CN114195896B

CN114195896B - BCMA chimeric antigen receptor based on single domain antibody and application thereof

Info

Publication number: CN114195896B
Application number: CN202111477662.4A
Authority: CN
Inventors: 王冰; 李治寰; 张文迪; 赵通介; 谭凤鸣
Original assignee: Dongguan Qingshi Biotechnology Co ltd
Current assignee: Dongguan Qingshi Biotechnology Co ltd
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2022-07-05
Anticipated expiration: 2041-12-06
Also published as: CN114195896A

Abstract

The present invention provides a BCMA chimeric antigen receptor based on a single domain antibody whose heavy chain complementarity determining regions comprise CDR1, CDR2, CDR 3; the CDR1 is SEQ ID NO.1 or a sequence with more than 80% of identity with SEQ ID NO.1, the CDR2 is SEQ ID NO.2 or a sequence with more than 80% of identity with SEQ ID NO.2, and the CDR3 is SEQ ID NO.3 or a sequence with more than 80% of identity with SEQ ID NO. 3. The BCMA chimeric antigen receptor provided by the invention is formed by connecting BCMA single domain antibody gene fragments and 2 generation CAR structural gene sequences in series, and 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 neoplastic disease of plasma cells characterized primarily by the malignant proliferation of monoclonal plasma cells, the presence of monoclonal immunoglobulins in the blood or urine, and associated end organ damage including hypercalcemia, impairment of renal function, anemia, and osteolytic destruction. It is presently believed that all MM patients have an unknown stage of Monoclonal Gammopathy (MGUS), also known as a premyeloma state. MGUS progressed to the next stage as myeloma with a probability of progression of approximately 1%/year. Myeloma is divided into 2 stages: smoldering Multiple Myeloma (SMM) and active myeloma. SMM is an intermediate stage between MGUS and active myeloma, also known as early myeloma. SMM patients do not require treatment, while active myeloma patients do.

MM accounts for about 1% of neoplastic diseases and about 13-18% of hematological malignancies. The etiology of MM has not been clear to date and may be associated with viral infection, ionizing radiation, exposure to certain chemicals, and genetics. MM has a short survival time, and if no treatment is given, the median survival time for progressive patients is 6 months. In recent years, with the increasing awareness of diseases and the application of proteasome inhibitors, immunomodulators, autologous stem cell transplantation, daratuzumab (CD38 target), erlotinib (SLAMF7 target) and the like, the remission rate and the survival time of MM patients are obviously improved, and the survival rate of MM patients for more than 5 years reaches 49%. However, most patients eventually develop drug resistance or relapse, and die, so MM is still considered to be an incurable disease until now.

In the innate immune system, both the cytotoxic and immunoregulatory functions of NK cells in MM patients are impaired. Antigen-presenting cells dendritic cells in MM patients have reduced phagocytic and antigen-presenting abilities, are not efficiently stimulated by tumor antigens and up-regulate co-stimulatory signaling molecules of T cell activation. In the acquired immune system, the function of T cell secretion and differentiation disorder and B cells of MM patients is weakened, and the Treg/Th17 imbalance promotes the continuous generation of inflammation and the growth of tumors. Furthermore, tumor heterogeneity and the effects of the bone marrow microenvironment on disease progression also make MM refractory, and therefore, the development of new approaches to the treatment of multiple myeloma is imminent.

A Chimeric Antigen Receptor (CAR) is an artificial molecule consisting of an Antigen recognition domain, an extracellular Hinge region (hinde), a Transmembrane region (Transmembrane region), one to two costimulatory domains (e.g., CD28, 4-1BB, etc.), and an Immunoreceptor tyrosine-based activation motif (ITAM, e.g., a fragment from CD3 zeta chain, etc.); wherein the antigen recognition domain is typically derived from a single-chain variable fragment (scFv) derived from the antigen binding region of a monoclonal antibody.

T lymphocytes can artificially express CAR by genetic engineering means, become chimeric antigen receptor T cells (CAR-T), and the CAR-T recognizes a specific cell surface antigen by means of scFv in an antigen-antibody binding manner, and then activates the CAR-T cells through a downstream costimulatory domain of the CAR and an immunoreceptor tyrosine activation motif, releases granulin, perforase, IFN-gamma and the like, so as to specifically target and kill tumor cells expressing the antigen.

Since the CAR-T molecule recognizes antigen by means of antigen-antibody binding, it is different from natural T cells in that tcr (T cell receptor) recognizes MHC I-peptide complex of tumor cells. Thus, the killing effect of CAR-T cells on target cells is not restricted by MHC molecules, so that the mechanism by which tumor cells escape immune attack by down-regulating MHC molecules can be overcome, and the same CAR can be applied to different patients without being HLA-restricted unlike TCR-T technology. Furthermore, the CAR molecule can recognize any type of antigen on the cell surface, including proteins, carbohydrates, glycolipids, etc., such that the CAR greatly increases the range of tumor surface markers that T cells can recognize relative to TCR which can only recognize MHC-peptide complexes. The binding of the scFv segment of CAR-T to the tumor surface antigen is affected by factors such as antibody affinity, epitope structure, number and density of tumor cell surface antigens, pH in the local environment of the tumor (tumor cells usually adopt anaerobic glycolysis to generate ATP due to rapid proliferation and hypoxia, so the local environment of the tumor is usually acidic, pH is about 6), local temperature (for example, local fever usually occurs in the local environment of the tumor due to increased tumor cell metabolism, cell necrosis, vascular proliferation, increased blood circulation, etc.), and ionic strength (for example, intracellular metal ions released after cell necrosis).

The originally designed scFv of the CAR were derived from the light chain variable region and the heavy chain variable region of a conventional antibody, which were linked by a linking region (linker). These traditional antibodies can be from mice, rabbits, etc., where the scFv is derived from a mouse anti-human CD19 monoclonal antibody (mab, clone number FMC63), as represented by the CD19CAR-T cell drugs that are marketed. With the compelling success of CD19CAR-T in acute B-lymphocyte leukemia, more and more research institutes have designed CARs that target numerous targets. However, after the tetrameric 3D space structure of the traditional antibody is modified to be a linear structure of a light chain variable region-linker-heavy chain variable region (i.e., scFv), and the traditional antibody is subjected to complex humanization (the traditional murine monoclonal antibody has a large difference from a human antibody, so that the immunogenicity is strong, and the in vivo retention time of the traditional murine monoclonal antibody can be increased to be applied by generally modifying and reducing the immunogenicity of the traditional murine monoclonal antibody), many scfvs may have reduced affinity or even disappear. There is therefore a need in the art for a simpler and more efficient form of antibody to obtain scFv.

In 1993, Hamers-Casterman et al, Brussel university of liberty, first reported that there is another type of antibody in camel blood in addition to the traditional tetrameric antibody, which naturally lacks the light and heavy chain constant region 1(CH1) regions of the traditional antibody but still has strong binding force to antigen, unlike the molecular structure of the traditional mammalian IgG antibody, and is called heavy-chain antibody (HCAbs). The variable region of HCAbs consists only of the variable region of the antibody heavy chain, which can bind specifically to antigens, similar to the Fab of traditional antibodies. The antigen recognition region fragment of heavy chain antibodies is called VHH (variable domain of the heavy chain of the heavy-chain antibody, VHH), and researchers developed single domain antibodies (sdabs) containing only the VHH domain of HCabs. VHH crystals are also called nanobodies (Nb) since they are 4nm long and 2.5nm in diameter.

Compared with traditional antibodies, the single domain antibody has the following advantages: (1) the molecular weight is small, so that the targeting drug is easier to carry into the cell or penetrate the blood brain barrier to take effect. Since no light chain is present, VHHs are only around 12-15kDa in size, the smallest antigen binding unit known; (2) VHH has better solubility and stability (including thermal stability, protein degradation resistance, strong acid resistance and the like); (3) the CDR3 of VHH is longer, which, in addition to increasing antibody diversity, allows VHH to bind to cryptic epitopes that conventional antibodies do not bind to; (4) compared with the murine antibody, the VHH of the HCAbs has high similarity with the VH of the human antibody, low immunogenicity and is easier for humanized modification and clinical transformation application.

Disclosure of Invention

The invention aims to solve the problem that no effective treatment means exists in clinical treatment of patients with relapsed/drug-resistant multiple myeloma, and the BCMA CAR-T 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 constructs a VHH-CAR aiming at BCMA antigen by a gene recombination method after screening HCAbs specifically aiming at the BCMA antigen, inserts the VHH-CAR into the genome of human T lymphocytes in a gene transduction mode, causes the surface of the cell membrane to express a specific single-domain antibody-derived chimeric antigen receptor (nano BCMA CAR-T) aiming at the BCMA antigen, and transfuses the cell membrane into a patient after in vitro amplification of CAR-T cells, thereby achieving the specific adoptive immune cell therapy aiming at tumor cells (myeloma cells) expressing the BCMA antigen.

In one aspect, the invention provides a BCMA single domain antibody.

The BCMA single domain antibody comprises a heavy chain variable region comprising a complementarity determining region.

The complementarity determining regions of the heavy chain variable region include CDR1, CDR2 and CDR 3.

The sequence of the CDR1 is selected from SEQ ID NO.1 or a sequence with more than 80% of identity with SEQ ID NO. 1;

the sequence of the CDR2 is selected from SEQ ID NO.2 or a sequence with more than 80% of identity with SEQ ID NO. 2;

the CDR3 sequence is selected from SEQ ID NO.3 or a sequence with more than 80% identity with SEQ ID NO. 3.

The sequences with more than 80% identity include but are not limited to: sequences with more than 85% identity, sequences with more than 90% identity, sequences with more than 95% identity, sequences with 80% -90% identity, and sequences with 85% -95% identity; preferably a sequence with 90% identity or more; more preferably 95% or more; further, the sequence has 98% or more identity.

Preferably, the heavy chain variable region amino acid sequence is one of SEQ ID NO.4-6 or a sequence with more than 80% identity with SEQ ID NO. 4-6.

Or the heavy chain variable region amino acid sequence is a humanized sequence of any one of SEQ ID NO. 4-6; the humanized sequence is one of SEQ ID NO. 7-11; preferably SEQ ID NO. 10.

In another aspect, the invention provides a BCMA chimeric antigen receptor based on single domain antibodies.

The antigen recognition domain of the BCMA chimeric antigen receptor is one or more of the BCMA single-domain antibodies.

In yet another aspect, the invention provides a coding gene.

The coding gene codes the BCMA single-domain antibody or the BCMA chimeric antigen receptor.

Preferably, the sequence of the coding gene is one of SEQ ID NO.12-15 or a sequence which has more than 80% of identity with SEQ ID NO. 12-15; further preferred is SEQ ID NO. 15.

In yet another aspect, the invention provides a viral vector.

The viral vector carries the coding gene.

Preferably, the virus is a lentivirus.

Further preferably, the viral vector is a BCMA chimeric antigen receptor lentiviral vector.

Further, the BCMA chimeric antigen receptor lentiviral vector is pCDH-EF1a-BCMA CAR.

In yet another aspect, the invention provides an antibody conjugate.

The antibody conjugate comprises the aforementioned BCMA single domain antibody.

In yet another aspect, the invention provides a CAR-T cell.

The CAR-T cell is a human T lymphocyte transduced by the viral vector.

In a further aspect, the invention provides the use of a BCMA single domain antibody and/or a BCMA chimeric antigen receptor and/or a coding gene and/or a viral vector and/or an antibody conjugate and/or a CAR-T cell as hereinbefore described in the manufacture of a medicament for the treatment and/or prognosis and/or prevention of a tumour.

The tumor is a BCMA expressing tumor, preferably, BCMA expressing multiple myeloma.

In still another aspect, the present invention provides a kit for diagnosing tumor.

The kit comprises the aforementioned BCMA single domain antibody and/or BCMA chimeric antigen receptor and/or coding gene and/or viral vector and/or antibody conjugate and/or CAR-T cell.

Preferably, the neoplasm is multiple myeloma.

In yet another aspect, the invention provides a medicament for the treatment or prognosis of a tumour.

The medicament comprises the BCMA single domain antibody and/or the BCMA chimeric antigen receptor and/or the coding gene and/or the virus vector and/or the antibody conjugate and/or the CAR-T cell.

The invention has the beneficial effects that:

the invention adopts a chimeric antigen receptor gene combination different from the prior art, and is formed by connecting a BCMA single domain antibody gene and a 2-generation CAR structural gene sequence in series.

Using an amino acid sequence that differs from known BCMA single domain antibody structures; the used antibody is a single-domain antibody, and compared with the CAR derived from the traditional antibody, the antibody has stronger binding force with target cells, better killing effect and longer in-vivo duration.

Drawings

FIG. 1 shows the result of gel electrophoresis of the amplification of VHH + CH1 fragment.

FIG. 2 shows the results of the gel electrophoresis of the amplification of VHH fragments.

FIG. 3 is a graph showing that the binding capacity of the 16-B7 recombinant antibody to a recombinant cell line CHO-BCMA highly expressing BCMA is detected by flow cytometry.

FIG. 4 shows the ability of the 16-B7 recombinant antibody to bind to BCMA at different dilution.

Figure 5 is a flow cytometry assay for the ability of recombinant antibodies to bind to BCMA target protein.

FIG. 6 is a flow cytometry assay of the ability of purified 16-B7-HM4 recombinant antibody to bind to BCMA target protein.

FIG. 7 shows the SPR affinity assay results of 16-B7 and its humanized clone 16-B7-HM 4.

Figure 8 is the BCMA CAR lentivirus infection titer assay (flow assay).

FIG. 9 shows the results of the BCMA CAR lentiviral physical titer assay (RT-PCR assay).

Figure 10 flow cytometry detected BCMA CAR-T cell membrane surface BCMA CAR expression rate.

FIG. 11 is a method of RT-PCR to detect the average copy number of CAR on BCMA CAR-T cells.

FIG. 12 shows the BCMA positive rate of recombinant Nalm6-BCMA-LUC cell line detected by flow cytometry.

FIG. 13 is the killing effect of BCMA CAR-T cells on Nalm6-BCMA-LUC cells.

FIG. 14 shows BCMA CAR-T cell IFN- γ release.

FIG. 15 shows the amount of BCMA CAR-T cell IL2 released.

FIG. 16 is live imaging of tumor-bearing mice in BCMA CAR-T functional assessment in vivo.

FIG. 17 is a tumor-bearing mouse weight curve in the assessment of BCMA CAR-T function in vivo.

FIG. 18 is a tumor-bearing mouse survival curve in the functional assessment of BCMA CAR-T in vivo.

FIG. 19 is the CAR copy number/genomic DNA ratio measured in peripheral blood the first day after reinfusion of BCMA CAR-T in clinical patients (kappa light chain MM phase II).

FIG. 20 is a pre-treatment urinary kappa light chain assay report for clinical patients (kappa light chain type MM II).

FIG. 21 is a serum kappa light chain assay report for clinical patients (kappa light chain type MM II phase) prior to treatment.

FIG. 22 is a serum kappa light chain assay report after treatment of clinical patients (kappa light chain type MM phase II).

FIG. 23 is a report of urinary kappa light chain detection after treatment of clinical patients (kappa light chain type MM II).

FIG. 24 is a comparison of serum kappa light chain and urinary kappa light chain assays before and after treatment of clinical patients (kappa light form phase MM II).

It should be noted that, because some pictures are straight-out or scanned pictures of the instrument, details thereof are difficult to modify, but related data parts are clear and recognizable, and the determination of the result is not affected.

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.

The terms:

VHH: a heavy chain variable region.

Example 1 obtaining BCMA Single Domain antibody VHH genes

Construction of BCMA single domain antibody library: performing multiple subcutaneous injections of 5 immunizations each with a 14-day interval of BCMA antigenic protein (purchased from ACRO BIOSYSTEMS, Catalogue number BCA-H522y-1mg) to healthy adult alpaca; after the third immunization, collecting peripheral blood from jugular vein to measure the serum antibody titer, and after the fifth immunization, detecting the result as follows:

serum anti-BCMA antibody titers at the end of the fifth immunization > 1: 64000.

after completion of the 5 th immunization, 150mL of peripheral blood was collected, PBMC was isolated using lymphocyte isolate (Axis-Shield/Alere Technologies AS, cat # 1114546), total RNA was extracted and reverse-transcribed into cDNA, and variable region fragment VHH of HCAbs was amplified by two-round PCR: the upstream and downstream primers of the first round of PCR are the sequences of FR1 and CH2 (constant region 2) of VHH for HCabs respectively, the target fragment is VHH + CH2, the length is 750bp, the result is shown in FIG. 1, and the concentration is determined after gel electrophoresis and gel recovery; the first round of PCR product is used as a template, the upstream and downstream primers of the second round of PCR are respectively the FR1 start end and FR4 end sequences of VHH of HCabs, and the target fragment is 400bp, and the result is shown in FIG. 2.

The VHH fragment and pCANTAB 5E vector (Orno gene, cat # HG-VSW0284) are respectively subjected to enzyme digestion at 50 ℃ of SfiI overnight and ligation at 16 ℃ of T4DNA ligase, then the VHH fragment is cloned into a phage display vector, then the ligation product is electrically transferred into escherichia coli through electric transfer (2500V, 5ms), SOC culture medium (Solarbio, LA2500) is immediately added for re-suspension of the thallus, and shaking culture is carried out at 37 ℃ for 1 hour. After 1 hour, 15mL of bacterial solution was taken for phage rescue, and another 20. mu.L of bacterial solution was diluted and uniformly spread on an LB plate containing ampicillin, and cultured overnight at 37 ℃. The next day, the plate was removed, the number of clones that could be generated per ligation was counted, the library capacity was calculated, and 20 single clones on the plate were picked up into ampicillin-containing 2YT medium (Bio/industry, cat # B540126-0500), shake-cultured at 37 ℃ for about 6-8 hours, sent to the bacterial solution for sequencing (sequencing Universal primer M13R), and the library diversity was calculated.

Resuscitation and rescue of phage display libraries: culturing the transformed Escherichia coli to OD with 2YT₆₀₀After reaching 0.5, M13KO7(NEB, cat # N0315S) was added, the mixture was shaken and allowed to stand at 37 ℃ for 30min, cultured at 37 ℃ and 225rpm for 1h, centrifuged to remove the supernatant, resuspended in 2YT-AK medium (2YT plus 100. mu.g/mL ampicillin and 50. mu.g/mL kanamycin), cultured overnight, and the bacterial solution was concentrated in PEG6000/NaCl solution to obtain a phage pool.

Panning of phage library: uses Streptavidin magnetic beads (Thermo Scientific)^TMCat No. 88816) was incubated with biotinylated BCMA target antigen, followed by incubation with phage library to be panned, washing off non-specifically bound phage, eluting recombinant phage bound to target antigen using TEA, and amplification; after 4 rounds of panning, positive single clones were selected for Sanger sequencing to obtain VHH antibody sequences (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4): QARFVESGGGLVQSGGSLVLSCTVSDVTLDDYVTAWFRQAPGKEREGVSCISKRNFTSYVDSVKGRFTTSRDITKNTVLLQMNALTPEDTGVYYCATPILPSLAFGEVCSLQTEFRSWGPGTRVTVSS (SEQ ID NO.4, the underlined parts are the CDR regions);

the corresponding nucleotide sequence is SEQ ID NO. 12.

The gene of SEQ ID NO.12 was synthesized and cloned into the expression vector pcDNA3.1-hIgG1-Fc2 (Haematococcus Shanghai, cat # P8203). After the recombinant expression vector pcDNA3.4-16-B7-hIgG1-Fc2 is verified to be correct by sequencing, the antibody expression vector is obtained by greatly improving the quality of particles.

Expression verification of recombinant antibodies: the LVTransm transfection reagent (Aikangdi, Cat: LVTrans 100) and the antibody expression vector were removed from the-80 ℃ refrigerator, thawed at room temperature, and blown up and down by a pipette gun to mix well. The PBS buffer stored at 4 ℃ is taken out and re-warmed to room temperature. And adding 500 mu L of PBS into one hole of a 24-hole plate, adding 4 mu g of pcDNA3.4-16-B7-hIgG1-Fc2, blowing and beating the mixture up and down by a pipette gun, fully mixing the mixture, adding 12 mu L of LVTransm, immediately blowing and beating the mixture up and down by a pipette, mixing the mixture uniformly, and standing the mixture at room temperature for 10 minutes.

The above DNA/LVTransm complex was added to 1.5mL 293T cells (ATCC, cat # CRL-3216)^TM) In the middle, gently shake and mix well. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator at 130RPM for 6-8 hours, adding 1.5mL of fresh FreeStyle^TM293Expression Medium (Gibco)^TMAnd the goods number: 12338018), the cells are returned to the incubator for further culture.

After continuous culture for 7 days, the culture medium supernatant was collected by centrifugation, filtered through a 0.45 μm filter, and the filtrate was subjected to flow assay.

The binding capacity of the 16-B7 recombinant antibody and a recombinant cell strain CHO-BCMA with high BCMA expression is detected by flow cytometry: dividing the blank control cells CHO and CHO-BCMA into a plurality of parts, wherein the number of each part of cells is 5.0E +05 cells; respectively mixing the 16-B7 recombinant antibody with the total protein concentration of 1 mu g/mL with target cells and control cells uniformly, and then incubating for 1 hour at 4 ℃; after washing the cells for 3 times with PBS, 100. mu.L of PBS was added to resuspend the cells, 1. mu.L of PE Anti-human IgG Fc (BioLegend, cat # 409304) was added thereto, and after mixing well, incubation was carried out for 30 minutes at 4 ℃ in the dark; the results of the detection on the machine after 3 times of PBS washing of the cells are shown in FIG. 3, and the results show that the 16-B7 recombinant antibody can be combined with BCMA.

The obtained recombinant antibody was subjected to protein purification, and the concentration of the recombinant antibody was measured. Dividing the blank control cells CHO and CHO-BCMA into a plurality of parts, wherein the number of each part of cells is 5.0E +05 cells; respectively mixing purified recombinant antibodies 16-B7 with the concentrations of 10, 2, 0.4 and 0.08 mu g/mL with target cells and control cells uniformly, and incubating for 1 hour at 4 ℃; washing the cells with PBS for 3 times, adding 100 mu L of PBS to resuspend the cells, adding 1 mu L of PE Anti-human IgG, fully mixing, 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. 4: FIG. 4 shows that 16-B7 recombinant antibody was able to bind BCMA at different dilutions, and reached saturation at an antibody concentration of 2. mu.g/mL.

According to the screened single-domain antibody 16-B7 sequence, a CDR Grafting scheme is adopted for humanized design. The humanized antibody sequence is as follows:

16-B7-HM1	SEQ ID NO.7
		16-B7-HM2	SEQ ID NO.8
16-B7-HM3	SEQ ID NO.9
		16-B7-HM4	SEQ ID NO.10
16-B7-HM5	SEQ ID NO.11

the genes corresponding to SEQ. ID No.7-11 were synthesized and then subjected to antibody expression according to the preparation procedure of recombinant 16-B7 antibody, and then the binding ability to BCMA target protein was examined by flow cytometry, and the results are shown in FIG. 5. The results show that the binding capacity of the 16-B7-HM4 humanized recombinant antibody to the over-expressed BCMA cell line is equivalent to that of the original sequence 16-B7. To further confirm the difference between the humanized candidate antibody and the original antibody, 16-B7-HM4 and 16-B7 recombinant antibodies were purified according to the aforementioned method and then subjected to flow assay, and the results are shown in FIG. 6. The results show that the humanized recombinant antibody 16-B7-HM4 has equivalent binding capacity with the original sequence recombinant antibody 16-B7 and the target protein under different antibody concentrations.

Affinity detection of recombinant antibodies:

the target antigen BCMA (ACRO BIOSYSTEMS, cat # BCA-H5)22y-1mg) was immobilized on a chip using 10mM Acetate buffer, and the binding ability of the antibody to the target protein BCMA before and after humanization was examined using the recombinant antibody prepared above as a mobile phase. The experimental results show that the affinity of the recombinant antibody is in nM level, wherein the affinity of 16-B7 is 3.729X 10^- ⁹M; 16-B7-HM4 affinity of 1.060X 10^-9And M. (FIG. 7).

Example 2 construction of chimeric antigen receptor Gene vectors

The following VHH sequence genes were synthesized by huada gene:

a designed 2 generation CAR structural gene is synthesized, which comprises a CD8a hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain + CD3 zeta intracellular signaling domain, and the nucleotide sequence of the 2 generation CAR structural gene comprising the domains is SEQ ID NO. 17.

After two sections of synthetic genes are respectively obtained, vector construction of BCMA CAR is carried out, and the vector is obtained by PCR amplified fragments, EcoRI and BamHI double enzyme digestion and T4ligase enzyme ligation:

SEQ ID NO.18(B7CAR)、SEQ ID NO.19(16-B7HM4CAR)、SEQ ID NO.20(BB2121CAR)。

after EcoRI and BamHI (NEB, cat #1 0101S and R3136S) double digestion and T4DNA ligase ligation of lentiviral vectors pCDH-EF1a (addgene, cat #170446) and SEQ ID NO.18-20, respectively, transformed Stbl3 competent cells (Bomeide, cat # BC 108-01; step Stbl3 competent storage was taken out of a-80 ℃ freezer, thawed on ice, 1. mu.L of the ligation product was taken in a competent centrifuge tube, gently rotated to mix the contents, left to stand in an ice bath for 30 minutes, then rapidly placed on ice after heat shock for 90 seconds at 42 ℃ in a water bath for 5 minutes, 1mL of sterile LB medium (containing no antibiotic) was added to the microfuge tube, left to stand at 37 ℃ for 200rpm, shaking culture was carried out for 60 minutes, 100. mu.L of the bacterial solution was added to the surface of a resistant LB solid plate, then applied to the surface of a resistant LB medium plate, left to stand for 5 minutes by a single pass coating of a bar, and then completely absorbed in a clean plate, inverting the plate, culturing for 18-20 hours at 37 ℃), selecting single clone, after the sequencing of bacterial liquid is correct, extracting plasmid greatly to obtain lentiviral vectors pCDH-EF1a-BCMA-16-B7, pCDH-EF1a-BCMA-16-B7-HM4 and pCDH-EF1a-BCMA-BB2121 with correct sequence.

Example 3 BCMA CAR lentivirus preparation

Lentiviral packaging plasmid mixtures (comprising PSPAX2 (Fenghui biosome, cat # BR036) and VSVG (Fenghui biosome, cat # FH1657) mixed in a 1:1 molar ratio) were mixed with pCDH-EF1a-BCMA-16-B7 or pCDH-EF1a-BCMA-16-B7-HM4 or pCDH-EF1a-BCMA-BB2121, respectively, at a ratio of 1: 2, the transfection aid reagent was added and incubated at room temperature for 15 minutes. The transfection mixture was then added dropwise to 293T cells (ATCC, cat # CRL-3216)^TM). Collecting the culture medium supernatant after 1-3 days to obtain a crude lentivirus solution. The supernatant was collected after centrifugation at 500g for 10min at 4 ℃. The lentivirus was then concentrated by ultracentrifugation (82,700g, 3 hours at 4 ℃ C.). After the ultracentrifugation was complete, the supernatant was carefully discarded and used with precooled DPBS (Gibco)^TMAnd the cargo number: 14190144) carefully resuspending the lentiviral particles, resulting in lentiviral particles: LV-16-B7, LV-16-B7-HM4 and LV-BB 2121. The lentivirus was stored at-80 ℃ after packaging. The physical and infectious titers of lentiviruses were determined based on RT-PCR methods and flow cytometry. The results are shown in FIGS. 8-9.

Example 4 preparation of BCMA CAR-T cells

PBMC preparation:

venous blood 30mL was centrifuged at 800g at 20 ℃ for 30 minutes and after centrifugation, the upper plasma was transferred to another centrifuge tube. 15mL of human lymphocyte separation medium (Tianjin tertiary amino product, product number: HY2015, internal control number: LTS1077006) was added into a 50mL centrifuge tube, and the centrifuged blood was slowly added above the lymphocyte separation medium by an electric pipette at 250g and 20 ℃ for 10 minutes. The supernatant was discarded and counted in 10mL PBS for T cell purification.

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 counting PBMCs, the cells were centrifuged at 300g and 20 ℃ for 10 minutes. The supernatant was decanted and the cell pellet was administered at 10 intervals⁷After each 40. mu.L of buffer was resuspended in buffer, 10. mu.L/10 of buffer was added⁷Pan T cells biotin-antibody mixture, 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 the incubation, PBMC were washed with 1mL of buffer and centrifuged at 250g and 4 ℃ for 10 minutes. 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 T cells were then enriched by centrifugation and resuspended in lymphocyte culture medium +1000IU/mL IL-2 at 2X 10⁶Density of individual/well was seeded in 4 wells of a 6-well plate.

CAR-T preparation:

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

After 10. mu.g/mL polybrene was added to the activated T cell suspension (3 wells in a six-well plate), lentiviruses LV-16-B7, LV-16-B7-HM4 or LV-BB2121 were added to each well at an MOI of 10, and T cells in another well were not treated as blank control cells; after addition of the virus, the six-well plate was centrifuged at 1200g and 32 ℃ for 1 hour. And (4) after the centrifugation is finished, putting the six-hole plate into a cell culture box, and supplementing a proper amount of T cell culture medium every day.

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. 10. The results in figure 10 show that the efficiency of CAR expression in BCMA CAR-T cells prepared in example 4 was between 32-53%. Simultaneously 1 × 10⁶The DNA of individual BCMA CAR-T cells was extracted and then the number of copies of CAR on the BCMA CAR-T cells was determined by RT-PCR, and the results are shown in FIG. 11, and the results in FIG. 11 show that each BCMA of the BCMA CAR-T cells prepared in example 4 was presentThe CAR-T cells all carry 1.8-3.8 CAR molecules.

Example 5 BCMA CAR-T cell functional assessment

1) In vitro functional evaluation

The BCMA positive rate is detected by the flow cytometry of the recombinant Nalm6-BCMA-LUC cell line, and the result is shown in figure 12. The results show that BCMA molecules are expressed efficiently in Nalm6-BCMA-LUC recombinant cell strains, are not expressed in Nalm6 cells, and can be used as target cells and control cells of CAR-T killing experiments respectively.

After 16-B7BCMA CAR-T cells, 16-B7-HM4BCMA CAR-T cells, BB2121BCMA CAR-T cells, and T blank control cells prepared in example 4 were co-cultured with Nalm6-BCMA-LUC or Nalm6 cells for 20 hours by setting four gradients in effective target ratios of 10:1, 5:1, 2.5:1, and 1.25:1, respectively, the remaining luciferase activity in the wells was measured 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 is taken and IFN-gamma is detected by an IFN-gamma ELISA kit (Dake is), the result is shown in figures 13-15, wherein the killing effect on Nalm6-BCMA-LUC cells is shown in figure 13; IL2, IFN-. gamma.release is shown in FIGS. 14-15. The results demonstrate that BCMA CAR-T cells prepared in example 4 can kill BCMA positive cells specifically and efficiently. In fig. 13-15, P <0.05, compared to CTRL T.

2) Evaluation of in vivo function

Tumor cells Nalm6-BCMA-Luciferase cells 2 x 10 are infused through rat tail vein⁶Setting up a mouse tumor model at 200 mu L, and after 5 days of tumor inoculation to the tail vein of the mouse, imaging a first living body and recording as the 0 th day; mice were randomized into 2 groups and infused with 16-B7-HM4BCMA CAR-T or T cells, respectively, via the tail vein (remark: only the in vivo efficacy of humanized BCMA CART (16-B7-HM4) was verified in animal experiments, not the in vivo efficacy of the original sequence BCMA CART (16-B7), since animal experiments were prepared for clinical studies); on days D14 and D21, in vivo imaging was performed to monitor the proliferation of tumor cells in mice. The results showed that at day D14, tumors in the treated mice were substantially completely cleared by CAR-T cellsIn addition, the tumor burden in the mice of the control group is continuously increased; day D21, control mice survived only one mouse, CAR-T treated mice continued to survive, and there were no tumor cells in vivo (figure 16).

Mice infused with control T cells lost significantly in body weight from day 13, while mice infused with CAR-T cells maintained substantially in body weight, indicating that CAR-T cells inhibited tumor cell proliferation in the mice (figure 17). Mouse survival analysis showed that control mice died all at day 23 from modeling, whereas CAR-T treated mice were normal and continued to observe for 30+ days, humanized BCMA CAR-T injected mice were all alive, indicating that the single domain antibody sequence BCMA CAR-T was effective in eliminating tumor cells in tumor-bearing mice (figure 18).

Example 6 clinical study data

Clinical grade BCMA CAR-T cells prepared as in example 4 were selected for clinical study in this example.

The patients were the patients in the affiliated hospital of Guangdong medical university, and the patients had their conditions: multiple detections show that the kappa light chain in blood and urine is obviously increased, and the diagnosis is multiple myeloma and kappa light chain type (IIA stage).

Patients who failed chemotherapy at months 03-08 at 2021, entered BCMA CAR-T clinical study treatment group ID regimen chemotherapy (isozamide 4mg d1, 8, 15+ dexamethasone 40mg d1, 8, 15, 21) at month 09 at 2021; pretreatment is carried out on 09/month (06 mg of fludarabine (50 mg of d1-3+ cyclophosphamide (1470 mg)) in 2021, and BCMA CAR-T cells (total number of CAR-T cells: 2X 10) are returned to 30mL on 09/month (11) in 2021⁸cells, CAR positivity 30%, CAR + T Total 6X 10⁷cells), the treatment process is smooth, and the patient has no fever and other reactions.

Detection results before feedback: immunoglobulin: IgG 5.49g/L, IgA0.364g/L, IgM 0.511g/L, IgE 9.87 IU/mL; blood beta 2-MG 2.37MG/L, urine protein quantification (24h urine volume 1500 mL): M-TP 2156.6mg/24h urine; test for outward delivery-urine immuno-fixation electrophoresis: SP positive (+), κ light chain positive (+); inspection for outward deliverySerum free light chain combination: free form Kappa light chain 1134.986mg/LFree Kappa/free lambda 148.3477Ratio, free lambda light chain 7.65 mg/L;urine free Kappa light chain 15800mg/L(ii) a Test delivery-urine protein electrophoresis: m protein content, urine, calculation method 856.54mg/24h, gamma globulin, urine, agarose gel electrophoresis method 99.1%, 24 hours urine volume 1000.0mL/24 h.

Detection results after treatment: the CAR copy number/genomic DNA ratio in peripheral blood on the first day after reinfusion is shown in figure 19. Bone marrow examination series (blood laboratory): combined with history, increased erythroid proportion of MM treated patients were tested for free Kappa light chain 118.71mg/L in serum as detected in 2021, 9, 28 days; urinary free Kappa light chain 705.00 mg/L. Testing the patient at 10 months 2021; urinary free Kappa light chain 322.5 mg/L. Then the patient completes autologous hematopoietic stem cell transplantation, the current situation is good, and the periodical reexamination is carried out. Patient serum, urinary Kappa light chain assay reports are shown in FIGS. 20-23, and Kappa light chain dynamics before and after treatment are shown in FIG. 24.

Sequence listing

<110> Dongguan Qingshi Biotech Co., Ltd

<120> BCMA chimeric antigen receptor based on single domain antibody and application thereof

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Val Thr Leu Asp Asp Tyr Val

1 5

<210> 2

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ser Lys Arg Asn Phe Thr

1 5

<210> 3

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu

1 5 10 15

Gln Thr Glu Phe Arg Ser

20

<210> 4

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Ala Arg Phe Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Val Leu Ser Cys Thr Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

Val Thr Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

Gln Met Asn Ala Leu Thr Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala

85 90 95

Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln

100 105 110

Thr Glu Phe Arg Ser Trp Gly Pro Gly Thr Arg Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gln Ala Arg Phe Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Val Leu Ser Cys Thr Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

Val Thr Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

Gly Arg Ala Thr Thr Ser Arg Asp Ile Thr Lys Asn Thr Val Leu Leu

65 70 75 80

Gln Met Asn Ala Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln

100 105 110

Thr Glu Phe Arg Ser Trp Gly Pro Gly Thr Arg Val Thr Val Ser Ser

115 120 125

<210> 6

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Ala Arg Phe Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Val Leu Ser Cys Thr Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

Val Thr Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

Gln Met Asn Leu Thr Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Thr

85 90 95

Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln Thr

100 105 110

Glu Phe Arg Ser Trp Gly Pro Gly Thr Arg Val Thr Val Ala Ser Ser

115 120 125

<210> 7

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gln Ala Gln Phe Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

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

35 40 45

Ser Cys Ile Ser Lys Arg Asn Phe Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln

100 105 110

Thr Glu Phe Arg Ser Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 8

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gln Ala Gln Phe Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

Val Thr Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Cys Ile Ser Lys Arg Asn Phe Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln

100 105 110

Thr Glu Phe Arg Ser Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 9

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gln Ala Gln Phe Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

Val Thr Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Cys Ile Ser Lys Arg Asn Phe Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln

100 105 110

Thr Glu Phe Arg Ser Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 10

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Ala Arg Phe Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

Val Thr Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Lys Arg Asn Phe Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln

100 105 110

Thr Glu Phe Arg Ser Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 11

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Val Ser Asp Val Thr Leu Asp Asp Tyr

20 25 30

Val Thr Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Lys Arg Asn Phe Thr Ser Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Thr Pro Ile Leu Pro Ser Leu Ala Phe Gly Glu Val Cys Ser Leu Gln

100 105 110

Thr Glu Phe Arg Ser Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 12

<211> 384

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caggcgcgtt tcgtggagtc cgggggagga ttggtgcagt ctgggggatc tcttgtactc 60

tcctgtacag tttctgacgt cactttggat gattatgtca cagcgtggtt ccggcaggcc 120

ccagggaagg agcgtgaggg ggtctcctgt attagtaaga ggaatttcac gtcgtatgtg 180

gactccgtga agggccgatt caccacctcc agggacatca ctaagaatac ggtgcttcta 240

cagatgaacg ctctaacacc tgaggataca ggcgtttatt actgtgcgac gccgatttta 300

ccttcacttg cgttcggtga agtgtgctcc ctacagaccg agtttcgttc ctggggcccg 360

gggacccggg tcaccgtctc ctca 384

<210> 13

<211> 390

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

caggcggcac gtttcgtgga gtccggggga ggattggtgc agtctggggg atctcttgta 60

ctctcctgta cagtttctga cgtcactttg gatgattatg tcacagcgtg gttccggcag 120

gccccaggga aggagcgtga gggggtctcc tgtattagta agaggaattt cacgtcgtat 180

gtggactccg tgaagggccg attcaccacc tccagggaca tcactaagaa tacggtgctt 240

ctacagatga acgctctaac acctgaggat acaggcgttt attactgtgc gacgccgatt 300

ttaccttcac ttgcgttcgg tgaagtgtgc tccctacaga ccgagtttcg ttcctggggc 360

ccggggaccc gggtcaccgc ggtctcctca 390

<210> 14

<211> 384

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caggcgcgtt tcgtggagtc cgggggagga ttggtgcagt ctgggggatc tcttgtactc 60

tcctgtacag tttctgacgt cactttggat gattatgtca cagcgtggtt ccggcaggcc 120

ccagggaagg agcgtgaggg ggtctcctgt attagtaaga ggaatttcac gtcgtatgtg 180

gactccgtga agggccgatt caccacctcc agggacatca ctaagaatac ggtgcttcta 240

cagatgaacg ctctaacacc tgaggataca ggcgtttatt actgtgcgac gccgatttta 300

ccttcacttg cgttcggtga agtgtgctcc ctacagaccg agtttcgttc ctggggcccg 360

gggacccggg tcaccgtctc ctca 384

<210> 15

<211> 384

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

caggccagat tcgtggaatc tggcggagga ctggtgaagc ccggcggcag cctcagactg 60

agctgcaccg tgtccgacgt gacactggac gactatgtga ccgcctggtt ccggcaggct 120

cctggcaagg gccgggaagg cgtgtcttgt atcagcaagc ggaacttcac aagctacgcc 180

gatagcgtga aaggcagatt taccaccagc agagatatca ccaagaatac cgtgctgctg 240

cagatgaaca gcctgagggc cgaggacacc gctgtgtact actgcgccac accaatcctg 300

ccttctctgg ccttcggcga ggtgtgcagc ctgcaaacag agttcagaag ctggggacag 360

ggcaccaccg tcacagtttc cagc 384

<210> 16

<211> 815

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gccaccatgg cactccccgt caccgccctt ctcttgcccc tcgccctgct gctgcatgct 60

gccaggcccg acattgtgct cactcagtca cctcccagcc tggccatgag cctgggaaaa 120

agggccacca tctcctgtag agccagtgag tccgtcacaa tcttggggag ccatcttatt 180

cactggtatc agcagaagcc cgggcagcct ccaacccttc ttattcagct cgcgtcaaac 240

gtccagacgg gtgtacctgc cagattttct ggtagcgggt cccgcactga ttttacactg 300

accatagatc cagtggaaga agacgatgtg gccgtgtatt attgtctgca gagcagaacg 360

attcctcgca catttggtgg gggtactaag ctggagatta agggaagcac gtccggctca 420

gggaagccgg gctccggcga gggaagcacg aaggggcaaa ttcagctggt ccagagcgga 480

cctgagctga aaaaacccgg cgagactgtt aagatcagtt gtaaagcatc tggctatacc 540

ttcaccgact acagcataaa ttgggtgaaa cgggcccctg gaaagggcct caaatggatg 600

ggttggatca ataccgaaac tagggagcct gcttatgcat atgacttccg cgggagattc 660

gccttttcac tcgagacatc tgcctctact gcttacctcc aaataaacaa cctcaagtat 720

gaagatacag ccacttactt ttgcgccctc gactatagtt acgccatgga ctactgggga 780

cagggaacct ccgttaccgt cagttccgcg gccgc 815

<210> 17

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

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> 18

<211> 1053

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

caggcgcgtt tcgtggagtc cgggggagga ttggtgcagt ctgggggatc tcttgtactc 60

tcctgtacag tttctgacgt cactttggat gattatgtca cagcgtggtt ccggcaggcc 120

ccagggaagg agcgtgaggg ggtctcctgt attagtaaga ggaatttcac gtcgtatgtg 180

gactccgtga agggccgatt caccacctcc agggacatca ctaagaatac ggtgcttcta 240

cagatgaacg ctctaacacc tgaggataca ggcgtttatt actgtgcgac gccgatttta 300

ccttcacttg cgttcggtga agtgtgctcc ctacagaccg agtttcgttc ctggggcccg 360

gggacccggg tcaccgtctc ctcaaccacg acgccagcgc cgcgaccacc aacaccggcg 420

cccaccatcg cgtcgcagcc cctgtccctg cgcccagaag cgtgccggcc agcggcgggg 480

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatttg ggcccctctg 540

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt 600

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 660

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc 720

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac 780

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg 840

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag 900

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga 960

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat 1020

gacgctcttc acatgcaggc cctgccgcct cgg 1053

<210> 19

<211> 1053

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

caggccagat tcgtggaatc tggcggagga ctggtgaagc ccggcggcag cctcagactg 60

agctgcaccg tgtccgacgt gacactggac gactatgtga ccgcctggtt ccggcaggct 120

cctggcaagg gccgggaagg cgtgtcttgt atcagcaagc ggaacttcac aagctacgcc 180

gatagcgtga aaggcagatt taccaccagc agagatatca ccaagaatac cgtgctgctg 240

cagatgaaca gcctgagggc cgaggacacc gctgtgtact actgcgccac accaatcctg 300

ccttctctgg ccttcggcga ggtgtgcagc ctgcaaacag agttcagaag ctggggacag 360

ggcaccaccg tcacagtttc cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 420

cccaccatcg cgtcgcagcc cctgtccctg cgcccagaag cgtgccggcc agcggcgggg 480

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatttg ggcccctctg 540

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt 600

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 660

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc 720

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac 780

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg 840

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag 900

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga 960

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat 1020

gacgctcttc acatgcaggc cctgccgcct cgg 1053

<210> 20

<211> 1484

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gccaccatgg cactccccgt caccgccctt ctcttgcccc tcgccctgct gctgcatgct 60

gccaggcccg acattgtgct cactcagtca cctcccagcc tggccatgag cctgggaaaa 120

agggccacca tctcctgtag agccagtgag tccgtcacaa tcttggggag ccatcttatt 180

cactggtatc agcagaagcc cgggcagcct ccaacccttc ttattcagct cgcgtcaaac 240

gtccagacgg gtgtacctgc cagattttct ggtagcgggt cccgcactga ttttacactg 300

accatagatc cagtggaaga agacgatgtg gccgtgtatt attgtctgca gagcagaacg 360

attcctcgca catttggtgg gggtactaag ctggagatta agggaagcac gtccggctca 420

gggaagccgg gctccggcga gggaagcacg aaggggcaaa ttcagctggt ccagagcgga 480

cctgagctga aaaaacccgg cgagactgtt aagatcagtt gtaaagcatc tggctatacc 540

ttcaccgact acagcataaa ttgggtgaaa cgggcccctg gaaagggcct caaatggatg 600

ggttggatca ataccgaaac tagggagcct gcttatgcat atgacttccg cgggagattc 660

gccttttcac tcgagacatc tgcctctact gcttacctcc aaataaacaa cctcaagtat 720

gaagatacag ccacttactt ttgcgccctc gactatagtt acgccatgga ctactgggga 780

cagggaacct ccgttaccgt cagttccgcg gccgcaccac gacgccagcg ccgcgaccac 840

caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagaa gcgtgccggc 900

cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat atctacattt 960

gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc actctttact 1020

gtaagcgcgg tcggaagaag ctgctgtaca tctttaagca acccttcatg aggcctgtgc 1080

agactactca agaggaggac ggctgttcat gccggttccc agaggaggag gaaggcggct 1140

gcgaactgcg cgtgaaattc agccgcagcg cagatgctcc agcctacaag caggggcaga 1200

accagctcta caacgaactc aatcttggtc ggagagagga gtacgacgtg ctggacaagc 1260

ggagaggacg ggacccagaa atgggcggga agccgcgcag aaagaatccc caagagggcc 1320

tgtacaacga gctccaaaag gataagatgg cagaagccta tagcgagatt ggtatgaaag 1380

gggaacgcag aagaggcaaa ggccacgacg gactgtacca gggactcagc accgccacca 1440

aggacaccta tgacgctctt cacatgcagg ccctgccgcc tcgg 1484

Claims

1. A BCMA single domain antibody, wherein said BCMA single domain antibody comprises a heavy chain variable region, said heavy chain variable region comprising a complementarity determining region; the complementarity determining regions of the heavy chain variable region include CDR1, CDR2, CDR 3; the sequence of the CDR1 is SEQ ID NO. 1; the sequence of the CDR2 is SEQ ID NO. 2; the sequence of the CDR3 is SEQ ID NO. 3.

2. The BCMA single domain antibody according to claim 1, characterized in that the heavy chain variable region amino acid sequence is one of SEQ ID No. 4-6.

3. The BCMA single domain antibody according to claim 2, characterized in that the heavy chain variable region amino acid sequence is the humanized sequence of any one of SEQ ID No. 4-6; the humanized sequence is one of SEQ ID NO. 7-11.

4. A BCMA chimeric antigen receptor based on single domain antibodies, wherein said antigen recognition domain of said BCMA chimeric antigen receptor is one or more of the BCMA single domain antibodies of any one of claims 1 to 3.

5. A coding gene encoding the BCMA single domain antibody according to any one of claims 1 to 3 or the BCMA chimeric antigen receptor according to claim 4.

6. The encoding gene of claim 5, wherein the sequence of the encoding gene comprises one of SEQ ID nos. 12-15.

7. A viral vector carrying the coding gene of claim 5 or 6.

8. The viral vector according to claim 7, wherein the viral vector is a BCMA chimeric antigen receptor lentiviral vector; the BCMA chimeric antigen receptor lentiviral vector is a pCDH-EF1a-BCMA CAR viral vector.

9. An antibody conjugate comprising a BCMA single domain antibody according to any one of claims 1 to 3.

10. A CAR-T cell produced by transducing a human T lymphocyte with the viral vector of claim 7 or 8.

11. Use of a BCMA single domain antibody according to any of claims 1 to 3 and/or a BCMA chimeric antigen receptor according to claim 4 and/or a coding gene according to claim 5 or 6 and/or a viral vector according to claim 7 or 8 and/or an antibody conjugate according to claim 9 and/or a CAR-T cell according to claim 10 in the manufacture of a medicament for the treatment of a neoplasm which is multiple myeloma expressing BCMA.

12. A tumor diagnostic kit comprising the BCMA single domain antibody according to any one of claims 1 to 3 and/or the BCMA chimeric antigen receptor according to claim 4 and/or the coding gene according to claim 5 or 6 and/or the viral vector according to claim 7 or 8 and/or the antibody conjugate according to claim 9 and/or the CAR-T cell according to claim 10.

13. A medicament for the treatment and/or prognosis and/or prevention of tumors, comprising a BCMA single domain antibody according to any one of claims 1 to 3 and/or a BCMA chimeric antigen receptor according to claim 4 and/or a coding gene according to claim 5 or 6 and/or a viral vector according to claim 7 or 8 and/or an antibody conjugate according to claim 9 and/or a CAR-T cell according to claim 10.