CN111909271B

CN111909271B - BCMA chimeric antigen receptor based on single domain antibody and application thereof

Info

Publication number: CN111909271B
Application number: CN202010806805.0A
Authority: CN
Inventors: 姜舒; 王冰; 彭方理; 张芸
Original assignee: Shenzhen Wingor Bio Technology Co ltd
Current assignee: Shenzhen Wingor Bio Technology Co ltd
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2021-03-23
Anticipated expiration: 2040-08-12
Also published as: WO2022033057A1; CN111909271A

Abstract

The invention provides a BCMA chimeric antigen receptor based on a single domain antibody, wherein a BCMA antigen binding structural domain of the BCMA chimeric antigen receptor comprises an anti-BCMA single domain antibody, and the anti-BCMA single domain antibody comprises CDR1, CDR2 and CDR 3. Wherein CDR1 is SEQ ID NO: 15-17 or any combination of SEQ ID NOs: 15-17, CDR2 is SEQ ID NO: 18-20 or a variant of SEQ ID NO: 18-20, CDR3 is SEQ ID NO: 21-23 or any combination of SEQ ID NOs: 21-23 sequences with 80% identity. Compared with the traditional antibody-derived BCMA CAR, the antibody-derived BCMA CAR has stronger binding force with target cells, better killing effect and longer in-vivo duration.

Description

BCMA chimeric antigen receptor based on single domain antibody and application thereof

Technical Field

The invention belongs to the field of immune cell therapy, and particularly relates to a BCMA chimeric antigen receptor based on a single domain antibody and application thereof.

Background

Multiple Myeloma (MM) is a malignant proliferative disease of plasma cells, with abnormal proliferation of clonal plasma cells in the bone marrow and secretion of monoclonal immunoglobulins or fragments thereof (M protein) and resulting in damage to associated organs or tissues (ROTI). The common clinical manifestations are bone pain, anemia, renal insufficiency, infection, etc. Statistics show that nearly 86000 patients will be diagnosed with myeloma each year, while about 63000 patients die each year from disease-related complications. In recent years, although the application of new proteasome inhibitors bortezomib, immunoregulatory drugs thalidomide and lenalidomide and the like improves the remission rate and disease-free survival time of MM patients, the total survival time is not obviously different from that of the traditional treatment. After MM is relieved by chemotherapy, most patients relapse, and the patients are resistant to the original sensitive drugs, so that the patients cannot be relieved again by increasing the dosage, and side effects such as bone marrow suppression, secondary infection, liver function damage and the like are easily caused. No effective treatment means is provided clinically for patients with relapsed/drug-resistant multiple myeloma at present.

Chimeric Antigen Receptor T cells (CAR-T) are prepared by expressing an artificially synthesized CAR molecule (Chimeric Antigen Receptor) on a T cell membrane by means of genetic modification, so that the T cells can recognize and kill tumor cells in an Antigen-antibody binding manner. CAR molecules include an extracellular binding region, which is a single chain antibody (scFv) derived from a monoclonal antibody that specifically recognizes a target antigen, a hinge region, a transmembrane region, and an intracellular signal segment. This technique has several advantages, for example, the recognition of TAA (tumor associated antigen) on the tumor surface by CAR molecules is performed in an MHC (major histocompatibility complex) -independent manner, so CAR-T cells can overcome the immune attack of tumor cells by down-regulating MHC molecules, and CAR recognition of tumor antigens is not MHC-restricted, and 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, glycolipids, such that the CAR greatly increases the range of tumor surface markers that a T cell can recognize relative to tcr (T cell receptor) which can only recognize MHC-peptidic fragments. Like antigen-antibody reactions, the binding of the scFv segment of CAR-T to an antigen is also influenced by factors such as the affinity of the binding domain, the structure of the epitope, the number of tumor cell surface antigens, pH, temperature, ionic strength, etc.

Chinese patent 201580050638.9 discloses a chimeric antigen receptor comprising: an extracellular domain; a transmembrane domain; one or more intracellular co-stimulatory signaling domains; and a primary signaling domain, the extracellular domain comprising a humanized anti-BCMA antibody or antigen-binding fragment thereof that binds one or more epitopes of a human BCMA (B cell maturation antigen) polypeptide. The gene engineering technology is utilized to obtain the coding gene, the gene segment is inserted into a lentivirus expression vector, and the lentivirus is packaged to infect human T cells, so that the T cells express the chimeric antigen receptor. Such chimeric antigen receptor T cells can be used in the treatment of B cell-associated malignancies.

Single domain antibodies (sdabs), also known as nanobodies (Nb), 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 heavy chain-only antibody has a single heavy chain variable domain (VHH), and this antibody contains only one heavy chain variable domain VHH and two conventional CH2 and CH3 constant domain regions, and is only half the molecular weight of a conventional antibody. VHH can have high affinity for antigen without the help of light chain, is one of the smallest functional antigen binding fragments, and has a molecular weight of about 15 kD. VHH consists of 3 antigen Complementary Determining Regions (CDRs) and 4 Framework Regions (FRs), typically arranged from N-terminus to C-terminus in the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. 3 CDRs are the binding region of the sdAb to the antigen, whereas traditional antibodies require 6 CDRs to maintain binding to the antigen. In addition, the amino acid sequences of CDR1 and CDR3 of sdabs are longer, to some extent complementing the loss of antigen binding capacity due to light chain deletion. After receiving antigenic stimulation, sdAb production is mainly dependent on somatic hypermutation, so longer CDR sequences can also produce more antibody diversity. Antibody crystallography studies have shown that longer CDR3 regions confer greater antigen binding ability to sdabs, thereby enabling binding to epitopes that are inaccessible to traditional antibodies. Thus, Nb exhibits comparable or even stronger antigen binding capacity compared to monoclonal antibodies.

An additional advantage of Nb is that it is found to be highly homologous to the VH domain of human immunoglobulin IgG by sequence alignment, with only significant differences in the FR2 and CDR3 regions. Research shows that the repeated administration of Nb does not cause humoral and cellular immune responses, but the immunogenicity of the body caused by the repeated use of Nb drugs for a long time is still needed to be researched.

Chinese patent 201810972053.8 discloses a chimeric antigen receptor, the CAR comprising: a BCMA antigen binding domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain; wherein 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(HCDR 3). Immune cells (such as CAR-T cells) containing the chimeric antigen receptor have strong lethality and specificity on related tumors. However, the efficiency of CAR expression in BCMA CAR-T cells prepared from the chimeric antigen receptor is low and needs to be further improved.

Disclosure of Invention

The invention constructs a chimeric antigen receptor aiming at the BCMA antigen by screening a specific single domain antibody aiming at the BCMA antigen and carrying out gene recombination on VHH of the antibody, inserts a recombinant gene into the genome of a human T lymphocyte by using lentivirus in a gene transduction mode, leads the surface of the cell membrane of the chimeric antigen receptor (BCMA CAR-T) to express the specific BCMA antigen, and leads the cell membrane to be transfused into a patient after the BCMA CAR-T is amplified in vitro, thereby achieving the specific immune cell therapy aiming at tumor cells (myeloma cells) expressing the BCMA antigen, and simultaneously avoiding the defect that the therapy fails because the mouse antibody is easily generated by mouse-derived BCMA CAR-T cells of scFv derived from a mouse antibody.

The terms:

BCMA (antigen): b cell maturation antigen;

CAR: a chimeric antigen receptor;

CAR-T cells: chimeric antigen receptor T lymphocytes.

In one aspect, the invention provides an anti-BCMA single domain antibody.

The anti-BCMA single domain antibody comprises CDR1, CDR2 and CDR 3.

The CDR1 is SEQ ID NO: 15-17 or any combination of SEQ ID NOs: 15-17 sequences with 80% identity; the CDR2 is SEQ ID NO: 18-20 or a variant of SEQ ID NO: 18-20 sequences with 80% identity; the CDR3 is SEQ ID NO: 21-23 or any combination of SEQ ID NOs: 21-23 sequences with 80% identity.

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: 7.

In another aspect, the invention provides a chimeric antigen receptor.

The chimeric antigen receptor comprises the anti-BCMA single domain antibody.

The chimeric antigen receptor further 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 SEQ ID NO: 10, or a fragment thereof.

The structural gene of the chimeric antigen receptor comprises SEQ ID NO: 11.

The nucleotide sequence of the structural gene of the chimeric antigen receptor is SEQ ID NO: 12. SEQ ID NO: 13 or SEQ ID NO: 14, or a sequence shown in fig. 14.

In yet another aspect, the present invention provides a biomaterial.

The biological material is a recombinant vector, a recombinant cell or a biological product for treating or preventing.

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 chimeric antigen receptor or a gene encoding the chimeric antigen receptor.

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.

Preferably, the viral vector is a lentiviral vector.

In some embodiments, the viral vector is a pCDH-EF1a-BCMA CAR viral vector; the virus vector is a virus vector comprising SEQ ID NO: 12-14 or a lentiviral vector pCDH-EF1a having any of the nucleotide sequences shown.

Further, the biological material is immune cells, including but not limited to: t cells, NK cells, Peripheral Blood Mononuclear Cells (PBMCs), hematopoietic stem cells, pluripotent stem cells or embryonic stem cells, preferably human peripheral blood T cells.

Preferably, the number of chimeric antigen receptor molecules carried by each individual cell in said cell is 3 to 4.

In yet another aspect, the invention provides a medical formulation for treating myeloma.

The medical formulation comprises the aforementioned anti-BCMA single domain antibody or a gene encoding the anti-BCMA single domain antibody.

The medical formulation comprises the aforementioned chimeric antigen receptor or a gene encoding the chimeric antigen receptor.

The medical formulation comprises the aforementioned biomaterial.

Preferably, the medical formulation comprises T cells expressing a BCMA chimeric antigen receptor.

Further, the cell concentration of said BCMA chimeric antigen receptor expressing T cells in the medical formulation is: 1X 10⁸Individual positive BCMA CAR-T/100 mL.

The application rate of the medical preparation is 2 x 10⁶Individual positive BCMA CAR-T/kg body weight.

The dosage form of the medical preparation comprises, but is not limited to, infusion solution and injection.

The modes of application of the medical formulation include, but are not limited to: intravenous injection, intraperitoneal injection.

In a further aspect, the invention provides the use of an anti-BCMA single domain antibody, a chimeric antigen receptor or a biological material as hereinbefore described in the manufacture of a medicament for the treatment of myeloma.

Preferably, the myeloma is multiple myeloma.

The invention aims to solve the problem that patients with relapsed/drug-resistant multiple myeloma have no effective treatment means clinically at present, and the BCMA CART technology is applied to specifically kill myeloma cells in bone marrow, so that the aim of treating patients with relapsed/drug-resistant multiple myeloma is fulfilled.

The invention adopts the structure that different from the prior art, the BCMA single domain antibody gene and 2 generation CAR structure gene sequences are connected in series to form the BCMA single domain antibody gene. The invention uses a sequence different from the known BCMA antibody, and the used antibody is a single-domain antibody, which has stronger binding force with target cells, better killing effect and longer in-vivo duration compared with the CAR derived from the traditional antibody.

Drawings

FIG. 1 is a graph showing the results of flow cytometry to detect the binding capacity of BCMA VHH recombinant antibody and BCMA-highly expressing recombinant cell line CHO-BCMA.

FIG. 2 is a partial assay of the binding of humanized antibodies to BCMA using flow cytometry.

FIG. 3 shows the result of antibody affinity detection. Wherein A represents the selected anti-BCMA single domain antibody B-6-14; B. c represents humanized antibodies NW2-1222-6-14-1 and NW2-1222-6-14-2, respectively.

FIG. 4 is a flow cytometric assay of CAR-T cell membrane surface markers, wherein A is the CD8 positive rate, B is the CD4 positive rate, C is the BCMA negative control, D is the CAR positive rate of B-6-14BCMA CAR-T, E is the CAR positive rate of NW2-1222-6-14-1BCMA CAR-T, and F is the CAR positive rate of NW2-1222-6-14-2BCMA CAR-T.

FIG. 5 is the result of RT-PCR method to detect the copy number of CAR on BCMA CAR-T cells. Where A is the standard curve and B is the copy number of the CAR.

FIG. 6 shows the BCMA positive rate detected by flow cytometry of recombinant Nalm6-BCMA-GFP-LUC cell line, wherein A is FSC-SSC scatter diagram, B is Nalm6 negative control, and C is BCMA positive rate of recombinant Nalm6-BCMA-GFP-LUC cell (BCMA-GFP double positive). The data in the graph can determine the result.

FIG. 7 shows the results of BCMA CAR-T cell function evaluation in vitro. Wherein A is the killing effect on Nalm6-BCMA-GFP-LUC cells, and B is the IFN-gamma release detection result.

FIG. 8 shows the result of in vivo imaging of mice in the evaluation of the in vivo function of BCMA CAR-T cells, wherein the higher the color signal (the darker the color) in the in vivo image of mice indicates the higher the number of tumor cells in the mice.

FIG. 9 shows the results of body weight changes in mice in the functional evaluation of BCMA CAR-T cells in vivo.

FIG. 10 is a mouse survival curve in the assessment of BCMA CAR-T cell function in vivo.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Example 1 anti-BCMA Single Domain antibody VHH Gene acquisition

1. anti-BCMA single domain antibody library construction: adult healthy alpacas are immunized by subcutaneous multi-point injection on the back of the neck by utilizing self-made BCMA antigen protein. In the immunization, after fully and uniformly mixing the antigen and the equal volume of Gerbu adjuvant, injecting alpaca subcutaneously at multiple points for 5 times, wherein the time interval of each immunization is 14 days; at the end of the third immunization, the antigen immunization titer was determined after separating the serum from a portion of peripheral blood collected via the jugular vein, and the results are shown in the following table:

antibody titer > 1: 160000.

2. 150mL of peripheral blood is collected after the 5 th immunization is finished, PBMC is separated, total RNA is extracted and is reversely transcribed into cDNA, and the variable region segment VHH of the alpaca heavy chain antibody is amplified through two rounds of PCR. The VHH fragment and the antibody display vector are respectively subjected to enzyme digestion through SfiI, the VHH fragment is cloned into the display vector by adopting T4 DNA ligase, then a ligation product is electrically transferred into a Pichia pastoris cell by an electrotransformation method, and screening is carried out to obtain a single domain antibody yeast display library.

3. Adding methanol with the final concentration of 0.5% into the obtained VHH yeast display library culture medium, inducing a yeast display single domain antibody to the yeast cell wall, adding biotin-labeled BCMA recombinant protein, labeling positive yeast clones in the yeast library, separating the clones capable of being combined with target protein BCMA in the yeast display library by using streptidin-labeled magnetic beads, repeating the enrichment screening process for 2-3 times, coating the clones capable of being combined with the BCMA recombinant protein on a solid plate, and selecting single clones for clone identification.

4. Amplifying the selected monoclonal yeast, adding methanol with the final concentration of 0.5% into a culture medium, carrying out VHH induction expression, detecting whether the displayed VHH fragment can be combined with BCMA protein by adopting flow cytometry, extracting genomic DNA from the clone which is verified to be positive by the flow cytometry, carrying out PCR amplification on the VHH fragment, and carrying out Sanger sequencing to obtain a VHH antibody sequence.

5. VHH antibody sequences were separately subjected to gene synthesis and cloned into the expression vector Lenti-hIgG 1-Fc. After the vector is verified to be correct by sequencing, the plasmid is greatly extracted.

6. And (3) inoculating 293T to a 6-well plate, performing transient transfection on the 293T by using a Lenti-VHH-hIgG1-Fc expression vector, collecting a culture medium supernatant after 3 days, centrifuging the culture medium supernatant, and passing the supernatant through a 0.45-micrometer filter membrane to collect the BCMA VHH recombinant antibody.

7. The binding capacity of the BCMA VHH recombinant antibody to a BCMA-highly expressing recombinant cell line CHO-BCMA was examined using flow cytometry: the CHO and CHO-BCMA control cells were divided into several portions, each of which was 5X 10 cells⁵(ii) individual cells; uniformly mixing 100 mu L of BCMA VHH recombinant antibody with target cells and control cells respectively, and incubating for 1 hour at 4 ℃; washing cells with PBS for 3 times, adding 100 mu L PBS for resuspending the cells, adding 1 mu L PE-labeled anti-human IgG antibody, mixing uniformly, and incubating for 30 minutes at 4 ℃ in a dark place; the cells were washed 3 times with PBS and tested on the machine, and the results are shown in FIG. 1, where A-D are CHO-BCMA cells and E-H are CHO cells.

8. BCMA VHH humanization design

The present application screens a variety of anti-BCMA single domain antibodies in which the amino acid sequence of the CDR1 region is selected from the group consisting of SEQ ID NO: 15-17, the amino acid sequence of the CDR2 region is selected from SEQ ID NO: 18-20, the amino acid sequence of the CDR3 region is selected from SEQ ID NOs: 21-23.

The amino acid sequence of the selected anti-BCMA single domain antibody B-6-14 is shown as SEQ ID NO: 1 is shown.

According to SEQ ID NO: 1, adopting surface amino acid substitution design to carry out humanized design, wherein the sequence of a humanized antibody is shown as SEQ ID NO: 2-7.

The 6 humanized antibody fragments were synthesized and cloned into the expression vector Lenti-hIgG 1-Fc. After the vector is verified to be correct by sequencing, the plasmid is greatly extracted.

9. Following transient 293T transfer, supernatants were incubated with CHO and CHO-BCMA cells, respectively, and flow cytometry was used to detect binding of humanized antibodies to BCMA as in steps 6-7, and FIG. 2 shows partial results (NW2-1222-6-14-1, NW 2-1222-6-14-2).

10. The results show that the humanized sequences were all high affinity BCMA VHH antibody sequences, in this example, NW2-1222-6-14-1, NW2-1222-6-14-2 were selected for further experiments.

11. Detecting the affinity of the humanized antibody: the binding ability of the antibody to the target protein BCMA before and after humanization was examined by immobilizing the BCMA recombinant protein on a CM5 chip using 10mM Acetate buffer, using the above humanized antibodies NW2-1222-6-14-1, NW2-1222-6-14-2 and the original sequence antibody as mobile phases, respectively, and the results are shown in fig. 3, and show that: antibody B-6-14 has an affinity KD of 1.985X 10^-10M; humanized antibody B-6-14-1 with KD ═ 1.818X 10^-10M; KD of humanized antibody B-6-14-2 is 1.760X 10^-10M。

Example 2 construction of chimeric antigen receptor Gene vectors

The Huada Gene company synthesizes 2 segments of genes, one segment is SEQ ID NO: 1-7, three of which were selected for synthesis as shown in SEQ ID NO: 8-10. Another segment is a designed 2 generation CAR structural gene, comprising a CD8a hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain + CD3 ζ intracellular signaling domain, and a nucleotide sequence encoding the 2 generation CAR structural gene comprising these domains is shown in SEQ ID NO: shown at 11.

After two sections of synthetic genes are obtained respectively, vector construction of BCMA-CAR is carried out, and the sequence of SEQ ID NO: 12-14.

The lentiviral vector pCDH-EF1a and SEQ ID NO: 12-14, respectively carrying out double enzyme digestion and T4 DNA ligase connection, transforming competent cells, selecting single clones, carrying out bacterial liquid sequencing, and then carrying out large quality improvement on granules to respectively obtain three lentiviral vectors pCDH-EF1a-BCMA1#, 2#, 3#, wherein the 1# is an original sequence of the anti-BCMA single domain antibody, and the 2# and the 3# are two humanized anti-BCMA single domain antibody sequences with correct sequences.

Example 3BCMA CAR lentivirus preparation

After the lentiviral packaging plasmid mixture (including PSPAX2 and VSVG) was mixed with pCDH-EF1a-BCMA1#, 2#, 3# respectively in a pre-optimized ratio, the transfection aid was added and incubated at room temperature for 15 minutes. The transfection mixture was then added dropwise to 293T cells. Collecting culture medium supernatant after 1-3 days to obtain crude lentivirus solution. The supernatant was collected after centrifugation at 500g for 10min at 4 ℃. And then ultracentrifuged to concentrate the lentivirus. After the ultracentrifugation was completed, the supernatant was carefully discarded and the lentiviral particles were carefully resuspended with precooled DPBS. The virus was stored at-80 ℃ after packaging. The physical and infectious titers of lentiviruses were determined based on RT-PCR methods and flow cytometry.

Example 4 preparation of BCMA CAR-T cells

1. PBMC preparation: 30mL of human venous blood was centrifuged at 800g at 20 ℃ for 30 minutes, and after centrifugation was completed, the upper plasma was transferred to another centrifuge tube. 15mL of human lymphocyte separation medium (a tertiary objective Tianjin organism) was added to a 50mL centrifuge tube, and the centrifuged blood was slowly added to the upper part of the lymphocyte separation medium by an electric pipette, and centrifuged at 250g and 20 ℃ for 10 minutes. The supernatant was discarded and counted in 10ml PBS for T cell purification.

2. T cell purification: human T cells were purified from PBMCs using the Miltenyi Pan T cell isolation kit (catalog No. 130-096-535) according to the manufacturer's protocol described below. After PBMC counting, centrifugation was carried out at 300g and 20 ℃ for 10 minutes. The supernatant was decanted and the cell pellet was administered at 10 intervals ⁷40 μ L buffer resuspended in bufferAfter the solution was added, 10. mu.L/10⁷The Pan T cell biotin-antibody mixture was mixed well and incubated at 4 ℃ for 5 minutes. Then 30. mu.L of buffer, and 20. mu.L of Pan T cell microbead mix were added. After mixing well, incubate at 4 ℃ for 10 minutes. After incubation, PBMC were washed with 1mL of buffer, 250g, and centrifuged at 4 ℃ for 10 min. After centrifugation the cells were resuspended in 500. mu.L of buffer, passed through the LS column, the cell suspension flowed out with gravity, the effluent (i.e., T cell fraction) was collected, and washing of the LS column with buffer continued and the effluent collected. The enriched T cells were then centrifuged and resuspended in lymphocyte culture medium +1000IU/mL IL-2.

3. CAR-T preparation: lentivirus infection was performed 24-96 hours after pre-activation of the prepared T cells with the human T cell activation/amplification kit (Miltenyi # 130-.

B-6-14BCMA CAR-T cells: pCDH-EF1a-BCMA1# lentivirus infection;

b-6-14-2BCMA CAR-T cells: pCDH-EF1a-BCMA 2# lentivirus infection;

b-6-14-3BCMA CAR-T cells: pCDH-EF1a-BCMA 3# Lentiviral infection.

After 10. mu.g/mL of polybrene was added to the activated T cell suspension, lentivirus was added at an MOI of 10, 1200g, and centrifuged at 32 ℃ for 1 hour. After centrifugation, the transduced T cells were placed in a cell incubator and supplemented daily with appropriate amounts of T cell culture medium.

After 7 days of infection, after incubation of BCMA Fc protein with BCMA CAR-T, the expression rate of CAR on the surface of T cell membrane was measured with flow cytometry.

The results are shown in FIG. 4. The results in fig. 4 show that the BCMA CAR-T cells prepared in example 4 all had CAR expression efficiencies above 90% for all three sequences. After three BCMA CAR-T are simultaneously extracted with DNA, the copy number of CAR on the BCMA CAR-T cells is detected by using an RT-PCR method, and the result is shown in FIG. 5, and the related data are as follows:

the results in FIG. 5 show that the BCMA CAR-T cells prepared in example 4 carry 3-4 CAR molecules per BCMA CAR-T cell.

Example 5 BCMA CAR-T cell functional assessment

Functional evaluation experiments were performed on the BCMA CAR-T cells prepared in example 4 as follows:

1. in vitro functional evaluation

The BCMA positive rate was measured by flow cytometry using the recombinant Nalm6-BCMA-GFP-LUC cell line (obtained from Aikangdi biomedical technology (Suzhou)) and the results are shown in FIG. 6. The results show that BCMA molecules are efficiently expressed in Nalm6-BCMA-LUC recombinant cell strain, and are not expressed in Nalm6 cells (purchased from Guangzhou Securio Biotechnology Co., Ltd.), and can be used as target cells and control cells of CAR-T killing experiments respectively.

The BCMA CAR-T cells prepared in example 4 and Nalm6-BCMA-GFP-LUC or Nalm6 cells were subjected to four gradients at effective target ratios of 10:1, 5:1, 2.5:1,1.25:1, and after 20 hours of total culture, the residual luciferase activity in the wells was detected using a luciferase assay kit (Promega # E6110). Specific cytotoxicity was calculated using the following formula: specific cytotoxicity ═ 100% × (1- (RLU sample-RLU min)/(RLU max-RLU min)). Meanwhile, the supernatant was collected and IFN-. gamma.was detected using IFN-. gamma.ELISA kit (Dake is), and the results are shown in FIG. 7, in which the killing effect on Nalm6-BCMA-LUC cells is shown in A in FIG. 7; IFN- γ release is shown in B in FIG. 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 that of original BCMA single-domain antibody sequence CAR-T cells.

2. Evaluation of in vivo function

Tumor cells Nalm6-BCMA-Luciferase cells 1 x 10 are infused through vein of rat tail ⁶200 mu L, establishing a mouse tumor model, randomly dividing the mice into 4 groups, performing in-vivo imaging observation on 5 days, 11 days, 17 days and 25 days after modeling, infusing control T cells (separated and purified from the same peripheral blood PBMC sample) and two humanized single domain antibody sequence BCMA CAR-T cells (B-6-14-1BCMA CAR-T, B-6-14-2BCMA CAR-T) through tail vein on 5 days after modeling, and using mouse-derived BCMA CAR-T as a positive control, wherein the injection dosage of each cell is as followsThe following:

b-6-14-1BCMA CAR-T cells: 1X 10⁷A plurality of;

b-6-14-2BCMA CAR-T cells: 1X 10⁷A plurality of;

control T cells: 1X 10⁷A plurality of;

mouse-derived BCMA CAR-T: 1X 10⁷And (4) respectively.

The results are shown in fig. 8-10, and show that both humanized BCMA CAR-T cells and mouse-derived BCMA CAR-T cells have significant killing effect on tumor cells compared with T cell control group, tumor cells of mouse-derived BCMA CAR-T group begin to recur at 25 days of tumor cell injection, and humanized B-6-14-1/B-6-14-2BCMA CAR-T still has significant killing effect (fig. 8). Mice infused with control T cells continued to lose weight from day 20, while mice infused with CAR-T cells maintained weight and slightly increased, indicating that CAR-T cells inhibited tumor cell proliferation in the mice (figure 9). Mouse survival analysis showed that control mice had dead mice starting from about day 25 after modeling, CAR-T treated mice were normal and were continuously observed for 60+ days, mice injected with mouse-derived BCMA CAR-T all died, while most of mice injected with humanized single domain antibody sequence BCMA CAR-T survived, indicating that single domain antibody sequence BCMA CAR-T was longer lasting in vivo and more effective (fig. 10).

Sequence listing

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<213> Artificial sequence (artificial sequence)

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Met Phe Ser Thr Gly

20 25 30

Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Gln Phe Val

35 40 45

Ala Ala Ile Thr Arg Ser Asp Ile Arg Glu Asn Asp Gly Ile Thr Tyr

50 55 60

Tyr Gly Val Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala

65 70 75 80

Gly Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Ala Asp Ala Lys Leu Val Phe Thr Pro Thr

100 105 110

Pro Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 126

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Met Phe Ser Thr Gly

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Phe Val

35 40 45

Ala Ala Ile Thr Arg Ser Asp Ile Arg Glu Asn Asp Gly Ile Thr Tyr

50 55 60

Tyr Gly Val Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala

65 70 75 80

Gly Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

85 90 95

Ala Val Tyr Tyr Cys Ala Ala Asp Ala Lys Leu Val Phe Thr Pro Thr

100 105 110

Pro Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 6

<211> 126

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Met Phe Ser Thr Gly

20 25 30

Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Gln Phe Val

35 40 45

Ala Ala Ile Thr Arg Ser Asp Ile Arg Glu Asn Asp Gly Ile Thr Tyr

50 55 60

Tyr Gly Val Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Val Tyr Tyr Cys Ala Ala Asp Ala Lys Leu Val Phe Thr Pro Thr

100 105 110

Pro Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 7

<211> 126

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Met Phe Ser Thr Gly

20 25 30

Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Gln Phe Val

35 40 45

Ala Ala Ile Thr Arg Ser Asp Ile Arg Glu Asn Asp Gly Ile Thr Tyr

50 55 60

Tyr Gly Val Ser Val Lys Gly Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Val Tyr Tyr Cys Ala Ala Asp Ala Lys Leu Val Phe Thr Pro Thr

100 105 110

Pro Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 8

<211> 378

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

gaggtgcagc tggtggagtc tgggggagga ttagtgcagg ctgggggctc tctgagactc 60

tcctgtgtag cctctggacg catgttcagt actggtgccg tgggctggtt ccgccaggct 120

ccagggaagg agcgtcaatt tgtagcagct attacccgga gtgatattcg cgagaatgat 180

ggtataacat actatggagt ctccgtgaag ggccgattca ccatctcccg agacagcgcc 240

ggcaacacgg tgtatctgca aatgaacagc ctgaaacctg aggacacggc cgtttattac 300

tgtgcagcag acgctaagct cgtatttacg cctacacctc agtactgggg ccaggggacc 360

caggtcaccg tctccggc 378

<210> 9

<211> 378

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60

tcttgtgccg ccagcggccg gatgttttct actggtgccg tcggctggtt cagacaggcc 120

cctggaaaag gcctgcagtt cgtggccgcc atcaccagaa gcgacatcag agagaacgac 180

ggcatcacct actacggcgt gtccgtgaag ggcagattca ccatcagcag agacagcgcc 240

ggcaacaccg tgtacctgca gatgaacagc ctgaagcctg aggacaccgc cgtgtattac 300

tgtgccgccg atgccaagct ggtgttcacc cctacacctc agtattgggg ccagggcaca 360

ctggtcacag tgtctagt 378

<210> 10

<211> 378

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60

tcttgtgccg ccagcggccg gatgttttct actggtgctg tcggctgggt ccgacaggct 120

cctggaaaag gactgcagtt cgtggccgcc atcaccagaa gcgacatcag agagaacgac 180

ggcatcacct actacggcgt gtccgtgaag ggcagattca ccatcagcag agacagcgcc 240

ggcaacaccg tgtacctgca gatgaacagc ctgaagcctg aggacaccgc cgtgtattac 300

tgtgccgccg atgccaagct ggtgttcacc cctacacctc agtattgggg ccagggcaca 360

ctggtcacag tgtctagt 378

<210> 11

<211> 669

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaagcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catttgggcc cctctggctg gtacttgcgg ggtcctgctg 180

ctttcactcg tgatcactct ttactgtaag cgcggtcgga agaagctgct gtacatcttt 240

aagcaaccct tcatgaggcc tgtgcagact actcaagagg aggacggctg ttcatgccgg 300

ttcccagagg aggaggaagg cggctgcgaa ctgcgcgtga aattcagccg cagcgcagat 360

gctccagcct acaagcaggg gcagaaccag ctctacaacg aactcaatct tggtcggaga 420

gaggagtacg acgtgctgga caagcggaga ggacgggacc cagaaatggg cgggaagccg 480

cgcagaaaga atccccaaga gggcctgtac aacgagctcc aaaaggataa gatggcagaa 540

gcctatagcg agattggtat gaaaggggaa cgcagaagag gcaaaggcca cgacggactg 600

taccagggac tcagcaccgc caccaaggac acctatgacg ctcttcacat gcaggccctg 660

ccgcctcgg 669

<210> 12

<211> 1047

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

gaggtgcagc tggtggagtc tgggggagga ttagtgcagg ctgggggctc tctgagactc 60

tcctgtgtag cctctggacg catgttcagt actggtgccg tgggctggtt ccgccaggct 120

ccagggaagg agcgtcaatt tgtagcagct attacccgga gtgatattcg cgagaatgat 180

ggtataacat actatggagt ctccgtgaag ggccgattca ccatctcccg agacagcgcc 240

ggcaacacgg tgtatctgca aatgaacagc ctgaaacctg aggacacggc cgtttattac 300

tgtgcagcag acgctaagct cgtatttacg cctacacctc agtactgggg ccaggggacc 360

caggtcaccg tctccggcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 420

atcgcgtcgc agcccctgtc cctgcgccca gaagcgtgcc ggccagcggc ggggggcgca 480

gtgcacacga gggggctgga cttcgcctgt gatatctaca tttgggcccc tctggctggt 540

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 600

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 660

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 720

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 780

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 840

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 900

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 960

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1020

cttcacatgc aggccctgcc gcctcgg 1047

<210> 13

<211> 1047

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60

tcttgtgccg ccagcggccg gatgttttct actggtgccg tcggctggtt cagacaggcc 120

cctggaaaag gcctgcagtt cgtggccgcc atcaccagaa gcgacatcag agagaacgac 180

ggcatcacct actacggcgt gtccgtgaag ggcagattca ccatcagcag agacagcgcc 240

ggcaacaccg tgtacctgca gatgaacagc ctgaagcctg aggacaccgc cgtgtattac 300

tgtgccgccg atgccaagct ggtgttcacc cctacacctc agtattgggg ccagggcaca 360

ctggtcacag tgtctagtac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 420

atcgcgtcgc agcccctgtc cctgcgccca gaagcgtgcc ggccagcggc ggggggcgca 480

gtgcacacga gggggctgga cttcgcctgt gatatctaca tttgggcccc tctggctggt 540

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 600

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 660

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 720

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 780

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 840

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 900

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 960

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1020

cttcacatgc aggccctgcc gcctcgg 1047

<210> 14

<211> 1047

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 14

gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60

tcttgtgccg ccagcggccg gatgttttct actggtgctg tcggctgggt ccgacaggct 120

cctggaaaag gactgcagtt cgtggccgcc atcaccagaa gcgacatcag agagaacgac 180

ggcatcacct actacggcgt gtccgtgaag ggcagattca ccatcagcag agacagcgcc 240

ggcaacaccg tgtacctgca gatgaacagc ctgaagcctg aggacaccgc cgtgtattac 300

tgtgccgccg atgccaagct ggtgttcacc cctacacctc agtattgggg ccagggcaca 360

ctggtcacag tgtctagtac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 420

atcgcgtcgc agcccctgtc cctgcgccca gaagcgtgcc ggccagcggc ggggggcgca 480

gtgcacacga gggggctgga cttcgcctgt gatatctaca tttgggcccc tctggctggt 540

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 600

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 660

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 720

ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct ctacaacgaa 780

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 840

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 900

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 960

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1020

cttcacatgc aggccctgcc gcctcgg 1047

<210> 15

<211> 8

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 15

Gly Arg Met Phe Ser Thr Gly Ala

1 5

<210> 16

<211> 8

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 16

Gly Arg Ala Phe Ser Tyr Gly Ala

1 5

<210> 17

<211> 8

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 17

Gly Arg Met Gly Asn Thr Gly Ala

1 5

<210> 18

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 18

Ile Thr Arg Ser Asp Ile Arg Glu Asn Asp

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 19

Ile Thr Arg Thr Asp Ile Arg Val Asn Asp

1 5 10

<210> 20

<211> 10

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 20

Ile Gly Arg Ser Asp Asn Arg Glu Asn Asp

1 5 10

<210> 21

<211> 17

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 21

Tyr Tyr Cys Ala Ala Asp Ala Lys Leu Val Phe Thr Pro Thr Pro Gln

1 5 10 15

Tyr

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 22

Tyr Tyr Cys Thr Ala Asp Ala Lys Asn Val Phe Thr Pro Thr Pro Gln

1 5 10 15

Tyr

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 23

Cys Tyr Gln Ala Ala Asp Ala Lys Leu Val Phe Thr Pro Thr Asn Gln

1 5 10 15

Tyr

Claims

1. An anti-BCMA single domain antibody, comprising CDR1, CDR2 and CDR 3; the CDR1 is SEQ ID NO: 15, or a sequence shown in seq id no; the CDR2 is SEQ ID NO: 18, or a sequence shown in seq id no; the CDR3 is SEQ ID NO: 21, and (b) the sequence shown in figure 21.

2. The anti-BCMA single domain antibody according to claim 1, characterized in that said anti-BCMA single domain antibody has the amino acid sequence of 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: 7.

3. A chimeric antigen receptor comprising an anti-BCMA single domain antibody according to claim 1 or 2.

4. The chimeric antigen receptor according to claim 3, further comprising a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain.

5. The chimeric antigen receptor according to claim 3 or 4, wherein the structural gene of the chimeric antigen receptor comprises a 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 SEQ ID NO: 10, or a fragment thereof.

6. The chimeric antigen receptor according to claim 5, wherein the structural gene of said chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 11.

7. 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 SEQ ID NO: 14, or a sequence shown in fig. 14.

8. A biomaterial, wherein said biomaterial is a recombinant vector, a recombinant cell, or a biological product for therapeutic or prophylactic purposes; the biological material comprising the anti-BCMA single domain antibody or the gene encoding the anti-BCMA single domain antibody of claim 1 or 2, or the biological material comprising the chimeric antigen receptor or the gene encoding the chimeric antigen receptor of any one of claims 3 to 7.

9. A medical formulation for treating multiple myeloma, comprising an anti-BCMA single domain antibody according to claim 1 or 2 or a gene encoding said anti-BCMA single domain antibody, or a chimeric antigen receptor according to any one of claims 3 to 7 or a gene encoding said chimeric antigen receptor, or a biological material according to claim 8.

10. Use of an anti-BCMA single domain antibody according to claim 1 or 2, a chimeric antigen receptor according to any one of claims 3 to 7 or a biological material according to claim 8 in the manufacture of a medicament for the treatment of myeloma.