CN112812186B - Nanometer antibody for resisting B cell mature antigen and application thereof - Google Patents

Abstract

The invention provides a nano antibody for resisting a B cell maturation antigen and application thereof. The heavy chain variable region of the nano antibody for resisting the B cell maturation antigen comprises: complementarity determining region 1 shown in SEQ ID NO.1, complementarity determining region 2 shown in SEQ ID NO.2, and complementarity determining region 3 shown in SEQ ID NO. 3. The antibody obtained by screening the phage display nano antibody library has a specific CDR (complementary deoxyribonucleic acid) region, can be specifically combined with a BCMA (brain cell antigen) and has better affinity; the antigen is used as an antigen binding domain to construct a chimeric antigen receptor and a CAR-T cell, and has obvious killing activity on BCMA positive tumor cells. Therefore, the nano antibody has wide application prospect in the aspect of immunotherapy of multiple myeloma.

Description

Nanometer antibody for resisting B cell mature antigen and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a nano antibody for resisting a B cell maturation antigen and application thereof.

Background

Multiple Myeloma (MM) is a malignant proliferative disease of plasma cells characterized by abnormal proliferation of clonal plasma cells in the bone marrow, secretion of monoclonal immunoglobulins or fragments thereof (M protein), and resulting in damage to associated organs or tissues. The common clinical manifestations are bone pain, anemia, renal insufficiency and infection. MM accounts for approximately 10% of hematological malignancies that are the second most prevalent in many countries. China has no exact epidemiological survey data of the incidence rate of MM, generally, the number of the data is estimated to be about 1/10 ten thousand, and the median survival time of the multiple myeloma is 3.5.

Current induction therapy for MM, while showing some efficacy, eventually almost all patients relapse and die. Allogeneic hematopoietic stem cell transplantation can eliminate myeloma cells, but the side effects of this therapy are very significant and only a few benefit. In addition, nanobodies also show some potential in the treatment of myeloma, but there is currently insufficient clinical data to justify. Therefore, it is of great interest to develop new methods for the effective treatment of multiple myeloma.

The concept of Chimeric antigen receptor modified T lymphocytes (CAR-T) was developed as early as 1989, but the ideal effect has not been achieved in clinical trials. Over the next two decades scientists have continually tried and optimized this until 2011, CD19(B lymphocyte antigen CD19, CD19) targeted CAR-T cells were used in clinical studies of relapsed/refractory chronic B-lymphocyte leukemia and achieved dramatic efficacy in the cure. To this end, the use of CAR-T cells for tumor therapy opens new sections.

The chimeric antigen receptor is an artificially synthesized fusion protein with a function similar to that of a T cell receptor, and mainly comprises a signal peptide, an antigen recognition region, a hinge region, a transmembrane region and an intracellular signal region. Upon binding to the target antigen, the chimeric antigen receptor forms a dimer, activates T cells by intracellular signaling molecules, secretes perforin and granzyme B, etc., to achieve killing of the target cells. Thus, CAR-T cells recognize target cells independently of MHC (MHC), thereby avoiding immune escape due to down-regulation of tumor cell MHC molecules.

In recent years, chimeric antigen receptor T lymphocytes (CAR-T) are rapidly developed, two products are approved by FDA in the United states and are on the market, and a plurality of products are approved by drug clinical trials in China.

The B cell maturation antigen (BCMA, also known as CD269), which is expressed only on the surface of mature B cells, is an important B cell biomarker. The gene encoding BCMA belongs to a member of the TNF receptor superfamily, which is preferentially expressed in mature B lymphocytes and plays an important role in B cell development and autoimmune response. The receptor specifically binds to tumor necrosis factor (ligand) superfamily member 13B (TNFSF13B/TALL-1/BAFF) and causes activation of NF-. kappa.B and MAPK 8/JNK. This receptor also binds to various members of the TRAF family, mediating signals for cell survival and proliferation. In multiple myeloma, BCMA expression increases, a proliferation promoting signal increases, and finally canceration occurs.

In multiple myeloma cells, BCMA is expressed at levels significantly higher than healthy plasma cells and is an ideal target. Therefore, the BCMA CAR-T prepared by introducing the BCMA-resistant chimeric antigen receptor gene into the T cell by a genetic engineering method can ensure that the BCMA CAR-T cell specifically recognizes and kills the BCMA-expressing multiple myeloma cells, thereby realizing the anti-tumor effect of the BCMA-expressing multiple myeloma cells, and having great clinical transformation value on a BCMA-targeted immunotherapy method.

Disclosure of Invention

In view of the problems in the prior art, the present invention aims to provide a nanobody against B cell maturation antigen and its application. The anti-BCMA monoclonal antibody has high affinity, can be used as an antigen binding domain of a chimeric antigen receptor molecule to construct CAR-T cells, and the obtained CAR-T cells have good application prospects in the aspect of tumors.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a nanobody against B Cell Maturation Antigen (BCMA), the heavy chain variable region of which comprises:

(1) CDR1 comprising an amino acid sequence shown in SEQ ID No.1, wherein the sequence of SEQ ID No.1 is GYSDSNYC;

(2) CDR2 comprising the amino acid sequence shown in SEQ ID No.2, wherein the sequence of SEQ ID No.2 is INGDGVI;

(3) CDR3 comprising the amino acid sequence shown in SEQ ID No.3, the sequence of SEQ ID No.3 being AALTAGCVRYAA.

In the invention, the plasmid containing BCMA protein extracellular region gene segment is introduced into eukaryotic cell Freestyle by a transient transfection mode ^TM 293F cells are expressed, and the recombinant protein is used for immunizing an unimmunized bactrian camel to construct a phage display nano antibody library. Screening anti-BCMA antibodies according to the phage display nano antibody library, and when CDR regions of heavy chain variable regions of the nano antibodies are respectively amino acid sequences shown in SEQ ID No. 1-3, the obtained monoclonal antibodies can be specifically combined with the BCMA antibodies And (6) originally.

As a preferred technical scheme of the invention, the heavy chain variable region of the nano antibody comprises any one or the combination of at least two of the amino acid sequences described by SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7.

Preferably, the heavy chain variable region of the nanobody further comprises: framework region 1(FR1) shown by SEQ ID NO.4, framework region 2(FR2) shown by SEQ ID NO.5, framework region 3(FR3) shown by SEQ ID NO.6, and framework region 4(FR4) shown by SEQ ID NO. 7.

Preferably, the heavy chain variable region of the nanobody comprises the amino acid sequence shown in SEQ ID NO. 8.

In the invention, a BCMA immune camel VHH library is screened by utilizing a phage display technology to obtain the nano antibody shown in SEQ ID NO.8, the affinity of the nano antibody to BCMA is high, and the nano antibody has an important application prospect in the aspect of constructing a chimeric antigen receptor targeting BCMA.

Wherein, the sequences of SEQ ID No. 4-7 are shown in the following table 1:

TABLE 1

SEQ ID No.8：

EVQLVESGGGPVQAGGSLRLSCTASGYSDSNYCMAWFRQAPGKARQGVAFINGDGVITYTDSVKGRFTISKDNAQKTLDLQMNSLKPEDTAMYYCAALTAGCVRYAAWGQGTQVTVSS

In a second aspect, the present invention provides a nucleic acid molecule encoding the nanobody of the first aspect.

Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO. 9.

SEQ ID No.9：

GAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCCCGTGCAGGCCGGCGGCAGCCTGAGACTGAGCTGCACCGCCAGCGGCTACAGCGACAGCAACTACTGCATGGCCTGGTTCAGACAGGCCCCCGGCAAGGCCAGACAGGGCGTGGCCTTCATCAACGGCGACGGCGTGATCACCTACACCGACAGCGTGAAGGGCAGATTCACCATTAGCAAAGATAATGCCCAGAAAACACTGGATCTGCAGATGAATAGCCTGAAACCTGAAGATACAGCCATGTATTATTGTGCCGCCCTGACAGCCGGATGTGTGAGATATGCCGCCTGGGGACAGGGCACACAGGTGACAGTGTCTAGC。

In a third aspect, the present invention also provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, and a signaling domain; the antigen binding domain is a nanobody according to the first aspect.

Preferably, the signal peptide comprises a CD8 a signal peptide.

Preferably, the hinge region comprises a CD8 a hinge region.

Preferably, the transmembrane region comprises any one of or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region.

Preferably, the signaling domain comprises an immunoreceptor tyrosine activation motif (CD3 ζ).

Preferably, the signaling domain further comprises a co-stimulatory molecule comprising any one of the 4-1BB, CD28 intracellular region, OX40, ICOS or DAP10 intracellular region or a combination of at least two thereof.

Preferably, the chimeric antigen receptor comprises a CD8 a signal peptide, the nanobody of the first aspect, a CD8 a hinge region, a CD8 a transmembrane region, and an immunoreceptor tyrosine-activation motif.

In a fourth aspect, the present invention also provides an expression vector comprising a gene encoding the chimeric antigen receptor of the third aspect.

Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to the third aspect, preferably a lentiviral vector.

In a fifth aspect, the present invention provides a recombinant lentivirus prepared from a mammalian cell transfected with the expression vector and helper plasmid of the fourth aspect.

In a sixth aspect, the present invention provides a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the third aspect.

Preferably, the chimeric antigen receptor immune cell comprises the expression vector of the fourth aspect and/or the recombinant lentivirus of the fifth aspect.

Preferably, the immune cells comprise any one of T cells, B cells, NK cells, mast cells or macrophages or a combination of at least two thereof.

In a seventh aspect, the present invention also provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell of the sixth aspect.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

In an eighth aspect, the nanobody of the first aspect, the nucleic acid molecule of the second aspect, the chimeric antigen receptor of the third aspect, the expression vector of the fourth aspect, the recombinant lentivirus of the fifth aspect, the chimeric antigen receptor immune cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect is used for preparing a tumor therapeutic drug.

Preferably, the neoplasm comprises multiple myeloma.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) according to the invention, a BCMA recombinant protein is used for immunizing unimmunized bactrian camel to construct a phage display nano antibody library in a transient transfection manner, anti-BCMA antibodies are screened according to the phage display nano antibody library, when CDR regions of heavy chain variable regions of the nano antibodies are respectively amino acid sequences shown in SEQ ID No. 1-3, the obtained monoclonal antibodies can be specifically combined with BCMA antigens, the affinity is good, and the determination of the antibody affinity shows that the antibody has the advantages that the Ka (1/Ms) is 2.70E +06, the Kd (1/s) is 2.43E-04, and the KD (M) is 9.01E-11;

(2) the anti-BCMA nano antibody provided by the invention has better affinity, is used as an antigen binding structural domain to construct a chimeric antigen receptor, and utilizes the chimeric antigen receptor to prepare a T cell, and experiments verify that the constructed CAR-T cell has killing activity on BCMA positive tumor cells, and efficiently secretes cell factors IL-2, TNF-alpha and IFN-gamma after being co-cultured with the BCMA positive cells, so that the nano antibody, the chimeric antigen receptor constructed by the nano antibody and the CAR-T cell can improve the killing capacity on tumor cells, particularly multiple myeloma.

Drawings

FIG. 1 is a graph of the affinity of Biacore for detecting BCMA nanobodies in example 3.

FIG. 2 is a graph showing the results of FACS detection of BCMA antigen recognition by BCMA nanobodies in example 4.

FIG. 3 is a plasmid map of pSIN-HD BCMACAR 8# lentiviral vector in example 5.

FIG. 4 is a schematic diagram of the structure of a chimeric antigen receptor expressing BCMA in example 5.

FIG. 5 is a graph showing the results of flow-based assay of the expression rate of chimeric antigen receptor of T lymphocytes in example 7, wherein the graph I is a blank control and the graph II is a BCMA-8-containing CAR T cells.

FIG. 6 is a graph of the results obtained by FACS detection of T cells and BCMA CAR-T cell phenotypes in example 7.

Figure 7A is a graph of the killing effect of BCMA CAR-T cells on K562 cells in example 8.

Figure 7B is a graph of the killing effect of BCMA CAR-T cells on RPMI 8226 cells in example 8.

Figure 7C is a graph of the killing effect of BCMA CAR-T cells on U266 cells in example 8.

FIG. 8 is a histogram of IL-2 secretion by BCMACAR-T cells in example 9.

FIG. 9 is a histogram of TNF α secretion by BCMA CAR-T cells of example 9.

Figure 10 is a histogram of the secretion of IFN γ by BCMA CAR-T cells in example 9.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1

This example was used for phage nanobody library construction and panning, and for primary screening using ELISA. The method comprises the following specific steps:

(1) construction of phage Nanobody libraries

Adopting BCMA-Fc expressing the extracellular region to immunize bactrian camel, and extracting 200mL of peripheral blood after the titer is verified by ELISA; sorting lymphocytes to obtain peripheral blood mononuclear lymphocyte sediment and extracting RNA; by using

III, synthesizing first strand cDNA by using reverse transcriptase with RNA as a template, and then amplifying a VHH gene by using nested PCR; inserting VHH gene into pMECS phage display carrier, after electrotransformation of TG1 competent cell, taking appropriate amount of bacterial liquid for library identification, uniformly coating the rest culture on LB/AMPGLU flat plate, collecting bacterial lawn after bacterial growth, adding 1/3 volume of 50% glycerol, uniformly mixing and subpackaging, and storing at-80 deg.C to obtain the final product with storage capacity greater than 10 ⁹ The phage display camelid VHH immune library of (a).

(2) Panning of phage Nanobody libraries

3 rounds of solid-phase screening are carried out on the immune nano antibody library in vitro, and phage clones with binding activity are enriched; after prokaryotic induction expression is carried out on the monoclonal phage, phage clones capable of combining with BCMA antigen extracellular regions are further screened out through ELISA. The method comprises the following specific steps:

firstly, diluting the purified BCMA-His recombinant protein to 4 mu g/mL by using PBS buffer solution, taking a 96-well enzyme label plate, selecting 3 wells, adding 100 mu L (400 ng/well) into each well, coating overnight at 4 ℃, and using PBS as negative control; discarding the coating solution, adding 150 mu L of 2% skimmed milk powder into each hole, sealing at room temperature for 1h to obtain an ELISA plate coated with BCMA-His recombinant protein;

then, the enzyme-linked plate was washed 4 times with PBST, and the prepared phage solution was diluted to 5X 10 with 2% skim milk powder ¹¹ PFU/mL, at 1Adding the enzyme label plate with the amount of 00 mu L/hole, and incubating for 2h at room temperature;

then, discarding the phage sample, washing with PBST for 10 times, then washing with PBS for 5 times, adding 100 μ L of freshly prepared 0.1M triethylamine to each well, standing at room temperature for 10min, sucking out the eluate, and rapidly neutralizing with an equal volume of 1M Tris-HCl (pH 7.4);

measuring bacteriophage titer with part of eluate, and infecting with 4mL fresh culture log-phase TG1 bacterial solution (OD) with 400 μ L eluate ₆₀₀ About 0.6), incubating at 37 deg.C for 30min, adding 16mL 2 XYT/ampicillin/glucose (2 XYT/AMP-GLU), culturing at 37 deg.C for 200r/min to OD ₆₀₀ 0.6 to 0.8.

Taking 100 mu L of bacterial suspension, performing gradient dilution, and uniformly smearing the bacterial suspension on a 2 XYT/AMP-GLU agar plate so as to perform library capacity and diversity determination; inoculating 100 μ L bacterial suspension, namely phage display carrier library, into a 2 XYT/AMP-GLU culture medium, culturing to logarithmic phase, adding auxiliary phage, performing library rescue, obtaining phage titer to be measured of phage particles, and concentrating and purifying to obtain phage particles for next round of screening; the screening operation was repeated 3 times;

the remaining bacterial liquid was centrifuged and resuspended in 2 XYT medium of appropriate volume, spread on a plate with screening resistance for overnight culture, scraped from the plate with appropriate volume of liquid medium, resuspended in 2 XYT medium containing 1/3 volume of 50% glycerol, and then aliquoted and stored at-80 ℃.

(3) Phage packaging

100 μ L of the frozen stock was added to 100mL of 2 XYT/AMP-GLU medium and cultured at 37 ℃ with shaking (200rpm) until logarithmic phase (OD) ₆₀₀ The value is 0.6 to 0.8); add 90. mu.L of the helper phage M13K07 (1.7X 10) ¹³ PFU/mL), standing at 37 ℃ for 30min, centrifuging at 2800g for 10min to collect the thallus, resuspending with 200mL of 2 XYT/AMP-KAN medium, and culturing at 37 ℃ for 12h with shaking (200 rpm);

centrifuging at 4 deg.C for 30min at 3800g for 30min to remove thallus and collect supernatant, adding 1/5 volume of precooled PEG/NaCl, mixing, and precipitating bacteriophage for 2 h; centrifuging at 4 ℃ and 3800g for 30min, collecting the phage, resuspending with a final volume of 2mL PBS solution and transferring to a 15mL centrifuge tube; centrifuging at 4 ℃ and 12000g for 15min, collecting supernatant, adding 1/5 volumes of precooled PEG/NaCl solution, turning upside down and mixing uniformly, and standing on ice for 2 h; centrifuging at 4 deg.C and 10000g for 10min, discarding supernatant, resuspending phage precipitate with 1mL PBS, incubating overnight at 4 deg.C with shaking table to dissolve phage particles completely, mixing phage solution with equal volume of 60% glycerol, packaging into 1.5mL EP tube, and storing at-80 deg.C.

As the BCMA antigen is adopted to carry out 3 rounds of panning on the phage library in the step (2), in order to avoid losing the diversity of sequences, the preliminary ELISA screening is carried out on products of panning of the 2 nd round and the 3 rd round, positive clones are randomly selected from the panning products and are induced to express, the expression supernatant is a crude VHH antibody, the VHH antibody sequence of the monoclonal strain is determined by sequencing, the antibody sequence is marked as BCMA-8, and the sequence is shown as SEQ ID NO. 8.

Example 2

Candidate clones were screened using flow cytometric activated Cell Sorting (FACS) in this example.

Performing cell culture according to a standard cell culture scheme, and preparing BCMA positive and negative cell suspensions by using pancreatin digestive cells; the culture medium was removed by centrifugation at 300g for 5min, and the cells were resuspended to 2X 10 with a Flow Buffer ⁶ cell/mL; adding 2X 10 to each well in a V-bottom 96 well plate ⁵ Centrifuging 300g of each cell for 5min, removing supernatant, adding a VHH antibody crude extract to resuspend the cells, and incubating at 4 ℃ for 1 h;

centrifuging 300g for 5min, removing supernatant, resuspending cells by using Flow Buffer, diluting the APC anti-his antibody to 2 μ g/mL by using the Flow Buffer, resuspending cells by 100 μ L per well, and incubating for 1h at 4 ℃; after 3 times of washing the cells with Flow Buffer, 200. mu.L of Flow Buffer was used to resuspend the cells and examined by Flow cytometry.

Example 3

In this example, VHH-mIgG2a Fc nanobody was expressed and purified, and the affinity of the antibody was determined. To further identify the antibodies screened, it is necessary to express the antibodies by mammalian cells. Therefore, a plasmid vector for expressing VHH with a mouse Fc tag is firstly constructed and is marked as C-4 pCP.Stuffer-mCg2a-FC, and the specific steps are as follows:

1. BCMA VHH B8 was amplified using PCR with primers:

HDB8-F(SEQ ID NO.10)：

CGCGATTCTTAAGGGTGTCCAGTGCGAGGTGCAGCTGGTGGA；

HD-B8-R(SEQ ID NO.11)：

GCATGGAGGACAGGGCTTGATTGTGGGGCTAGACACTGTCACCTG

the reaction system is shown in Table 2, and the amplification procedure is shown in Table 3 below:

TABLE 2

TABLE 3

2. The enzyme digestion system is shown in Table 4, the enzyme digestion temperature is 37 ℃, the time is 6h, the carrier after enzyme digestion is used

The PCR purification kit was purified, and the recovered DNA was dissolved in 45. mu.L of water to detect the concentration of the DNA.

TABLE 4

Reagent	Amount of the composition used
		C-4pCP.Stuffer-mCg2a-FC	～5μg
10 Xdigestion Buffer (10 Xreaction Buffer)	5μL
		FspA I	2μL
PfI 23II	2μL
		ddH 2 O	Make up to 50. mu.L

3. The PCR amplification product was ligated into the enzyme-digested linearized vector by homologous recombination, as shown in Table 5, in a 37 ℃ water bath for 30 min.

TABLE 5

Reagent	Volume (μ L)
		Exnase II	1
2×Exnase II buffer	2
		Linearized vector	4
Insert (Insert fragment)	3

4. All the homologous recombination reaction systems are added into DH5 alpha competent cells, and DH5 alpha competent cells are transformed, and the transformation conditions are shown in Table 6.

TABLE 6

Procedure	Temperature of	Time
			Ice bath
	0℃	5min
			Heat shock	42℃	1min
Ice bath	0℃	3min
			Adding 500. mu.L LB medium, shaking at 220rpm	37℃	1.5h
Pipette
	200. mu.L of the solution and spread on LB/Amp plates	37℃	Overnight

5. Selecting monoclonal PCR for pre-identification by the transformation plate, wherein the conditions of a PCR identification system are shown in Table 7; the conditions are pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, extension at 72 ℃ for 5min, storage at 4 ℃, and sending to a sequencing company for sequencing and identification. The sequencing result is in line with the expectation, and the plasmid vector with the mouse Fc tag for expressing VHH is successfully constructed in the example of the specification.

TABLE 7

293E cells were passaged to a cell density of about 0.6X 10 approximately 24h prior to plasmid transfection ⁶ cells/mL; when the cell density is (1.0-1.2) × 10 ⁶ cells/mL, viability>At 95%, 293E cells were transfected with PEI at a ratio of 0.15. mu.g scFV-mIgG1/100mL 293E, wherein the ratio of plasmid DNA to PEI was 1: 2;

37℃、130rpm、8％CO ₂ the cell culture supernatant was collected by shaking for 5 days at 3000rpm for 30min, filtered through a Millex-GP Filter Unit 0.45 μm Sterile, and subjected to MabSelect ^TM SuRe ^TM Concentrate by centrifugation, wash the column with 1 × PBS, elute protein with 0.1M Gly-HCl, and neutralize with 1/10 volumes of Tris-HCl at pH 8.5.

Protein dialysis overnight at 4 ℃ A was measured using NanoDrop 2000 ₂₈₀ The method of (3) is quantitative, and the SEC-HPLC is used for determining the purity of the antibody.

In addition, in this example, affinity of purified BCMA VHH antibody was measured by Biacore.

Biacore is a bioanalytical sensing technology developed based on Surface Plasmon Resonance (SPR), and can detect the whole process of change of binding and dissociation of molecules in a tracking solution and molecules fixed on the surface of a chip, record the change in the form of a sensorgram, and provide kinetic and affinity data.

In the measurement process, the antibody is immobilized on the surface of the chip, and the mobile phase is a solution containing the antigen. The measurement results are shown in table 8 and fig. 1.

TABLE 8

Example 4

In this example, single chain antibodies against BCMA VHH were subjected to flow assay.

Three tumor cells, K562(BCMA-), RPMI 8226(BCMA +), and U266(BCMA +), were mixed with purified recombinant anti-BCMA VHH-mIgG2 antibody, incubated for 30min under ice bath, then incubated for 30min with APC-labeled goat anti-mouse IgG antibody, and detected by flow cytometry.

As shown in FIG. 2, the single-chain antibody recognized BCMA antigen on the cell surface, which was 84.9% and 95.2% in RPMI 8226(BCMA +) and U266(BCMA +), respectively.

Example 5

This example was used to prepare lentiviral vectors expressing chimeric antigen receptors for BCMA VHH.

First, a lentiviral vector pSIN-HD BCMA CAR 8# carrying a BCMA VHH chimeric antigen receptor was constructed, the vector map is shown in FIG. 3, and the schematic representation of the chimeric antigen receptor is shown in FIG. 4, comprising the CD8 α signal peptide, BCMA VHH, CD8 α hinge region, transmembrane region, and immunoreceptor tyrosine activation motif (CD3 ζ).

Wherein the amino acid sequence of the signal peptide (SEQ ID No.12) is:

MALPVTALLLPLALLLHAARP

the amino acid sequence of BCMA VHH is shown in SEQ ID No. 8;

The amino acid sequence of the CD8 α hinge and transmembrane regions (SEQ ID No.13) is:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

the amino acid sequence of the 4-1BB intracellular domain (SEQ ID No.14) is:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

the CD3 ζ amino acid sequence (SEQ ID No.15) is:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

the preparation method comprises the following steps:

1. a PCR reaction system was prepared according to table 9 to amplify BCMA VHH fragments using primers:

B8(op)-FR1F-F(E)(SEQ ID NO.16)：

CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGGAAAGCGGCGGCGGCC

B8(op)-R(SEQ ID NO.17)：

GCGCTGGCGTCGTGGTGCTAGACACTGTCACC

TABLE 9

The above reagent is from TOYOBO Inc.

After preparation, the reaction was performed according to the PCR procedure shown in Table 10.

Watch 10

2. PCR reaction systems were prepared according to table 11, and the CD8 α signal peptide was added before the amplification product obtained, using the primers:

BamH-CD8αsig-F(SEQ ID NO.18)：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC

B8(op)-R(SEQ ID NO.17)；

TABLE 11

Reagent	Volume (μ L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO 4	3
10 μ M upstream primer (BamH-CD8 α sig-F)	1
		10 μ M downstream primer	1
Template DNA (VHH fragment PCR reaction solution)	4
		Sterile deionized water (PCR grade water)	30
KOD-Plus-Neo high fidelity PCR enzyme	1

After preparation, PCR reactions were performed according to the PCR procedure shown in Table 10.

After the reaction is finished, carrying out 1% agarose gel electrophoresis on the PCR product, recovering fragments of about 500bp, and quantifying by an ultraviolet absorption method.

3. The PCR reaction system was prepared in accordance with Table 12, and after the preparation, the PCR reaction was carried out in accordance with the PCR procedure shown in Table 10. The CD8 α hinge-TM-41BB-CD3 ζ fragment was amplified using the following primers:

CD8αH-F(SEQ ID NO.19)：ACCACGACGCCAGCGCCGCGAC

Vector-R(SEQ ID NO.20)：TCGATAAGCTTGATATCG

TABLE 12

Reagent	Volume (μ L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO 4	3
10 μ M of the forward primer CD8 α H-F	1
		10 mu M downstream primer Vector-R	1
Template DNA (HD CD19CAR)	1
		Sterile deionized water (PCR grade water)	33
KOD-Plus-Neo high fidelity PCR enzyme	1

After the PCR is finished, 1% agarose gel electrophoresis is carried out, fragments of about 780bp are recovered, and the quantification is carried out by an ultraviolet absorption method.

4. Mu.g of the laboratory-constructed HD CD19CAR plasmid was digested with BamHI and EcoRI, reacted in a water bath at 37 ℃ for 2 hours, and then the vector was recovered.

The 3 fragments were ligated with a recombinase, and the recombination reaction system was as shown in Table 13, followed by a reaction in water bath at 37 ℃ for 0.5 hour after the preparation, and transformation into Escherichia coli stbl3 competent cells by a conventional method.

Watch 13

Reagent	Amount of the composition used
		HD CD19 CAR	184.54ng
CD8αsingal BCMA VHH	31.32ng
		CD8αhinge-TM-41BB-CD3ζ	29.72ng
5×CE buffer	2μL
		Exnase TM II	1μL
Sterile deionizationWater (PCR grade water)	Make up to 10. mu.L

Single clones were selected from the solid medium, cultured overnight, and identified by PCR, the PCR reaction formulations are shown in Table 14, and the PCR procedures are shown in Table 15. After the PCR is finished, the positive clone is selected for further sequencing identification, and the sequencing result is in line with expectation.

TABLE 14

Reagent	Volume (μ L)
		Taq PCR Master Mix	10
10μM F Seq-trEF1a-F	1
		10μM R Vector-R	1
Template DNA bacterial liquid	1
		Sterile deionized water (PCR grade water)	7

Watch 15

Separating the two gene fragments of the obtained signal peptide-VHH, CD8 alpha hinge region, transmembrane region and immunoreceptor tyrosine activation motif by agarose gel electrophoresis, and then recovering, purifying and quantifying by using an agarose gel DNA fragment recovery kit;

The lentiviral expression vector pRRL. EF1. alpha. -BCMA CAR-WPRE was cut with the restriction enzymes BamHI and EcoRI (from NEB) and manipulated as described. Separating the enzyme digestion product by agarose gel electrophoresis, and then recovering, purifying and quantifying by using an agarose gel DNA fragment recovery kit;

then, the two target fragments and the vector are cloned into a lentiviral vector by using a recombinase, sequencing verification is carried out, and the sequencing result is in accordance with expectation.

Example 6

In this example, lentiviral vector pSIN-HD BCMA CAR 8# prepared in example 5 was subjected to lentiviral packaging, concentration and titer detection.

(1) Lentiviral packaging

At 1.6X 10 ⁷ Cell number 293T cells were plated in 15cm dishes at 37 ℃ with 5% CO ₂ Culturing overnight to prepare packaged virus, wherein the culture medium contains DMEM and 10% Fetal Bovine Serum (FBS); dissolving lentiviral vector 30 μ g pSIN-HD BCMA CAR 8#, helper plasmid gag/pol 12.5 μ g and envelope plasmid VSVg 10 μ g into 2000 μ L serum-free DMEM culture solution, and mixing;

mu.g PEI (1. mu.g/. mu.L) was dissolved in 2000. mu.L serum free DMEM medium, gently mixed (or vortexed at 1000rpm for 5 seconds) and incubated at room temperature for 5 min; adding the PEI mixed solution into the DNA mixed solution, immediately mixing by vortex or mixing lightly, and incubating for 20min at room temperature to form a transfection compound; dripping 4mL of the transfection compound into 25mL of DMEM medium containing 293T cells, and replacing the fresh medium after 4-5 h; after 48h, the virus supernatant was collected.

(2) Lentiviral concentration

Filtering the virus supernatant with a 0.45-micron filter membrane, collecting the filtrate in a 50mL centrifuge tube, adding 1/4 PEG-NaCl virus concentrated solution, turning upside down, mixing uniformly, and standing at 4 ℃ overnight; centrifuging at 4 deg.C and 3500rpm for 30 min; removing supernatant, adding an appropriate amount of RPMI 1640 medium (containing 10% of FBS by mass), and dissolving the heavy suspension virus precipitate; subpackaging the concentrated lentivirus suspension into 50 μ L portions, storing in a finished product tube, and storing at-80 deg.C;

(3) lentiviral titer detection

500 μ L K562 cells (1X 10) ⁵ Individual cells) were seeded in 24-well culture plates, and the concentrated lentivirus was added to the cell suspension in volumes of 1. mu.L, 0.2. mu.L, and 0.04. mu.L, respectively, and polybrene was added to a final concentration of 5. mu.g/mL, 37 ℃, 5% CO ₂ After overnight culture, the fresh medium was replaced;

after infection for 72h, centrifuging for 5min at 400g, discarding the supernatant to collect cells, adding 100 μ L PBS + 2% FBS to resuspend the cells, adding 1 μ g hBCMA-EcD-Fc antibody, and incubating on ice for 30 min; washing with PBS (containing FBS with mass fraction of 2%) buffer solution for 1 time, adding 100 μ L buffer solution to resuspend cells, adding APCanti-human IgG Fc antibody, and incubating on ice for 30 min; after being washed for 2 times by PBS (containing FBS with the mass fraction of 2%), 300 mu L of buffer solution is added to resuspend cells, and a flow cytometer is adopted to detect the infection efficiency; preferably, a cell sample with the positive rate of 5-20% is taken, the titer is calculated, and the titer calculation formula is as follows: titer (TU/mL) is cell number (10) ⁵ ) X positive rate/virus volume (mL).

Example 7

In this example, T lymphocytes were transduced with the lentiviruses prepared in example 6.

(1) Diluting anti-human CD3 antibody and anti-human CD28 antibody with PBS to final concentrations of 1 μ g/mL and 0.5 μ g/mL respectively, coating the well plate, and standing overnight in a refrigerator at 4 deg.C; discarding the antibody coating solution in the pore plate, and washing twice with 1mL of PBS;

(2) human PBMC were adjusted to a density of 1X 10 with T cell culture medium (X-VIVO + 10% FBS +300U/mL IL-2) ⁶ Activation for 48 h/mL, inoculated into CD3 and CD28 antibody coated well plates; collecting activated T cells, adjusting cell density to 1 × 10 ⁶ (iv)/mL, lentivirus was added at a multiplicity of infection (MOI) of 10, polybrene was added to a final concentration of 5. mu.g/mL; at 37 ℃ with 5% CO ₂ After overnight culture in the environment, replacing a fresh culture medium, and carrying out passage every 2-3 days;

(3) after 5 days of T cell infection, 3X 10 cells were taken ⁵ Centrifuging the T cells at 4 ℃ for 5min at 400g, discarding the supernatant, and washing the cells once with PBS (containing FBS with the mass fraction of 2%); adding 100. mu.L buffer solution to resuspend the cells, adding 1. mu.g of hBCMA-EcD-Fc antibody, and incubating on ice for 30 min; washing with buffer solution for 1 time, adding 100 μ L buffer solution to resuspend cells, adding APCanti-human IgG Fc antibody, and incubating on ice for 30 min; after washing for 2 times by the buffer solution, adding 300 mu L of the buffer solution to resuspend the cells;

The expression rate of the chimeric antigen receptor of the T lymphocytes was measured by flow cytometry, and the result is shown in FIG. 5, in which FIG. I is a blank control and FIG. II is an experimental group to which BCMA-8 was added, the expression rate of the chimeric antigen receptor was 59.9%.

In addition, flow cytometry was also used to detect lymphocyte phenotypes in this example.

(1) After 5 days of T cell infection, 3X 10 cells were taken ⁵ Centrifuging the T cells at 4 ℃ for 5min at 400g, discarding the supernatant, and washing the cells once with PBS (containing FBS with the mass fraction of 2%);

(2) add 50 u L buffer heavy suspension cells, add 1 u L FITC labeled Anti-CD3 Ab, Percp-Cy5.5 labeled Anti-CD4 Ab and PE-Cy7 labeled Anti-CD8 Ab, ice incubation for 30 min; after the buffer solution is washed twice, 300 mu L of buffer solution is added to resuspend the cells, and a flow cytometer is adopted to detect the cell phenotype;

the results are shown in FIG. 6, where the proportion of CD3+ CD4+ cells was 8.66% and the proportion of CD3+ CD8+ cells was 90.6% in the untransfected T cells; the proportion of T lymphocytes CD3+ CD4+ cells in the experimental group was 29.1%, and the proportion of CD3+ CD8+ cells was 68.5%.

Example 8

In vitro toxicity experiments were performed on CAR-T cells in this example.

1. Target cell seeding

The target cell concentrations were adjusted to 1X 10 using K562(BCMA-), 8266(BCMA +), and U266(BCMA +) as target cells ⁵ mL, 100. mu.L of the suspension was inoculated into a 96-well plate;

2. effector cell inoculation:

the BCMA CAR-T and the control T cells are effector cells, and the CAR-T cells and the control T cells are added into a 96-well plate according to an effective target ratio of 0.3:1, 1:1 and 3: 1;

3. each group was set with 3 replicate wells, and the average of the 3 replicate wells was taken. Wherein each experimental group and each control group are as follows:

experimental groups: each target cell + CAR-T;

control group 1: maximal release of LDH by target cells;

control group 2: target cells spontaneously release LDH;

control group 3: the effector cells spontaneously release LDH;

4. the detection method comprises the following steps:

after the effector cells and the target cells were co-cultured for 18 hours, they were subjected to CytoTox 96 nonradioactive cytotoxicity assay kit (Promega).

The method is a detection method based on a colorimetric method, and reflects the cracking degree of cells by detecting the content of Lactate Dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released upon cell lysis in a manner substantially identical to that of 51Cr in a radioactive assay. The released LDH medium supernatant can be detected by a coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed; reference is made in particular to the instructions of the CytoTox 96 nonradioactive cytotoxicity detection kit.

5. The cytotoxicity was calculated as:

the results are shown in fig. 7A, the constructed CAR-T cells did not have a killing effect on BCMA negative cells, and as shown in fig. 7B and 7C, BCMA positive tumor cells had killing activity.

Example 9

The secretion of CAR-T cytokines was examined in this example.

1. Cell culture supernatant

400g of cell culture with an effective target ratio of 1:1 is centrifuged for 10 minutes to remove precipitates, and the supernatant is taken and stored at-80 ℃ for detection.

2. Reagent preparation

Before detection, all reagents and samples are returned to room temperature, and if the concentrated reagents are crystallized, the temperature of the concentrated reagents is warmed at 37 ℃ until all crystals are dissolved. The 1 × wash solution, 1 × detection buffer, detection antibody were prepared according to the instructions and diluted detection antibody was used within 30 minutes.

3. Standard and sample preparation

And (3) standard substance: the standard stock was diluted 2-fold using 5% 1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample preparation: samples were diluted proportionally using 5% 1640 medium.

4. Detection step

(1) Soaking the enzyme label plate: add 300. mu.L of 1 Xlotion and allow to stand for 30 seconds. Soaking is necessary to obtain the desired experimental results. After discarding the wash solution, the microplate was patted dry on absorbent paper. After the plate wash was complete, please use the plate immediately without allowing the plate to dry.

(2) Adding a standard substance: the standard was added to 100. mu.L of 2 fold diluted standard. The blank wells were filled with 100 μ L of standard dilutions (serum/plasma samples) or culture medium (cell culture supernatant samples).

(3) Adding a sample: cell culture supernatant: 100 μ L of cell culture supernatant was added to the wells.

(4) Adding a detection antibody: add 50. mu.L of diluted detection antibody per well (1:100 dilution). Ensuring continuous sample adding in the steps 4, 5 and 6 without interruption. The loading process was completed in 15 minutes.

(5) And (3) incubation: a closure plate membrane is used to close the plate. Shaking at 300 rpm, and incubating at room temperature for 2 hours.

(6) Washing: the liquid was discarded and the plate washed 6 times by adding 300. mu.L of wash solution per well. The plate was washed each time and patted dry on absorbent paper. To obtain the desired experimental performance, the residual liquid must be thoroughly removed.

(7) Adding enzyme for incubation: mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) was added to each well.

(8) And (3) incubation: the plates were sealed with a new sealing plate, shaken at 300 rpm, incubated at room temperature for 45 minutes and washed.

(9) Adding a substrate for color development: add 100. mu.L chromogenic substrate TMB to each well, protect from light, incubate for 20min at room temperature.

(10) Adding a stop solution: add 100. mu.L of stop buffer to each well. The color changed from blue to yellow. If the color is green or the color change is obviously uneven, the plate frame is tapped lightly and the mixture is mixed well.

(11) And (3) detection reading: within 30 minutes, using an enzyme-labeling instrument to carry out dual-wavelength detection, and measuring OD values under the maximum absorption wavelength of 450nm and the reference wavelength; the OD value after calibration was the value measured at 450nm minus the value measured at the reference wavelength.

The secretion results of IL-2, TNF-alpha and IFN-gamma factors are respectively shown in figure 8, figure 9 and figure 10, and the constructed CAR-T cells release cytokines for BCMA positive tumor cells, but do not have obvious cytokine secretion for BCMA negative cells.

In conclusion, the anti-BCMA antibody is obtained by screening a phage display nano antibody library, has better antibody affinity, is used as an antigen binding domain to construct a chimeric antigen receptor and a CAR-T cell, has the proportion of CD3+ CD4+ cells of 68.5% and CD3+ CD8+ cells of 66.4%, and has killing activity on BCMA positive tumor cells through cytotoxicity test detection.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Huadao (Shanghai) biopharmaceutical Co., Ltd

<120> nanometer antibody for resisting B cell maturation antigen and application thereof

<130> 20210127

<160> 20

<170> PatentIn version 3.3

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Gly Tyr Ser Asp Ser Asn Tyr Cys

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Ile Asn Gly Asp Gly Val Ile

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Ala Ala Leu Thr Ala Gly Cys Val Arg Tyr Ala Ala

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

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Ser Leu Arg Leu Ser Cys Thr Ala Ser

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

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gaggtgcagc tggtggaaag cggcggcggc cccgtgcagg ccggcggcag cctgagactg 60

agctgcaccg ccagcggcta cagcgacagc aactactgca tggcctggtt cagacaggcc 120

cccggcaagg ccagacaggg cgtggccttc atcaacggcg acggcgtgat cacctacacc 180

gacagcgtga agggcagatt caccattagc aaagataatg cccagaaaac actggatctg 240

cagatgaata gcctgaaacc tgaagataca gccatgtatt attgtgccgc cctgacagcc 300

ggatgtgtga gatatgccgc ctggggacag ggcacacagg tgacagtgtc tagc 354

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cgcgattctt aagggtgtcc agtgcgaggt gcagctggtg ga 42

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gcatggagga cagggcttga ttgtggggct agacactgtc acctg 45

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Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

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Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

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ctgccgctgg ccttgctgct ccacgccgcc aggccggagg tgcagctggt ggaaagcggc 60

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gctgcaggtc gactctagag gatcccgcca ccatggcctt accagtgacc gccttgctcc 60

tgccgctggc cttgc 75

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accacgacgc cagcgccgcg ac 22

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tcgataagct tgatatcg 18

Claims (16)

1. A nanobody against B cell maturation antigen, wherein the heavy chain variable region of the nanobody comprises:

(1) CDR1 of an amino acid sequence shown in SEQ ID NO.1, wherein the sequence of SEQ ID NO.1 is GYSDSNYC;

(2) CDR2 of the amino acid sequence shown in SEQ ID NO.2, wherein the sequence of SEQ ID NO.2 is INGDGVI;

(3) CDR3 of the amino acid sequence shown in SEQ ID NO.3, wherein the sequence of SEQ ID NO.3 is AALTAGCVRYAA.

2. The nanobody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the nanobody is represented by SEQ ID No.8, and the sequence of SEQ ID No.8 is:

EVQLVESGGGPVQAGGSLRLSCTASGYSDSNYCMAWFRQAPGKARQGVAFINGDGVITYTDSVKGRFTISKDNAQKTLDLQMNSLKPEDTAMYYCAALTAGCVRYAAWGQGTQVTVSS。

3. A nucleic acid molecule encoding the nanobody of any one of claims 1-2.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence of said nucleic acid molecule is set forth as SEQ ID No. 9.

5. A chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, and a signaling domain;

the antigen binding domain is a nanobody according to any one of claims 1-2.

6. The chimeric antigen receptor according to claim 5, wherein the signal peptide comprises a CD8 a signal peptide;

the hinge region comprises a CD8 a hinge region;

the transmembrane region comprises any one of or a combination of at least two of a CD8 alpha transmembrane region, a CD28 transmembrane region or a DAP10 transmembrane region;

the signaling domain comprises an immunoreceptor tyrosine activation motif;

the signaling domains also include co-stimulatory molecules comprising any one of the 4-1BB, CD28 intracellular region, OX40, ICOS or DAP10 intracellular region, or a combination of at least two thereof.

7. The chimeric antigen receptor according to claim 6, wherein the chimeric antigen receptor comprises a CD8 a signal peptide, the nanobody of claim 1 or 2, a CD8 a hinge region, a CD8 a transmembrane region, and an immunoreceptor tyrosine activation motif.

8. An expression vector comprising a gene encoding the chimeric antigen receptor of any one of claims 5-7.

9. The expression vector according to claim 8, wherein the expression vector is any one of a lentiviral vector, a retroviral vector or an adenoviral vector comprising the gene encoding the chimeric antigen receptor according to any one of claims 5 to 7.

10. The expression vector of claim 9, wherein the expression vector is a lentiviral vector.

11. A recombinant lentivirus produced from a mammalian cell transfected with the expression vector of any one of claims 8 to 10 and a helper plasmid.

12. A chimeric antigen receptor immune cell, wherein said chimeric antigen receptor immune cell expresses the chimeric antigen receptor of any one of claims 5-7.

13. The chimeric antigen receptor immune cell according to claim 12, wherein the chimeric antigen receptor immune cell comprises the expression vector of any one of claims 8-10 and/or the recombinant lentivirus of claim 11;

the immune cells include any one of T cells, B cells, NK cells, mast cells or macrophages or a combination of at least two of the same.

14. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of claim 12 or 13.

15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable excipient.

16. Use of a nanobody according to any one of claims 1 to 2, a nucleic acid molecule according to claim 3 or 4, a chimeric antigen receptor according to any one of claims 5 to 7, an expression vector according to any one of claims 8 to 10, a recombinant lentivirus according to claim 11, a chimeric antigen receptor immune cell according to claim 12 or 13 or a pharmaceutical composition according to claim 14 or 15 for the preparation of a medicament for the treatment of tumors;

the tumor is multiple myeloma.

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