CN112062851A

CN112062851A - Antibody targeting BCMA chimeric antigen receptor and application thereof

Info

Publication number: CN112062851A
Application number: CN201910500680.6A
Authority: CN
Inventors: 周剑锋; 谭涛超; 戴振宇; 谢萌; 汪胜义; 魏振
Original assignee: Nanjing Iaso Biotherapeutics Technology Co ltd
Current assignee: Nanjing Reindeer Biotechnology Co ltd
Priority date: 2019-06-11
Filing date: 2019-06-11
Publication date: 2020-12-11
Anticipated expiration: 2039-06-11
Also published as: CN112062851B

Abstract

The invention discloses an antibody, or scFv or fragment thereof, which specifically binds to the extracellular binding domain scFv of a Chimeric Antigen Receptor (CAR) targeting BCMA without cross-reacting with other scFv or antibody sequences. The invention also discloses the application of the antibody in detecting the CAR.

Description

Antibody targeting BCMA chimeric antigen receptor and application thereof

Technical Field

The present invention relates to antibodies targeting BCMA chimeric antigen receptors, to nucleic acid molecules encoding the antibodies and to uses thereof.

Background

Multiple myeloma (mul)tiple myeloomas, MM) is the second most common hematological malignancy next to non-hodgkin lymphoma, whose tumor cells originate from plasma cells in the bone marrow, which are currently incurable by traditional methods, progress rapidly, and have a median survival time of only 3 to 4 years^[1-2]. Adoptive therapy of Chimeric Antigen Receptor (CAR) T cells is a breakthrough new therapy for hematological malignancies. B Cell Maturation Antigen (BCMA), also known as CD269 or TNFRSF17, is a member of the tumor necrosis factor receptor family and has been identified as an important Multiple Myeloma (MM) specific target for Chimeric Antigen Receptor (CAR) T cell therapy^[3]. Previously, my company developed a chimeric antigen receptor targeting BCMA (RD103A CAR, whose extracellular domain is referred to as RD103A CAR scFv or RD103A scFv), using patient's own T cells, and after in vitro genetic modification, CAR-T cells expressing fully human 103A scFv were infused back into the patient. 103A CAR-T was shown to be very effective in preclinical and clinical studies. This product is preparing to be submitted for a first clinical trial in china. In view of the excellent clinical treatment effect, the RD103 CAR-T is expected to be widely applied, and has huge clinical application and market value.

In clinical application, the expression of the CAR gene, the expression efficiency and the activity and purity of the CAR-T cells directly influence the cancer cell clearance efficiency of the CAR-T cells. Clinical studies prove that the proliferative capacity and the curative effect of CAR-T cells in peripheral blood of patients after the CAR-T cells are back-transfused have strong correlation^[4]。

Currently, the mainstream clinical test adopts a real-time fluorescent quantitative PCR method to detect the CAR gene copy number in the peripheral blood genome, so as to indirectly estimate the CAR-T cell number. The qPCR method has several problems: 1) the qPCR detects the number of CAR genes in the genomic DNA of peripheral blood PBMC, and the expression level of CAR is not directly related to the content of the CAR genes in the genome, even the gene is not expressed due to the possibility of gene silencing; 2) the copy number of CAR in the genome can be in a dynamically changing process due to proliferation or apoptosis of different CAR-T cell clones, further leading to errors in the estimation of CAR-T cells by detecting CAR content in the peripheral blood genome; 3) the qPCR method has difficulty in further analyzing the state of the CAR-T cells in vivo, such as T cell subtypes, and brings difficulty to the deep research of CAR-T.

There is also great uncertainty in using fluorescently labeled target antigens (e.g., FITC-BCMA) to estimate CAR-T cell numbers. The main reason is the false positive signal generated by antibodies against BCMA antigens, or natural ligands of BCMA expressed on cells, which may be present in the peripheral blood of the patient. These factors can seriously affect the experimental results. There is also a case where patients, after infusing multiple CAR-ts due to the complex disease condition, cannot differentiate the number of different CAR-T cells with fluorescently labeled antigen. Therefore, the development of a technology for accurately and rapidly detecting the expression condition of the CAR is of great significance to the implementation, monitoring and deep research of the therapy.

On the other hand, the rate of recurrence within one year after complete remission with CAR-T therapy is high and literature^[5]The recurrence rate for the different CD19CAR-T therapies was (4.6% to 33.9%). Recurrence is largely divided into target antigen-negative recurrence and target antigen-positive recurrence. The former is due to failure of CAR-T therapy by the development of antigen-downregulation or loss of tumor cells; the latter is often associated with CAR-T cells expanding and having a short lifetime in vivo. It is currently believed that in addition to the patient's T cell being deficient in itself, the production of antibodies (ADA) and killer T Cells (CTL) against CAR-T cells in the patient is the main possible cause of rapid CAR-T failure^[5]. Therefore, detecting whether ADA is generated in a reinfused patient and aiming at CAR-T scFv, and quantifying the concentration of ADA can analyze the correlation between the concentration of ADA and the survival of CAR-T cells, and is of great significance for the research of CAR-T products. At present ADA detection is mainly based on enzyme linked immunosorbent assay (ELISA). The ELISA for quantitative detection of ADA requires a standard reference, i.e. a control antibody that specifically binds to the antigen to be detected. We can only quantitatively assess the ADA concentration in a sample by making a standard curve based on ELISA readings of different concentrations of control antibodies.

Disclosure of Invention

In one aspect, provided herein is an antibody, or scFv or fragment thereof, having a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are: the amino acid sequences of GFTFSSYA, ISGSGGST and ARGYMHAEHEKMEKSSRSDP, LCDR1, LCDR2 and LCDR3 are respectively: SLRSTY, GKT, and SSRDSSGYHYV.

In some embodiments, the light chain variable region of the antibody comprises SEQ ID NO: 5, and the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments, the scFv comprises SEQ ID NO: 4.

In another aspect, provided herein is the use of the antibody, or scFv or fragment thereof, for the detection of an antigen, wherein the antigen comprises SEQ ID NO: 12.

In some embodiments, the antigen is part of a fusion protein.

In some embodiments, the fusion protein is a chimeric antigen receptor that targets BCMA, the antigen being the extracellular binding domain of the chimeric antigen receptor or a portion thereof.

In some embodiments, the application comprises:

1) performing qualitative and quantitative detection of the cultured T cells expressing the chimeric antigen receptor;

2) detecting the concentration of T cells expressing the chimeric antigen receptor in a patient, wherein the patient is infused with a treatment prior to the detecting via the T cells expressing the chimeric antigen receptor; or

3) As a standard for detecting antibodies raised against the chimeric antigen receptor in a patient, wherein the patient is treated by infusion of T cells expressing the chimeric antigen receptor prior to the detection.

In another aspect, provided herein is a nucleic acid molecule encoding an antibody, or an scFv or fragment thereof, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are: the amino acid sequences of GFTFSSYA, ISGSGGST, and ARGYMHAEHEKMEKSSRSDP, LCDR1, LCDR2 and LCDR3 are: SLRSTY, GKT, and SSRDSSGYHYV.

In some embodiments, the sequence encoding the light chain variable region of the antibody comprises SEQ ID NO: 2, and the sequence encoding the heavy chain variable region of the antibody comprises the nucleotide sequence shown in SEQ ID NO: 3.

In some embodiments, the sequence encoding the scFv comprises SEQ ID NO: 1.

In another aspect, provided herein is an expression vector comprising the nucleic acid molecule described above.

The antibodies provided herein specifically target RD103A scFv as an idiotypic antibody, without cross-reacting with other scFv or antibody sequences, and are useful for pharmaceutical studies on RD103A CAR-T products.

Drawings

Fig. 1 shows a general study flow diagram of the present invention.

FIG. 2 shows the results of an ELISA preliminary screening of enriched phage antibody clones.

FIG. 3 shows the results of FACS measurements of the binding of phage antibody clones to the cell surface target antigen (Jurkat-103A scFv).

FIG. 4 shows the results of an ELISA assay in which the antibody molecules in protein form bind to the target antigen and to unrelated antigens.

FIG. 5 shows the results of FACS measurement of the binding of the antibody molecule in protein form to the cell surface target antigen (Jurkat-103A scFv).

Figure 6 shows the results of FACS measurements of binding of the antibody molecule in protein form to the CAR T cell surface target antigen (RD103A CAR).

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Antibodies refer to immunoglobulins which are secreted by plasma cells (effector B cells) and which are used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). The foreign substance is accordingly referred to as an antigen. The basic structure of an antibody molecule is a 4-mer consisting of 2 identical heavy chains and 2 identical light chains. Heavy and light chains are divided into variable (V) regions at the amino terminus and constant (C) regions at the carboxy terminus based on conservative differences in amino acid sequence. The variable regions of one heavy and one light chain interact to form an antigen binding site (Fv). In the variable region, the composition and arrangement order of amino acid residues in some regions are more variable than those in other regions (framework regions, FRs) within the variable region, and are called hypervariable regions (HVRs), which are actually key sites for binding of an antibody to an antigen. These hypervariable region sequences are also called complementarity-determining regions (CDRs) because they are complementary to antigenic determinants. Both the heavy and light chains have three complementarity determining regions, designated HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively. An antibody fragment refers to an antibody portion obtained by treatment with enzymatic, chemical, or genetic engineering means, which may or may not have an antigen-binding ability.

Single chain antibodies (scFv) are composed of an antibody heavy chain variable region and an antibody light chain variable region connected together by a short peptide to form a single peptide chain. By proper folding, the variable regions from the heavy and light chains interact through non-covalent bonds to form the Fv fragment, and thus the scFv is able to better retain its affinity activity for antigen.

Chimeric Antigen Receptors (CARs), also known as chimeric T cell receptors, chimeric immunoreceptors, are an engineered protein receptor molecule that can confer a desired specificity to immune effector cells, e.g., the ability to bind to a particular tumor antigen. Chimeric antigen receptors are generally composed of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In most cases, the antigen binding domain is a scFv sequence that is responsible for recognizing and binding to a particular antigen. Intracellular signaling domains typically include an Immunoreceptor Tyrosine Activation Motif (ITAM), such as the signaling domain derived from the CD3 ζ molecule, which is responsible for activating immune effector cells to produce killing. In addition, the chimeric antigen receptor may also include a signal peptide at the amino terminus responsible for intracellular localization of the nascent protein, and a hinge region between the antigen binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also include, for example, a costimulatory domain derived from a 4-1BB molecule. RD103A CAR is a BCMA-targeting CAR previously developed by the applicant, whose scFv has the amino acid sequence of SEQ ID NO: 12.

CAR-T cells refer to T cells expressing a CAR, typically obtained by transducing T cells with an expression vector encoding the CAR. Commonly used expression vectors are viral vectors, such as lentiviral expression vectors. Chimeric antigen receptor modified T cells (CAR-T) are not limited by the essential histocompatibility complex, have specific targeted killing activity and the ability to expand persistently.

Brief description of the research procedure

1. Elutriation: using phage antibody library, positive panning was performed with RD103A scFv-rFc protein, negative panning was performed with RD103B scFv-rFc protein (another fully human scFv clone) and B53scFv-rFc (murine scFv clone), enriching for antibody clones that specifically bind to RD103A scFv protein. Here, RD103B scFv-rFc is another BCMA-targeting fully human single chain antibody with different CDR sequences from 103A, and B53scFv is a murine BCMA single chain antibody with large differences in both framework regions and CDRs, wherein the amino acid sequence of RD103A scFv is as shown in SEQ ID NO: 12, the amino acid sequence of RD103B scFv is shown in SEQ ID NO: shown at 13. Meanwhile, in each round of the positive panning step, we added Human serum (Human serum) at a final concentration of 5% as a competitive negative panning. Human serum contains a large amount of IgG and IgM antibody proteins, so that antibodies bound with unrelated human IgG can be further reduced, and RD103A scFv special antibodies can be better enriched.

2. Primary screening of monoclonal: isolating monoclonal antibodies from the enriched antibody library, packaging the monoclonal antibodies into monoclonal phage, and screening clones which can bind to RD103A scFv-rFc protein but not to RD103B scFv-rFc protein by ELISA; DNA sequencing to determine the variable region sequence of the antibody contained in the primary screening specific clones;

3. monoclonal flow cytometry (FACS) identification at phage level: single clones obtained by sequencing were packaged into phage, and clones capable of specifically binding to cell surface RD103A-scFv were selected by FACS to identify their binding to Jurkat-RD103A and Jurkat (negative control cell line). The Jurkat-RD103A is a Jurkat cell line transfected by RD103A CAR lentivirus, the cell surface of the Jurkat cell line highly expresses RD103A scFv, the Jurkat cell line can be used as a good model cell line representing RD103A scFv in a cell surface state, and whether the special antibodies screened by the Jurkat cell surface state can be combined with RD103A scFv in a natural conformation in the cell surface state is identified.

Monoclonal identification of IgG levels:

4.1 ELISA identification: 3 specific clones were selected, constructed into mammalian cell expression vectors, and expressed as scFv-rFc in 293F cell line. After the expressed proteins were purified by the AKTA Protein purification system (Protein a affinity purification), ELISA detected the binding of these IgG-like proteins to RD103A scFv, RD103B scFv proteins, and 6 healthy human sera (containing a large amount of unrelated IgG proteins), and the specificity of these clones was compared at the IgG level;

4.2 FACS identification: these clones were expressed as scFv-rFc protein and, after affinity purification by protein A, were identified by flow cytometry to Jurkat-103A (a positive cell line highly expressing RD103A scFv), Jurkat (a negative control cell line); RD103A CAR-T (positive sample)/Mock-T (negative control) binding was assessed and its consistency with the fluorescently labeled antigen FITC-BCMA to detect the proportion of CAR-T positive cells. The general study procedure is shown in figure 1.

The present invention will be described in detail below with reference to specific examples.

Example 1 phage antibody library panning to enrich RD103A scFv specific phage clones

Negative panning antigens RD103B scFv-rFc, B53scFv-rFc and positive panning target antigen RD103A scFv-rFc are used for alternative panning, and in the step of positive panning, human serum with the final concentration of 5% is added as competition negative panning to enrich the specific phage clone targeting RD103B scFv. Table 1 lists the elutriation process.

Brief experimental procedure:

1) coating the Fc-tagged target antigen RD103A scFv-rFc or the control antigens RD103B scFv-rFc, B53scFv-rFc onto a high-binding 96-well ELISA plate (Corning Incorporated 96well EIA/RIA plate, Costar,3590), and blocking the ELISA plate with blocking solution;

2) incubating the phage antibody library with the coated negative antigen RD103B scFv-rFc or B53scFv-rFc, and subtracting phage antibody clones that non-specifically bind Fc tag or blocking fluid components;

3) transferring the supernatant after the last incubation step to a plate coated with a target antigen RD103A scFv-rFc, adding human serum (containing a large amount of human antibody proteins) with the final concentration of 5%, and continuing incubation to combine the phage and the target antigen;

4) washing the surface of the solid carrier with a washing solution to wash away unbound phage;

5) eluting the positive phage from the target antigen with an eluent;

6) re-infecting host bacteria XL1-blue with the eluted phage and amplifying the recovered phage;

7) repeating steps 1) to 6), it is usually necessary to perform 2 to 3 rounds of panning until a significant increase in phage recovery (number of eluted phage/number of input phage) is observed.

The enriched phage Pool (Pool) can be used for the next monoclonal selection and ELISA/FACS identification.

Main materials and reagents:

a fully human phage antibody library comprising a natural library and a semi-synthetic library;

helper phage KO7, Thermo/Invitrogen, 18311019;

RD103A scFv-rFc protein;

RD103B scFv-rFc protein;

b53scFv-rFc protein;

High binding ELSIA plate,Costar，#3590；

sealing liquid: PBS + 3% BSA;

rinsing liquid: PBS + 0.1% Tween 20;

eluent: 1mg/mL Trypsin PBS.

Table 1 phage panning procedure.

The experimental results are as follows:

table 2 shows the recovery (phage recovered/phage input) and fold enrichment (recovery in this round/recovery in the previous round) for 3 rounds of panning for different antibody libraries. The 3rd round elutriation recovery rate of the antibody libraries XL-N1-3, XL-N4-6 and XL-SS1 is obviously improved, which indicates that specific phage clones are enriched; the 3rd round of elutriation recovery of XL-SS2 did not increase but decreased, indicating that no specific clone was enriched.

TABLE 2 recovery and fold enrichment of 3 rounds of panning for different antibody pools

Example 2 ELISA Primary screening of phage monoclonals

Monoclonal antibodies were randomly selected from the pool of enriched phage antibodies of example 1, packaged into phage, and the binding of the monoclonal phage to RD103A scFv-rFc protein, 103B scFv-rFc protein was detected by phage ELISA to find phage antibody clones that specifically bind 103A scFv. These clones were sequenced to determine their scFv sequences.

Brief experimental procedure:

1) carrying out gradient dilution on the enriched phage solution, infecting host bacteria XL1-blue, and coating a flat plate containing Amp and Tet;

2) selecting a monoclonal in a 96-well plate, culturing at 37 ℃ and 250rpm until logarithmic phase, adding helper phage KO7, continuously culturing, and amplifying to obtain a monoclonal phage solution;

3) immobilizing the target antigen and the control antigen directly or indirectly in a 96-well plate;

4) adding the monoclonal phage supernatant into a 96-well plate coated with the antigen for incubation;

5) washing the ELISA plate 6 times with washing solution, washing away unbound phage;

6) adding an antibody of a murine Anti-phage capsid Protein PIII [ Anti-M13Bacteriophage Coat Protein g8p antibody (clone: RL-ph2), abcam, ab9225], and incubating at room temperature for 45 min;

7) washing the ELISA plate 6 times with washing solution, washing away unbound phage;

8) adding HRP coupled Goat anti-mouse Antibody [ HRP Goat anti-mouse IgG (minor x-reactivity) Antibody, BioLegend ], incubating at room temperature for 45 min;

9) washing the ELISA plate 6 times with washing solution, adding TMB (soluble single-component TMB substrate solution, TIANGEN, PA107-02), and reading after color development;

10) single clones specific for 103A scFv-rFc were picked for sequencing and numbered.

Main materials and reagents:

helper phage KO7, Thermo/Invitrogen, 18311019;

High binding ELSIA plate,Costa，#3590；

sealing liquid: PBS + 3% BSA;

rinsing liquid: PBS + 0.1% Tween 20;

soluble single component TMB substrate solution, Tiangen, PA-107-02;

Anti-M13Bacteriophage Coat Protein g8p antibody,abcam,ab9225；

HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306。

the experimental results are as follows:

the ELISA results of the partial clones are shown in FIG. 2. Control 1 is HRP Donkey anti rabbitIgG. Control 2 was the negative Control phage KO 7. Clones #1 to #10 bound well to 103A scFv-rFc and did not bind to 103B scFv-rFc, with good specificity. ITRD103-43 and helper phage KO7 did not bind to either 103A scFv-rFc or 103B scFv-rFc and were negative clones. ITRD103-21 binds to 103A scFv-rFc, but also to 103B scFv-rFc, and is a non-specific clone.

EXAMPLE 3 FACS identification of phage monoclonals

By preliminary screening with ELISA in example 2, we have demonstrated that clones #1 to #10 bind specifically to the 103A scFv-rFc protein, but in actual testing we need to ensure that they can bind efficiently to the scFv domain of the CAR molecule to be able to function to detect CAR-T cells. Therefore, we determined the binding of these clones to Jurkat-103A scFv and Jurkat cells by flow cytometry (FACS).

Brief experimental procedure:

1. incubating the monoclonal phage antibody to be detected with Jurkat-103A scFv and Jurkat cells respectively at 4 ℃ for 1h, and centrifuging to obtain a first precipitate;

2. washing the first precipitate, then combining with a primary antibody [ Anti-M13Bacteriophage Coat Protein g8p antibody, abcam, ab9225], incubating for 30min, washing, then combining with a secondary antibody [ FITC horse antibody mouse-IgG (H + L), vector, FI2000], incubating for 30min, and centrifuging to obtain a second precipitate;

3. the second pellet was washed and then resuspended in flow buffer on the flow machine, analyzed for binding to Jurkat-103A scFv and Jurkat cells, and judged if the percent positive rate of CAR matches the actual positive rate of CAR (the positive rate measured by the positive control FITC-BCMA antigen).

Main materials and reagents:

helper phage KO7, Thermo/Invitrogen,18311019

Jurkat cell line and Jurkat-103A cell line;

rinsing liquid: PBS + 0.1% Tween 20;

FITC horse anti mouse-IgG(H+L),Vector,FI2000；

Anti-M13Bacteriophage Coat Protein g8p antibody,abcam,ab9225

FITC-BCMA antigen, ACROYOSystems, BCA-HF 254.

The experimental results are as follows:

as shown in FIG. 3, 10 of the primary-screened monoclonal antibodies that bound to 103A scFv-rFc protein were able to bind to Jurkat-103A scFv cells, while not binding to Jurkat cells, and the percentage of positive rate of the tested CAR was close to the actual positive rate of the CAR. Control 1 was helper phage KO7 (negative Control). Control 2 is FITC-BCMA antigen (positive Control).

Example 4 monoclonal identification of IgG levels

Monoclonal antibody ELISA verification:

in order to verify that the clone obtained in the above example can still be combined with 103A CAR molecule after being expressed in the form of antibody molecule and is not interfered by human serum, 3 single clones #4, #9 and #10 with better specificity in the previous stage are constructed into scFv-rFc structure, protein expression is carried out in 293F cells, and antibody protein with higher purity is obtained through protein A affinity purification. We then tested the scFv-rFc protein versions of these clones by ELISA for binding to RD103A scFv (target antigen), RD103B scFv (control antigen), and 6 healthy human sera (containing large amounts of antibody-like proteins).

Brief experimental procedure:

1) target antigen RD103A scFv-Streptag protein in scFv format (2. mu.g/mL), control antigen RD103BscFv-Streptag protein (2. mu.g/mL), and serum Human sera from different healthy donors (5% in PBS) were coated on high binding 96-well ELISA plates (2 h at room temperature), 100. mu.L/well;

2) blocking the ELISA plate with 250 μ L of blocking solution, and blocking for 2h at 4 ℃;

3) after washing 5 times with 200. mu.L of washing solution, 100. mu.L of monoclonal antibodies #4, #9, #10 and positive and negative controls (5. mu.g/mL) were added and incubated, respectively. Positive control wells: the positive controls for RD103A scFv and RD103B scFv were BCMA-hFc added during this step, and the serum samples were added to the rinse; adding PBST into the negative control, and incubating for 30min at room temperature;

4) after washing 5 times with 200. mu.L of washing solution, 100. mu.L of Anti-Rabbit IgG-HRP (1:2000) as a secondary antibody was added to the detection sample wells and the negative control wells; adding Anti-Human IgG-HRP (1:2000) as a secondary antibody into the positive control hole, and incubating for 30min at room temperature;

5) the ELISA plate was washed 6 times with 200. mu.L of wash solution, TMB substrate was added, color developed, and read after termination.

Main materials and reagents:

RD103A scFv-Strep tag protein and RD103B scFv-Strep tag protein;

healthy donor serum;

High binding ELSIA plate,Costar,#3590；

sealing liquid: PBS + 3% BSA;

rinsing liquid: PBS + 0.1% Tween 20;

soluble single component TMB substrate solution, Tiangen, PA-107-02;

BCMA-hFC-Biotin,ACRObiosystems,BC7-H82FO；

Anti-Rabbit IgG-HRP,Biolegend,406401；

Anti-Human IgG-HRP,Biolegend,410902。

the experimental results are as follows:

the results are shown in FIG. 4. The #4, #9, #10scFv-rFc protein was able to bind to the target antigen RD103A scFv-strep tag, while not binding to the control antigen RD103B scFv-strep tag and to serum components of 6 healthy persons. The results show that after the 3 clones are converted into the form of scFv-rFc protein, the RD103scFv protein can be recognized, and the specificity is better. Control 1 is a positive Control, BCMA-hFc + anti human IgG-HRP is added to the wells of 103A scFv and 103B scFv, and only anti human-HRP is added to the serum sample; PBST + anti rabbitIgG-HRP was added to the negative control wells.

FACS validation of monoclonal antibodies against Jurkat-103A/Jurkat cells:

to further verify that the clones expressed as antibodies still bound to the RD103A CAR molecule in its native conformation on the cells, we constructed a Jurkat-103A CAR cell line highly expressing RD103CAR by lentivirus infection, with Jurkat itself as the negative cell. We then examined the binding ability of these 3 cloned scFv-rFc proteins to the 103A CAR molecule on cells by FACS.

Brief experimental procedure:

1) Jurkat-103A scFv and Jurkat cells were washed 2 times with PBS and resuspended at 1X10 with PBS⁷At a concentration of 50. mu.L/mL, dispensed into tubes;

2) 50 mu L of antibodies to be detected #4, #9 and #10(10 mu g/ml) are put into each tube in the experimental group, the other groups are supplemented with PBS with the same volume, and after mixing, the antibodies are combined for 1h at 4 ℃;

3) wash 2 times with 200 μ L PBS;

4) a1: 400 dilution of Goat Anti-Rabbit IgG, 100. mu.L/well, was added to the experimental group, and a 1: 100 diluted FITC-BCMA, 100 mu L/hole, blank control Cell Only, supplementing PBS with equal volume, blowing, mixing, and binding at 4 deg.C for 30 min;

5) wash 2 times with 200 μ L PBS; finally resuspend the cells with 200 μ L PBS;

6) and detecting the fluorescence intensity of the Vioblue and FITC channels of the samples on a flow cytometer, and analyzing the results.

Main materials and reagents:

rinsing liquid: PBS + 0.1% Tween 20;

a scFv-rFc protein of #4, #9, # 10;

DyLight 405-AffiniPure F(ab)2Fragment Goat Anti-Rabbit IgG；

FITC-BCMA antigen, ACROYOSystems, BCA-HF 254.

The experimental results are as follows:

table 3 and FIG. 5 show the results of the cell flow analysis of the scFv-rFc proteins of clones #4, #9 and # 10. Monoclonal antibodies #4 and #9 did not bind significantly to both Jurkat-103A scFv and Jurkat cells, indicating that they do not bind the 103A CAR molecule in its native conformation on the cell. The #10 monoclonal antibody can specifically bind to 103A CAR molecules on Jurkat-103A scFv cells, the percentage (%) of 103A CAR positivity measured matches the percentage (%) of 103A CAR positivity measured by the positive control FITC-BCMA (difference < 5%) and does not bind to Jurkat cells, so the #10 monoclonal antibody has good specificity in this experiment, meets the needs of the experiment, and can further verify its detection effect on 103A CAR-T cells.

TABLE 3 flow assay results (percentage of positivity) for scFv-rFc protein binding to Jurkat-103A/Jurkat cells

Example 5 FACS binding of monoclonal antibodies to actual CAR-T samplesAuthentication

To verify the binding ability of the #10 monoclonal antibody (scFv-rFc protein) to the 103A CAR molecule on 103A CAR-T cells, we performed FACS analysis of the #10 monoclonal antibody using 103A CAR-T cells 6 days after lentiviral transfection with the same healthy donor T cells and untransfected negative control cells (Mock T).

The main experimental steps are similar to the FACS validation of cell lines of reagents and monoclonal antibodies in example 4.

And (3) judging standard: whether the percent (%) 103A CAR positive for the test antibody matches the percent (%) 103A CAR positive for the positive control FITC-BCMA (< 5% difference).

The experimental results are as follows:

the results are shown in table 4 and fig. 6. The #10 monoclonal antibody can specifically bind to 103A CAR molecules on 103A CAR-T cells, the percentage (%) of 103A CAR positivity matches the percentage (%) of 103ACAR positivity measured by the positive control FITC-BCMA (difference < 5%), and there is no non-specific binding to Mock T, so the #10 monoclonal antibody has good specificity in the experiment and can meet the detection requirements in the experiment and clinic.

TABLE 4 flow assay results ((percent positive))

The monoclonal antibody screened by the invention is an idiotypic antibody specifically recognizing RD103A scFv, can be specifically combined with 103A scFv protein and 103A CAR-T cells, is not non-specifically combined with other irrelevant antibodies and membrane protein, can be used for detecting CAR-T cells by flow cytometry or detecting RD103scFv protein by ELISA, and has important value for pharmaceutical research on RD103A CAR-T products.

Specific applications of the specific antibody obtained by the present invention may include, but are not limited to, the following 3 aspects: 1) quantitative analysis and detection of RD103A CAR-T product cells for flow cytometry (FACS), such as detection of the positivity rate of CAR-T products; 2) used for flow cytometry (FACS) to measure the RD103A CAR-T cell concentration in the blood of patients after transfusion. Compared with the method for detecting RD103A scFv by using fluorescence labeled BCMA protein, the idiotype antibody has better specificity, lower background and higher sensitivity; 3) this specific antibody was used as a standard reference in an ELISA assay to determine the concentration of anti-antibody (ADA) against RD103scFv in the patient's reinfused blood.

Some of the sequences mentioned herein are as follows:

SEQ ID NO: 1#10scFv DNA sequence: 771bp

TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAAGATCACATGCCGAGGAGACAGCCTCAGAAGCACTTACGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAACCAACCGGCCCTCAGGGATCCCAGACCGATTCTATGGTTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAGCTCCCGCGACAGCAGTGGTTACCATTATGTCTTCGGAAGTGGGACCAAGCTGACCGTCCTAGGTTCTAGAGGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCCCTCGAGATGGCCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCGGTTACATGCATGCAGAACATGAAAAAATGGAAAAATCTTCTCGTTCTGATCCATGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO: 2#10VL DNA sequence: 327bp

TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAAGATCACATGCCGAGGAGACAGCCTCAGAAGCACTTACGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAACCAACCGGCCCTCAGGGATCCCAGACCGATTCTATGGTTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAGCTCCCGCGACAGCAGTGGTTACCATTATGTCTTCGGAAGTGGGACCAAGCTGACCGTCCTAGGT

SEQ ID NO: 3#10VH DNA sequence: 387bp

ATGGCCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGCGGTTACATGCATGCAGAACATGAAAAAATGGAAAAATCTTCTCGTTCTGATCCATGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA

SEQ ID NO: 4#10scFv amino acid sequence: 256aa

SSELTQDPAVSVALGQTVKITCRGDSLRSTYASWYQQKPGQAPVLVIYGKTNRPSGIPDRFYGSSSGNTASLTITGAQAEDEADYYCSSRDSSGYHYVFGSGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYMHAEHEKMEKSSRSDPWGQGTLVTVS

SEQ ID NO: 5#10VL amino acid sequence: 109aa

SSELTQDPAVSVALGQTVKITCRGDSLRSTYASWYQQKPGQAPVLVIYGKTNRPSGIPDRFYGSSSGNTASLTITGAQAEDEADYYCSSRDSSGYHYVFGSGTKLTVLG

SEQ ID NO: 6#10VH amino acid sequence: 130aa

LEMAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYMHAEHEKMEKSSRSDPWGQGTLVTVS

Amino acid sequence of #10 monoclonal antibody determinant (CDR):

SEQ ID NO: 12RD103A scFv amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKYDLLTFGGGTKVEIKGSTSGSGKPGSGEGSTKGQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSISYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRGDTILDVWGQGT

SEQ ID NO: 13RD103B scFv amino acid sequence:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQASALPLTFGGGTKVEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKDSPRRDSFGSIAFDIWGQGT

reference documents:

1.Ms R.Podar k,Breitkreutz I,Richardson PG and Anderson kC:Multiple myeloma[J].Lancet,2009,374:324-339.

2.Shely R N,Ratliff P D.Carfilzomib-Associated Tumor Lysis Syndrome[J].Pharmacotherapy:The Journal of Human Pharmacology and Drug Therapy,2014,34(5).

3.Carpenter R O,Evbuomwan M O,Pittaluga S,et al.B-cell maturation antigen is apromising target for adoptive T-cell therapy of multiple myeloma[J].Clinical cancer research,2013,19(8):2048-2060.

4.Porter D L,Hwang W T,Frey N V,et al.Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia[J].Science translational medicine,2015,7(303):303ra139-303ra139.

5.Jiasheng Wang,Yongxian Hu,and He Huang，Acute lymphoblastic leukemia relapse after CD19-targeted chimeric antigen receptor T cell therapy，PP1-11，Volume 102,December 2017,Journal of Leukocyte Biology.

SEQUENCE LISTING

<110> Nanjing reindeer medical technology Co., Ltd

<120> antibodies targeting BCMA chimeric antigen receptor and uses thereof

<130> RD103

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 771

<212> DNA

<213> Artificial

<220>

<223> #10 scFv

<400> 1

tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaagatc 60

acatgccgag gagacagcct cagaagcact tacgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa accaaccggc cctcagggat cccagaccga 180

ttctatggtt ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg tagctcccgc gacagcagtg gttaccatta tgtcttcgga 300

agtgggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360

tctggtggtg gtggatccct cgagatggcc caggtgcagc tggtggagtc tgggggaggc 420

ttggtacagc ctggggggtc cctgagactc tcctgtgcag cctctggatt cacctttagc 480

agctatgcca tgagctgggt ccgccaggct ccagggaagg ggctggagtg ggtctcagct 540

attagtggta gtggtggtag cacatactac gcagactccg tgaagggccg gttcaccatc 600

tccagagaca attccaagaa cacgctgtat ctgcaaatga acagcctgag agccgaggac 660

acggccgtat attactgtgc gcgcggttac atgcatgcag aacatgaaaa aatggaaaaa 720

tcttctcgtt ctgatccatg gggtcaaggt actctggtga ccgtctcctc a 771

<210> 2

<211> 327

<212> DNA

<213> Artificial

<220>

<223> #10 VL

<400> 2

tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaagatc 60

acatgccgag gagacagcct cagaagcact tacgcaagct ggtaccagca gaagccagga 120

caggcccctg tacttgtcat ctatggtaaa accaaccggc cctcagggat cccagaccga 180

ttctatggtt ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240

gatgaggctg actattactg tagctcccgc gacagcagtg gttaccatta tgtcttcgga 300

agtgggacca agctgaccgt cctaggt 327

<210> 3

<211> 387

<212> DNA

<213> Artificial

<220>

<223> #10 VH

<400> 3

atggcccagg tgcagctggt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 120

caggctccag ggaaggggct ggagtgggtc tcagctatta gtggtagtgg tggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtatatta ctgtgcgcgc 300

ggttacatgc atgcagaaca tgaaaaaatg gaaaaatctt ctcgttctga tccatggggt 360

caaggtactc tggtgaccgt ctcctca 387

<210> 4

<211> 256

<212> PRT

<213> Artificial

<220>

<223> #10 scFv

<400> 4

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

1 5 10 15

Thr Val Lys Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser Thr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

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

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Tyr His

85 90 95

Tyr Val Phe Gly Ser Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu

115 120 125

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

130 135 140

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

145 150 155 160

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

165 170 175

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

180 185 190

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

195 200 205

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

210 215 220

Tyr Cys Ala Arg Gly Tyr Met His Ala Glu His Glu Lys Met Glu Lys

225 230 235 240

Ser Ser Arg Ser Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250 255

<210> 5

<211> 109

<212> PRT

<213> Artificial

<220>

<223> #10 VL

<400> 5

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

1 5 10 15

Thr Val Lys Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser Thr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

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

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser Gly Tyr His

85 90 95

Tyr Val Phe Gly Ser Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 6

<211> 130

<212> PRT

<213> Artificial

<220>

<223> #10 VH

<400> 6

Leu Glu Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

1 5 10 15

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

20 25 30

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

35 40 45

Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr

50 55 60

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

65 70 75 80

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

85 90 95

Val Tyr Tyr Cys Ala Arg Gly Tyr Met His Ala Glu His Glu Lys Met

100 105 110

Glu Lys Ser Ser Arg Ser Asp Pro Trp Gly Gln Gly Thr Leu Val Thr

115 120 125

Val Ser

130

<210> 7

<211> 8

<212> PRT

<213> Artificial

<220>

<223> #10 HCDR1

<400> 7

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial

<220>

<223> #10 HCDR2

<400> 8

Ile Ser Gly Ser Gly Gly Ser Thr

1 5

<210> 9

<211> 20

<212> PRT

<213> Artificial

<220>

<223> #10 HCDR3

<400> 9

Ala Arg Gly Tyr Met His Ala Glu His Glu Lys Met Glu Lys Ser Ser

1 5 10 15

Arg Ser Asp Pro

20

<210> 10

<211> 6

<212> PRT

<213> Artificial

<220>

<223> #10 LCDR1

<400> 10

Ser Leu Arg Ser Thr Tyr

1 5

<210> 11

<211> 11

<212> PRT

<213> Artificial

<220>

<223> #10 LCDR3

<400> 11

Ser Ser Arg Asp Ser Ser Gly Tyr His Tyr Val

1 5 10

<210> 12

<211> 237

<212> PRT

<213> Artificial

<220>

<223> RD103A scFv

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Lys Tyr Asp Leu Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser

100 105 110

Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Leu Gln Leu

115 120 125

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

130 135 140

Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp

145 150 155 160

Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser

165 170 175

Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg

180 185 190

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

195 200 205

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

210 215 220

Arg Gly Asp Thr Ile Leu Asp Val Trp Gly Gln Gly Thr

225 230 235

<210> 13

<211> 243

<212> PRT

<213> Artificial

<220>

<223> RD103B scFv

<400> 13

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ala Ser Ala Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln

115 120 125

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

130 135 140

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Ala Met His

145 150 155 160

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile

165 170 175

Ser Trp Ser Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys Gly Arg

180 185 190

Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met

195 200 205

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp

210 215 220

Ser Pro Arg Arg Asp Ser Phe Gly Ser Ile Ala Phe Asp Ile Trp Gly

225 230 235 240

Gln Gly Thr

Claims

1. An antibody, or an scFv or fragment thereof, having a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are: the amino acid sequences of GFTFSSYA, ISGSGGST and ARGYMHAEHEKMEKSSRSDP, LCDR1, LCDR2 and LCDR3 are respectively: SLRSTY, GKT, and SSRDSSGYHYV.

2. The antibody or scFv or fragment thereof of claim 1, wherein the light chain variable region of said antibody comprises SEQ ID NO: 5, and the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 6.

3. The antibody or scFv or fragment thereof of claim 1, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 4.

4. Use of the antibody or scFv or fragment thereof of any one of claims 1 to 3 for antigen detection wherein said antigen comprises the amino acid sequence of SEQ ID NO: 12.

5. The use of claim 4, wherein the antigen is part of a fusion protein.

6. The use according to claim 5, wherein the fusion protein is a chimeric antigen receptor targeting BCMA, the antigen being the extracellular binding domain of the chimeric antigen receptor or a part thereof.

7. The use of any of claims 4 to 6, comprising:

8. A nucleic acid molecule encoding an antibody or scFv or fragment thereof, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are: the amino acid sequences of GFTFSSYA, ISGSGGST and ARGYMHAEHEKMEKSSRSDP, LCDR1, LCDR2 and LCDR3 are respectively: SLRSTY, GKT, and SSRDSSGYHYV.

9. The nucleic acid molecule of claim 8, wherein the sequence encoding the light chain variable region of the antibody comprises SEQ ID NO: 2, and the sequence encoding the heavy chain variable region of the antibody comprises the nucleotide sequence shown in SEQ ID NO: 3.

10. The nucleic acid molecule of claim 8 or 9, wherein the sequence encoding the scFv comprises SEQ ID NO: 1.

11. An expression vector comprising the nucleic acid molecule of any one of claims 8 to 11.