA VHH recombinant antibody was mixed with target cells and control cells, respectively, and incubated at 4 degrees for 1 hour; PBS was washed 3 times, then 100 μL PBS was added to resuspend the cells, and 1 μL PE-labeled anti-human IgG antibody was added After fully mixing, incubate at 4 degrees for 30 minutes in the dark; after washing the cells 3 times with PBS, the cells were tested on the machine. The results are shown in Figure 1, where AD is CHO-BCMA cells, and EH is CHO cells.
8、BCMA VHH人源化设计8. BCMA VHH humanized design
本申请筛选了多种抗BCMA单域抗体，在这些抗BCMA单域抗体中，CDR1区氨基酸序列选自SEQ ID NO：15-17，CDR2区氨基酸序列选自SEQ ID NO：18-20，CDR3区氨基酸序列选自SEQ ID NO：21-23。This application has screened a variety of anti-BCMA single-domain antibodies. Among these anti-BCMA single-domain antibodies, the amino acid sequence of the CDR1 region is selected from SEQ ID NO: 15-17, the amino acid sequence of the CDR2 region is selected from SEQ ID NO: 18-20, and the CDR3 region is selected from SEQ ID NO: 18-20. The amino acid sequences of the regions are selected from SEQ ID NOs: 21-23.
本实施例以所筛选的抗BCMA单域抗体B-6-14为示例，其氨基酸序列如SEQ IDNO：1所示。This example takes the screened anti-BCMA single-domain antibody B-6-14 as an example, and its amino acid sequence is shown in SEQ ID NO: 1.
根据SEQ ID NO：1，采用表面氨基酸替换的设计进行人源化设计，人源化抗体序列如SEQ ID NO：2-7。According to SEQ ID NO: 1, the design of surface amino acid substitution is used to carry out humanization design, and the humanized antibody sequences are as shown in SEQ ID NO: 2-7.
将上述6条人源化抗体片段合成、克隆至表达载体Lenti-hIgG1-Fc中。载体经测序验证无误后，大提质粒。The above six humanized antibody fragments were synthesized and cloned into the expression vector Lenti-hlgG1-Fc. After the vector was verified by sequencing, the plasmid was extracted.
9、同步骤6-7，瞬转293T后，取上清分别与CHO和CHO-BCMA细胞孵育后流式细胞术检测人源化抗体与BCMA的结合力，图2展示部分结果（NW2-1222-6-14-1、NW2-1222-6-14-2）。9. The same as steps 6-7, after transiently transfecting 293T, the supernatant was incubated with CHO and CHO-BCMA cells, respectively, and the binding capacity of the humanized antibody to BCMA was detected by flow cytometry. Figure 2 shows part of the results (NW2-1222 -6-14-1, NW2-1222-6-14-2).
10、结果显示，人源化序列均为为高亲和力BCMA VHH抗体序列，本实施例中，选择了NW2-1222-6-14-1、NW2-1222-6-14-2继续进行后续实验。10. The results show that the humanized sequences are all high-affinity BCMA VHH antibody sequences. In this example, NW2-1222-6-14-1 and NW2-1222-6-14-2 were selected for subsequent experiments.
11、人源化抗体亲和力检测：将BCMA重组蛋白使用10mM Acetate缓冲液固定在CM5芯片上，分别以上述人源化抗体NW2-1222-6-14-1、NW2-1222-6-14-2及原始序列抗体作为流动相，检测人源化前后抗体与靶蛋白BCMA的结合能力，结果见图3，结果显示：抗体B-6-14的亲和力KD=1.985×10-10M；人源化抗体B-6-14-1的KD==1.818×10-10M；人源化抗体B-6-14-2的KD=1.760×10-10M。11. Humanized antibody affinity detection: The BCMA recombinant protein was immobilized on the CM5 chip using 10 mM Acetate buffer, and the humanized antibodies NW2-1222-6-14-1 and NW2-1222-6-14-2 were used for the above-mentioned humanized antibodies respectively. and the original sequence antibody as the mobile phase to detect the binding ability of the antibody to the target protein BCMA before and after humanization. KD=1.818×10-10M of B-6-14-1; KD=1.760×10-10M of humanized antibody B-6-14-2.
实施例2 嵌合抗原受体基因载体构建Example 2 Construction of chimeric antigen receptor gene vector
由华大基因公司合成2段基因，一段为SEQ ID NO：1-7所示的任一条氨基酸序列对应的核苷酸序列，本实施例选择了其中三条进行合成，如SEQ ID NO：8-10所示。另外一段为设计的2代CAR结构基因，包括CD8a铰链区、CD8跨膜结构域、4-1BB共刺激结构域+CD3ζ胞内信号传导结构域，编码包含这些结构域的2代CAR结构基因的核苷酸序列如SEQ ID NO：11所示。Two sections of genes were synthesized by BGI, and one section was the nucleotide sequence corresponding to any amino acid sequence shown in SEQ ID NO: 1-7. In this example, three of them were selected for synthesis, as shown in SEQ ID NO: 1-7. ID NO: 8-10. Another segment is the designed second-generation CAR structural gene, including CD8a hinge region, CD8 transmembrane domain, 4-1BB costimulatory domain + CD3ζ intracellular signaling domain, encoding the second-generation CAR structural gene containing these domains. The nucleotide sequence is shown in SEQ ID NO:11.
分别得到两段合成基因后，进行BCMA-CAR的载体构建，首先通过PCR、酶切、酶连得到SEQ ID NO：12-14。After obtaining two synthetic genes respectively, the vector construction of BCMA-CAR was carried out. First, SEQ ID NOs: 12-14 were obtained by PCR, enzyme digestion and enzyme ligation.
将慢病毒载体pCDH-EF1a和SEQ ID NO：12-14，分别双酶切、T4 DNA连接酶连接后，转化感受态细胞、挑单克隆，菌液测序无误后，大提质粒，分别得到序列正确的三个慢病毒载体pCDH-EF1a-BCMA 1#，2#，3#，其中1#为抗BCMA单域抗体原始序列，2#，3#为两个人源化抗BCMA单域抗体序列。The lentiviral vector pCDH-EF1a and SEQ ID NOs: 12-14 were double digested and ligated with T4 DNA ligase, respectively, and then transformed into competent cells and single clones were picked. After the bacterial liquid was sequenced correctly, the plasmids were extracted and sequences were obtained. Correct three lentiviral vectors pCDH-EF1a-BCMA 1#, 2#, 3#, of which 1# is the original sequence of anti-BCMA single-domain antibody, and 2# and 3# are the sequences of two humanized anti-BCMA single-domain antibodies.
实施例3 BCMA CAR慢病毒制备Example 3 Preparation of BCMA CAR lentivirus
慢病毒包装质粒混合物（包括PSPAX2和VSVG）分别与pCDH-EF1a-BCMA 1#，2#，3#按预优化的比例混合后，加入助转染试剂，室温下孵育15分钟。然后将转染混合物逐滴加入293T细胞。1-3天后收集培养基上清得到慢病毒粗液。在4℃下500g离心10min后收集上清液。然后超离心以进行慢病毒浓缩。在超离心结束后，小心弃去上清液，并用预冷的DPBS小心重悬慢病毒颗粒。将病毒分装后储存在-80℃下。基于RT-PCR方法和流式细胞术测定慢病毒的物理滴度和感染滴度。The lentiviral packaging plasmid mixture (including PSPAX2 and VSVG) was mixed with pCDH-EF1a-BCMA 1#, 2#, and 3# according to the pre-optimized ratio, and then the co-transfection reagent was added, and incubated at room temperature for 15 minutes. The transfection mixture was then added dropwise to the 293T cells. After 1-3 days, the medium supernatant was collected to obtain the crude lentivirus solution. The supernatant was collected after centrifugation at 500 g for 10 min at 4°C. Ultracentrifugation was then performed for lentiviral concentration. After ultracentrifugation, the supernatant was carefully discarded and the lentiviral particles were carefully resuspended in pre-chilled DPBS. The virus was aliquoted and stored at -80°C. Physical and infectious titers of lentivirus were determined based on RT-PCR method and flow cytometry.
实施例4 BCMA CAR-T细胞的制备Example 4 Preparation of BCMA CAR-T cells
1、PBMC制备：将人静脉血30mL在800g，20℃离心30分钟，离心结束后，将上层血浆转移入另外一个离心管。在50mL离心管中加入15mL人淋巴细胞分离液（天津灏洋生物），用电动移液枪将离心后的血液缓慢加入淋巴细胞分离液上方，250g，20℃，离心10分钟。弃掉上层液体，用10 ml PBS重悬计数，用于T细胞纯化。1. Preparation of PBMC: Centrifuge 30 mL of human venous blood at 800 g for 30 minutes at 20°C. After the centrifugation, transfer the upper plasma into another centrifuge tube. Add 15mL of human lymphocyte separation solution (Tianjin Haoyang Biotechnology) into a 50mL centrifuge tube, and slowly add the centrifuged blood above the lymphocyte separation solution with an electric pipette, centrifuge at 250g, 20°C for 10 minutes. Discard the supernatant and resuspend in 10 ml of PBS for T cell purification.
2、T细胞纯化：根据以下所述的制造商的方案，使用Miltenyi Pan T细胞分离试剂盒（目录号130-096-535）从PBMC纯化人T细胞。PBMC计数后，300g，20℃，离心10分钟。倒掉上清液，将细胞沉淀按每107/40μL缓冲液重悬于缓冲液中后，加入10μL/107 Pan T细胞生物素-抗体混合物，充分混匀后4℃孵育5分钟。然后加入30μL缓冲液，和20μL Pan T细胞微珠粒混合物。充分混匀后在4℃孵育10分钟。孵育结束后用1mL缓冲液，洗涤PBMC，250g，4℃离心10分钟。离心后细胞用500μL的缓冲液重悬，通过LS柱，细胞悬液随重力流出，收集流出物（即T细胞组分），继续通过缓冲剂洗涤LS柱并收集流出液。然后离心富集T细胞并重悬于淋巴细胞培养基+1000IU/mL IL-2中。2. T cell purification: Miltenyi was used according to the manufacturer's protocol as described below Pan T Cell Isolation Kit (Cat. No. 130-096-535) purifies human T cells from PBMC. After PBMC counting, centrifuge at 300g for 10 minutes at 20°C. Pour off the supernatant, resuspend the cell pellet in buffer per 107/40μL buffer, add 10μL/107 Pan T cell biotin-antibody mixture, mix well and incubate at 4°C for 5 minutes. Then add 30 μL of buffer, and 20 μL of Pan T cell bead mix. Mix well and incubate at 4°C for 10 minutes. After the incubation, PBMC were washed with 1 mL of buffer, centrifuged at 250 g for 10 minutes at 4°C. After centrifugation, cells were resuspended in 500 μL of buffer and passed through the LS column. The cell suspension flowed out with gravity. The effluent (ie, the T cell fraction) was collected. Continue to wash the LS column with buffer and collect the effluent. T cells were then enriched by centrifugation and resuspended in lymphocyte medium + 1000 IU/mL IL-2.
3、CAR-T制备：用人T细胞活化/扩增试剂盒（Miltenyi#130-091-441）预活化所制备的T细胞24-96小时后，进行慢病毒感染。3. CAR-T preparation: After pre-activating the prepared T cells with a human T cell activation/expansion kit (Miltenyi #130-091-441) for 24-96 hours, lentivirus infection was performed.
B-6-14 BCMA CAR-T细胞：pCDH-EF1a-BCMA 1#慢病毒感染；B-6-14 BCMA CAR-T cells: pCDH-EF1a-BCMA 1# lentivirus infection;
B-6-14-2 BCMA CAR-T细胞：pCDH-EF1a-BCMA 2#慢病毒感染；B-6-14-2 BCMA CAR-T cells: pCDH-EF1a-BCMA 2# lentivirus infection;
B-6-14-3 BCMA CAR-T细胞：pCDH-EF1a-BCMA 3#慢病毒感染。B-6-14-3 BCMA CAR-T cells: pCDH-EF1a-BCMA 3# lentivirus infection.
在活化后的T细胞悬液中加入10μg/mL聚凝胺后，按MOI=10 加入慢病毒，1200g，32℃，离心1小时。离心结束后将转导的T细胞放入细胞培养箱，每天补加适量T细胞培养基。After adding 10 μg/mL polybrene to the activated T cell suspension, lentivirus was added at MOI=10, 1200 g, 32° C., and centrifuged for 1 hour. After centrifugation, the transduced T cells were placed in a cell culture incubator, and an appropriate amount of T cell culture medium was supplemented every day.
感染7天后，将BCMA Fc蛋白与BCMA CAR-T孵育后，用流式细胞仪检测T细胞膜表面CAR的表达率。Seven days after infection, after BCMA Fc protein was incubated with BCMA CAR-T, the expression rate of CAR on the surface of T cell membrane was detected by flow cytometry.
检测结果见图4。图4的结果显示，实施例4制备的BCMA CAR-T细胞中，三种序列的CAR表达效率均在90％以上。同时将三种BCMA CAR-T 提取DNA后用RT-PCR的方法检测BCMA CAR-T细胞上CAR的拷贝数，结果见图5，相关数据如下：The test results are shown in Figure 4. The results in Figure 4 show that in the BCMA CAR-T cells prepared in Example 4, the CAR expression efficiencies of the three sequences are all above 90%. At the same time, the DNA of the three BCMA CAR-T cells was extracted by RT-PCR to detect the copy number of CAR on the BCMA CAR-T cells. The results are shown in Figure 5. The relevant data are as follows:
Figure 25993dest_path_image002
Figure 25993dest_path_image002
图5的结果显示，实施例4制备的BCMA CAR-T细胞中，每个BCMA CAR-T细胞上均带3-4个CAR分子。The results in Figure 5 show that in the BCMA CAR-T cells prepared in Example 4, each BCMA CAR-T cells all carry 3-4 CAR molecules.
实施例5 BCMA CAR-T细胞功能评价Example 5 BCMA CAR-T cell function evaluation
对实施例4制备的BCMA CAR-T细胞进行功能评价实验如下：The functional evaluation experiment of the BCMA CAR-T cells prepared in Example 4 is as follows:
1、体外功能评价1. In vitro functional evaluation
重组Nalm6-BCMA-GFP-LUC细胞株（自爱康得生物医学技术（苏州）有限公司获赠）流式细胞术检测BCMA阳性率，结果见图6。结果显示，BCMA分子在Nalm6-BCMA-LUC重组细胞株中高效表达，在Nalm6细胞（购自广州赛库生物技术有限公司）中不表达，可分别作为CAR-T杀伤实验的靶细胞和对照细胞。Recombinant Nalm6-BCMA-GFP-LUC cell line (gifted from Elken Biomedical Technology (Suzhou) Co., Ltd.) was used to detect the positive rate of BCMA by flow cytometry. The results are shown in Figure 6. The results show that BCMA molecules are highly expressed in Nalm6-BCMA-LUC recombinant cell line, but not expressed in Nalm6 cells (purchased from Guangzhou Saiku Biotechnology Co., Ltd.), which can be used as target cells and control cells for CAR-T killing experiments respectively. .
将实施例4制备的BCMA CAR-T细胞和Nalm6-BCMA-GFP-LUC或Nalm6细胞按照效靶比10:1、5:1、2.5:1,1.25:1设置四个梯度，共培养20小时后，使用发光荧光素酶测定试剂盒(Promega#E6110)，检测孔中的剩余荧光素酶活性。用下列公式计算特异性细胞毒性：特异性细胞毒性％＝100％×(1-(RLU样品-RLU最小)/(RLU最大-RLU最小))。同时，取上清用IFN-γ ELISA试剂盒（达科为）检测IFN-γ，结果见图7，其中对Nalm6-BCMA-LUC细胞的杀伤作用参见图7中的A；IFN-γ释放见图7中的B。结果说明，实施例4制备的BCMA CAR-T细胞可以特异高效地杀伤BCMA阳性的细胞，两个人源化BCMA CAR-T细胞杀伤活性不低于甚至略高于原始BCMA 单域抗体序列CAR-T细胞。The BCMA CAR-T cells prepared in Example 4 and Nalm6-BCMA-GFP-LUC or Nalm6 cells were set up in four gradients according to the effect-target ratio of 10:1, 5:1, 2.5:1, 1.25:1, and co-cultured for 20 hours Afterwards, the remaining luciferase activity in the wells was detected using a luminescent luciferase assay kit (Promega #E6110). Specific cytotoxicity was calculated using the following formula: % specific cytotoxicity = 100% x (1-(RLU sample-RLU min)/(RLU max-RLU min)). At the same time, the supernatant was taken to detect IFN-γ with IFN-γ ELISA kit (Daktronics), and the results are shown in Figure 7. The killing effect on Nalm6-BCMA-LUC cells is shown in Figure 7 A; the release of IFN-γ is shown in Figure 7. B in Figure 7. The results show that the BCMA CAR-T cells prepared in Example 4 can kill BCMA-positive cells specifically and efficiently, and the killing activity of the two humanized BCMA CAR-T cells is not lower than or even slightly higher than the original BCMA single-domain antibody sequence CAR-T cell.
2、体内功能评价2. In vivo function evaluation
通过小鼠尾静脉输注肿瘤细胞Nalm6-BCMA-Luciferase细胞1×106/200μL，建立小鼠肿瘤模型，将小鼠随机分为4组，分别于建模后5天，11天，17天以及25天进行活体成像观察，并于建模后第5天分别通过尾静脉输注对照T细胞(分离纯化自同一外周血PBMC样本)及两个人源化单域抗体序列BCMA CAR-T细胞（B-6-14-1 BCMA CAR-T， B-6-14-2 BCMA CAR-T），并用小鼠源性BCMA CAR-T 作为阳性对照，各细胞注射用量如下：Tumor cell Nalm6-BCMA-Luciferase cells 1×106/200μL were infused through the tail vein of mice to establish a mouse tumor model. The mice were randomly divided into 4 groups, 5 days, 11 days, 17 days and In vivo imaging observation was performed on 25 days, and control T cells (isolated and purified from the same peripheral blood PBMC sample) and two humanized single-domain antibody sequence BCMA CAR-T cells (B) were infused through the tail vein on the 5th day after modeling. -6-14-1 BCMA CAR-T, B-6-14-2 BCMA CAR-T), and mouse-derived BCMA CAR-T was used as a positive control, and the injection dose of each cell was as follows:
B-6-14-1 BCMA CAR-T细胞：1×107个；B-6-14-1 BCMA CAR-T cells: 1×107;
B-6-14-2 BCMA CAR-T细胞：1×107个；B-6-14-2 BCMA CAR-T cells: 1×107;
对照T细胞：1×107个；Control T cells: 1×107;
小鼠源性BCMA CAR-T：1×107个。Mouse-derived BCMA CAR-T: 1×107.
结果见图8-10，结果显示，与T细胞对照组相比，人源化BCMA CAR-T细胞、小鼠源性BCMA CAR-T细胞对肿瘤细胞均有显著地杀伤效果，肿瘤细胞注射第25天，小鼠源性BCMA CAR-T组的肿瘤细胞开始出现复发，而人源化B-6-14-1/B-6-14-2 BCMA CAR-T仍具有显著的杀伤效果（图8）。输注对照T细胞的小鼠从第20天开始体重持续下降，而输注CAR-T细胞的小鼠体重维持，并有轻微上升，表明CAR-T细胞抑制了小鼠体内的肿瘤细胞增殖（图9）。小鼠生存分析显示对照组小鼠从建模后第25天左右开始有死亡小鼠，而CAR-T处理组小鼠状态正常，进行持续观察至60+天，小鼠源性BCMA CAR-T注射小鼠全部死亡，而人源化单域抗体序列BCMA CAR-T注射小鼠大部分存活，表明单域抗体序列BCMA CAR-T 体内持续时间更久，效果更好（图10）。The results are shown in Figure 8-10. The results show that compared with the T cell control group, humanized BCMA CAR-T cells, mouse-derived BCMA CAR-T cells have a significant killing effect on tumor cells. On the 25th day of tumor cell injection, tumor cells in the mouse-derived BCMA CAR-T group began to recur, while humanized B-6-14-1/B -6-14-2 BCMA CAR-T still had a significant killing effect (Fig. 8). The weight of mice infused with control T cells continued to decrease from day 20, while the weight of mice infused with CAR-T cells maintained and increased slightly, indicating that CAR-T cells inhibited tumor cell proliferation in mice ( Figure 9). The mouse survival analysis showed that the mice in the control group had dead mice from about 25 days after modeling, while the mice in the CAR-T treatment group were in a normal state and continued to be observed for 60+ days. The mouse-derived BCMA All the mice injected with CAR-T died, while most of the mice injected with the humanized single-domain antibody sequence BCMA CAR-T survived, indicating that the single-domain antibody sequence BCMA CAR-T lasted longer in vivo and had better effects (Figure 10). .

Claims (10)

  1. 一种抗BCMA单域抗体，其特征在于，所述的抗BCMA单域抗体包含CDR1、CDR2和CDR3；所述的CDR1为SEQ ID NO：15-17中的一种或与SEQ ID NO：15-17有80%同一性的序列；所述的CDR2为SEQ ID NO：18-20中的一种或与SEQ ID NO：18-20有80%同一性的序列；所述的CDR3为SEQ ID NO：21-23中的一种或与SEQ ID NO：21-23有80%同一性的序列。 An anti-BCMA single-domain antibody, characterized in that the anti-BCMA single-domain antibody comprises CDR1, CDR2 and CDR3; the CDR1 is one of SEQ ID NOs: 15-17 or the same as SEQ ID NO: 15 -17 is a sequence with 80% identity; said CDR2 is one of SEQ ID NO: 18-20 or a sequence with 80% identity with SEQ ID NO: 18-20; said CDR3 is SEQ ID One of NO: 21-23 or a sequence 80% identical to SEQ ID NO: 21-23.
  2. 根据权利要求1所述的抗BCMA单域抗体，其特征在于，所述的抗BCMA单域抗体的氨基酸序列为SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的氨基酸序列。 The anti-BCMA single-domain antibody according to claim 1, wherein the amino acid sequence of the anti-BCMA single-domain antibody is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID The amino acid sequence shown in NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
  3. 一种嵌合抗原受体，其特征在于，所述的嵌合抗原受体包含权利要求1或2所述的抗BCMA单域抗体。 A chimeric antigen receptor, characterized in that the chimeric antigen receptor comprises the anti-BCMA single domain antibody of claim 1 or 2.
  4. 根据权利要求3所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体还包含跨膜结构域、一个或多个共刺激结构域、以及胞内信号传导结构域。 The chimeric antigen receptor according to claim 3, wherein the chimeric antigen receptor further comprises a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain.
  5. 根据权利要求3或4所述的嵌合抗原受体，其特征在于，嵌合抗原受体的结构基因包含所述的抗BCMA单域抗体的编码基因，所述的抗BCMA单域抗体的编码基因的核苷酸序列为SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10所示的序列。 The chimeric antigen receptor according to claim 3 or 4, wherein the structural gene of the chimeric antigen receptor comprises the coding gene of the anti-BCMA single domain antibody, the coding of the anti-BCMA single domain antibody The nucleotide sequence of the gene is SEQ ID NO: 8, SEQ ID NO:9 or the sequence shown in SEQ ID NO:10.
  6. 根据权利要求5所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体的结构基因包含SEQ ID NO：11所示的核苷酸序列。 The chimeric antigen receptor according to claim 5, wherein the structural gene of the chimeric antigen receptor comprises the nucleotide sequence shown in SEQ ID NO: 11.
  7. 根据权利要求6所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体的结构基因的核苷酸序列为SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14所示的序列。 The chimeric antigen receptor according to claim 6, wherein the nucleotide sequence of the structural gene of the chimeric antigen receptor is SEQ ID NO: 12, SEQ ID NO: 13 or the sequence shown in SEQ ID NO: 14.
  8. 一种生物材料，其特征在于，所述的生物材料为重组载体、重组细胞或以治疗或预防为目的生物制品；所述的生物材料包含权利要求1或2所述的抗BCMA单域抗体或编码所述抗BCMA单域抗体的基因，或所述的生物材料包含权利要求3-7任一项所述的嵌合抗原受体或编码所述嵌合抗原受体的基因。 A biological material, characterized in that the biological material is a recombinant carrier, a recombinant cell or a biological product for the purpose of treatment or prevention; the biological material comprises the anti-BCMA single domain antibody according to claim 1 or 2 or The gene encoding the anti-BCMA single domain antibody, or the biological material comprises the chimeric antigen receptor described in any one of claims 3-7 or the gene encoding the chimeric antigen receptor.
  9. 一种用于治疗多发性骨髓瘤的医用配制品，其特征在于，所述的医用配制品包含权利要求1或2所述的抗BCMA单域抗体或编码所述抗BCMA单域抗体的基因、或权利要求3-7任一项所述的嵌合抗原受体或编码所述嵌合抗原受体的基因、或权利要求8所述的生物材料。 A medical preparation for the treatment of multiple myeloma, characterized in that the medical preparation comprises the anti-BCMA single-domain antibody of claim 1 or 2 or the gene encoding the anti-BCMA single-domain antibody, Or the chimeric antigen receptor of any one of claims 3-7 or the gene encoding the chimeric antigen receptor, or the biological material of claim 8.
  10. 权利要求1或2所述的抗BCMA单域抗体、权利要求3-7任一项所述的嵌合抗原受体或权利要求8所述的生物材料在制备具有治疗骨髓瘤的药物中的用途。 Use of the anti-BCMA single-domain antibody according to claim 1 or 2, the chimeric antigen receptor according to any one of claims 3-7, or the biological material according to claim 8 in the preparation of a drug for treating myeloma .
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