Monoclonal antibody for detecting anti-BCMA CAR expression level and application thereof

Abstract

The invention provides a monoclonal antibody of an anti-BCMA antibody and application of the monoclonal antibody. The monoclonal antibody aiming at the BCMA antibody is high in specificity and strong in affinity. The invention further provides a BCMA CAR-T cell detection reagent which can aim at an extracellular antigen binding region of the BCMA CAR-T cell and has the characteristics of high detection sensitivity, good specificity and the like.

Description

Monoclonal antibodies for detecting anti-BCMA CAR expression levels and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody for detecting the expression level of anti-BCMA CAR and its application. Background Art

[0002] With the development of tumor immunotherapy, chimeric antigen receptor (CAR)-T cell immunotherapy, which combines the advantages of antibodies and immune cells, has attracted great attention. Chimeric antigen receptor (CAR)-T cells are genetically modified T cells that can recognize specific target antigens in an MHC-unrestricted manner and continuously activate and proliferate. CARs are primarily composed of an extracellular antigen-binding region and an intracellular signaling region, connected by a hinge region and a transmembrane region. The extracellular antigen-binding region has the function of specifically recognizing and binding to target cell surface antigens. Its structure is derived from the single-chain variable fragment (scFv) domain of a monoclonal antibody and consists of a heavy chain variable region (VH) and a light chain variable region (VL). The intracellular signaling region is primarily composed of costimulatory signals and the CD3zeta chain of the T cell receptor (TCR). After CAR-expressing T cells (CAR-T) bind to target cell antigens through scFv, the intracellular signal transduction region transmits the signal to the T cells, thereby activating the T cells and secreting perforin, granzymes, and cytokines, etc., to exert a killing effect.

[0003] B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor (TNFR) superfamily that binds to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). BCMA is reportedly expressed primarily by plasma cells and a subset of mature B cells in normal cells, but is absent from most B cells or other organs. Therefore, it is an ideal target for CAR-T immunotherapy against multiple myeloma. Currently, Abecma (idecabtagene viclecuel, ide-cel, bb2121), jointly developed by Bristol-Myers Squibb (BMS) and Bluebird Bio, has received FDA approval, becoming the first cell therapy targeting BCMA. Janssen / Legend Biotech has also submitted a Biologics License Application (BLA) to the FDA for an anti-BCMA CAR-T cell therapy. Both drugs target BCMA-positive B cell tumors and utilize the same antigen-binding region, a scFv derived from the murine monoclonal antibody clone C11D5.3.

[0004] The construction of CAR-T cells requires the CAR gene to be transfected and integrated into the T cell genome via a viral or non-viral system. Only when the gene is expressed normally, forming a transmembrane CAR structure on the cell membrane, can CAR-T cells recognize and kill target cells. Therefore, accurate detection of CAR-positive T cells is a key step in the quality control of CAR-T drugs and an important part of clinical patient treatment dosage control, process monitoring, and auxiliary diagnosis. There are two common detection methods: detecting CAR gene-positive T cells and detecting CAR protein-positive T cells.

[0005] Quantitative real-time PCR (qPCR) is the primary method for detecting CAR gene-positive T cells. qPCR can detect the presence of CAR genes integrated into the cell genome and the number of copies of the CAR gene. However, this method cannot accurately reflect the presence of CAR-positive T cells, as some T cells with integrated CAR genes do not normally express the CAR (Jennifer N. Brudno, et al., Journal of Clinical Oncology, 2016). These false positives from these T cells reduce the accuracy of qPCR. More seriously, qPCR cannot distinguish between CAR-positive T cells and CAR-positive B cells, making it difficult to identify treatment-related and relapse risks associated with CAR-positive B cells (Marco Ruella, et al., Nature Medicine, 2018). Furthermore, qPCR cannot meet the requirement for simultaneous detection of multiple T cell surface markers (CD3, CD4, CD8, etc.), further limiting its application.

[0006] Existing reagents for detecting CAR protein-positive T cells include anti-mouse IgG (Fab')2 (Jackson ImmunoResearch) and Protein L (Zhili Zheng, et al., Journal of Translational Medicine, 2012). However, these reagents have low sensitivity for detecting BCMA CARs and are unable to distinguish between different CARs constructed with different mouse scFvs (e.g., CD19 CAR, CD22 CAR, etc.). These reagents, due to their respective limitations, have limited application.

[0007] Therefore, there is an urgent need in this field to develop anti-BCMA-CAR positive cell detection reagents and methods with high sensitivity and accuracy to meet the needs of anti-BCMA CAR-T drug quality control and clinical sample testing.

[0008] Summary of the Invention

[0009] The present invention aims to provide a monoclonal antibody that specifically binds to BCMA CAR and its use in detecting BCMA CAR-positive cells.

[0010] In a first aspect of the present invention, an antibody or antigen-binding fragment thereof that specifically targets BCMA CAR is provided, wherein the heavy chain variable region of the antibody comprises the following three complementarity determining regions (CDRs):

[0011] H-CDR1 as shown in SEQ ID NO: 4,

[0012] H-CDR2 as shown in SEQ ID NO: 5, and

[0013] H-CDR3 as shown in SEQ ID NO: 6; and

[0014] The light chain variable region of the antibody comprises the following three complementarity determining regions (CDRs):

[0015] L-CDR1 as shown in SEQ ID NO: 7,

[0016] The amino acid sequence of L-CDR2 is SAS, and

[0017] L-CDR3 as shown in SEQ ID NO:8.

[0018] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2.

[0019] In another preferred embodiment, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 3.

[0020] In another preferred embodiment, the antibody comprises: a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 2, and a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 3.

[0021] In another preferred embodiment, the heavy chain constant region and light chain constant region of the antibody are of rabbit origin.

[0022] In another preferred embodiment, the antibody includes: a monoclonal antibody, a single-chain antibody (scFv), a Fab or (Fab')2 fragment, and a double-chain antibody.

[0023] In another preferred embodiment, the antibody is a rabbit-derived monoclonal antibody.

[0024] In another preferred embodiment, the antibody specifically binds to the scFv in BCMA CAR.

[0025] In another preferred embodiment, the scFv in the BCMA CAR is C11D5.3, and its amino acid sequence is shown in SEQ ID NO: 1.

[0026] In another preferred embodiment, the equilibrium dissociation constant K between the antibody and the scFv in BCMA CAR is D ≤1nM, more preferably, K D ≤100pM, optimally, K D ≤50pM.

[0027] The second aspect of the present invention provides a recombinant protein, comprising:

[0028] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0029] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0030] In another preferred embodiment, the tag sequence includes a secretory peptide tag.

[0031] In another preferred embodiment, the tag sequence includes a 6*His tag and / or an Fc tag.

[0032] In another preferred embodiment, the recombinant protein is a specific anti-BCMA scFv antibody.

[0033] The third party of the present invention provides a polynucleotide encoding a polypeptide selected from the following group: the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; or the recombinant protein as described in the second aspect of the present invention.

[0034] In another preferred example, the polynucleotide encodes the heavy chain variable region of the antibody, and its sequence is shown in SEQ ID NO:9.

[0035] In another preferred example, the polynucleotide encodes the light chain variable region of the antibody, and its sequence is shown in SEQ ID NO: 10.

[0036] The nucleotide sequence of the heavy chain variable region of the A3 antibody of the present invention (SEQ ID NO: 9):

[0037] The nucleotide sequence of the light chain variable region of the A3 antibody of the present invention (SEQ ID NO: 10):

[0038] The fourth aspect of the present invention provides a vector comprising the polynucleotide according to the third aspect of the present invention.

[0039] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus (such as adenovirus, retrovirus, lentivirus, etc.), transposon or other vectors.

[0040] The fifth aspect of the present invention provides a genetically engineered host cell, wherein the cell contains the vector described in the fourth aspect of the present invention or the polynucleotide described in the third aspect of the present invention is integrated into its genome.

[0041] In another preferred embodiment, the cells include: prokaryotic cells or eukaryotic cells.

[0042] The sixth aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0043] (a) the antibody according to the first aspect of the present invention; and

[0044] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a magnetic bead, agarose, and the like.

[0045] In another preferred embodiment, the coupling portion is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, gold nanoparticles / nanorods, nanoparticles in any form, or particles of any form or other size (e.g., magnetic beads, agarose particles), etc.

[0046] The seventh aspect of the present invention provides a method for preparing the antibody according to the first aspect of the present invention, comprising the steps of:

[0047] (a) Providing BCMA CAR scFv recombinant protein;

[0048] (b) immunizing rabbits with the recombinant protein, taking spleen tissue from the immunized rabbits, extracting RNA therefrom and reverse transcribing it into cDNA;

[0049] (c) amplifying the antibody variable region sequence in the cDNA obtained in step (b), ligating the sequence to a phage vector, and transforming the vector into an Escherichia coli strain to construct a phage antibody library;

[0050] (d) Screening BCMA CAR scFv-specific positive monoclones from the constructed phage antibody library;

[0051] (e) sequencing the positive monoclonal antibody to obtain the light and heavy chain gene sequences of the monoclonal antibody;

[0052] (f) using the monoclonal antibody light / heavy chain gene sequence to construct an expression plasmid, and transfecting the plasmid into a eukaryotic host cell to obtain a transfected eukaryotic host cell;

[0053] (g) culturing the transfected eukaryotic host cell under conditions suitable for expression, thereby expressing the monoclonal antibody; and

[0054] (h) isolating and / or purifying the monoclonal antibody from the culture system.

[0055] In another preferred embodiment, the expression includes secretory expression (ie, secretion into the cell culture medium).

[0056] In another preferred embodiment, the eukaryotic host cell is selected from the following group: CHO cells, COS-1 cells, COS-7 cells, and HEK-293T cells.

[0057] In another preferred embodiment, the eukaryotic host cell is a CHO cell, specifically an ExpiCHO-S cell.

[0058] In another preferred embodiment, the BCMA CAR scFv recombinant protein is a C11D5.3-hFc single chain, wherein the amino acid sequence of C11D5.3 is shown in SEQ ID NO: 1.

[0059] In another preferred embodiment, the method further comprises the optional steps of:

[0060] (h) performing performance tests on the isolated and / or purified monoclonal antibody.

[0061] In another preferred embodiment, the performance measurement comprises one or more tests selected from the following group:

[0062] (1) Specificity detection;

[0063] (2) potency testing;

[0064] (3) Sensitivity testing;

[0065] (4) Precision testing;

[0066] (5) neutralization blocking assay; and

[0067] (6) Binding kinetics detection.

[0068] In an eighth aspect of the present invention, a detection reagent for detecting BCMA CAR-positive cells is provided, the detection reagent comprising:

[0069] The first detection reagent is the antibody described in the first aspect of the present invention as the first antibody.

[0070] In another preferred embodiment, the detection reagent further includes a second detection reagent, and the second detection reagent specifically binds to the first antibody or a label coupled to the first antibody.

[0071] In another preferred embodiment, the first antibody is a monoclonal antibody coupled to a fluorescent or luminescent marker.

[0072] In another preferred embodiment, the first antibody is an unmodified monoclonal antibody.

[0073] In another preferred embodiment, the second detection reagent is a second antibody specific to the first antibody.

[0074] In another preferred embodiment, the first antibody is a biotinylated monoclonal antibody, and the second detection reagent is streptavidin with a detectable label.

[0075] In another preferred embodiment, the second antibody carries a detectable label, and the detectable label includes a fluorescent or luminescent label.

[0076] In another preferred example, the detection sensitivity of the detection reagent is ≥1 BCMA CAR-positive cell / 1000 cells, more preferably ≥5 BCMA CAR-positive cells / 10000 cells, more preferably ≥1 BCMA CAR-positive cell / 10000 cells, and more preferably ≥2 BCMA CAR-positive cells / 100000 cells.

[0077] In another preferred embodiment, the monoclonal antibody is a rabbit monoclonal antibody.

[0078] In another preferred embodiment, the first antibody and the second antibody are from different mammalian species.

[0079] In another preferred embodiment, the first antibody is from rabbit, and the second antibody is from donkey or sheep.

[0080] In another preferred embodiment, the CAR-positive cells include CAR immune cells and CAR non-immune cells.

[0081] In another preferred embodiment, the CAR-positive cells are selected from the following group: T cells, NK cells, CIK cells, NKT cells, or a combination thereof.

[0082] In another preferred embodiment, the CAR-positive cells include: HEK-293T cells, Jurkat cells, or a combination thereof.

[0083] In another preferred embodiment, the monoclonal antibody specifically recognizes and binds to the BCMA CAR constructed based on the scFv of the anti-BCMA monoclonal antibody C11D5.3.

[0084] In another preferred embodiment, the BCMA CAR scFv recombinant protein is a C11D5.3-hFc single chain, wherein the amino acid sequence of C11D5.3 is shown in SEQ ID NO: 1.

[0085] A ninth aspect of the present invention provides a kit for detecting BCMA CAR-positive cells, the kit comprising:

[0086] (t1) a first container, wherein the first container contains the antibody according to the first aspect of the present invention, and the antibody specifically binds to the extracellular antigen-binding domain of BCMA CAR,

[0087] and optionally (t0) instructions.

[0088] In another preferred embodiment, the antibody is a monoclonal antibody, and the monoclonal antibody carries a detectable label.

[0089] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0090] In another preferred embodiment, the antibody is a fluorescently labeled monoclonal antibody.

[0091] In another preferred embodiment, the detection kit further comprises (t2) a second container, wherein the second container comprises a second antibody specific to the monoclonal antibody.

[0092] In another preferred embodiment, the second antibody is an anti-rabbit IgG antibody.

[0093] In another preferred embodiment, the second antibody is selected from the following group: goat anti-rabbit IgG antibody, donkey anti-rabbit IgG antibody.

[0094] In another preferred embodiment, the second antibody carries a detectable label.

[0095] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0096] In another preferred embodiment, the second antibody is a fluorescently labeled secondary antibody.

[0097] In another preferred embodiment, the first container and the second container may be integrated or separated.

[0098] In another preferred embodiment, the detection kit further comprises one or more additional detection reagents selected from the following group:

[0099] (t3) a third container, and a sealing agent located within the third container; and

[0100] (t4) a fourth container, and a positive control (such as BCMA CAR scFv recombinant protein) located in the fourth container; and

[0101] (t5) a fifth container, and a negative control reagent located in the fifth container.

[0102] In another preferred embodiment, the additional detection reagents are located in the same or different containers.

[0103] In another preferred embodiment, the instructions describe a method for detecting BCMA CAR-positive cells.

[0104] In a tenth aspect, the present invention provides a use of the antibody described in the first aspect of the present invention for preparing a detection reagent or kit for detecting BCMA CAR-positive cells.

[0105] In another preferred embodiment, the CAR-positive cells include CAR immune cells and CAR non-immune cells.

[0106] In another preferred embodiment, the CAR-positive cells are selected from the following group: T cells, NK cells, CIK cells, NKT cells, or a combination thereof.

[0107] In another preferred embodiment, the CAR-positive cells include: HEK-293T cells, Jurkat cells, or a combination thereof.

[0108] In another preferred embodiment, the BCMA CAR-positive cells comprise a BCMA CAR constructed based on the scFv of the anti-BCMA monoclonal antibody C11D5.3.

[0109] In an eleventh aspect of the present invention, a method for detecting BCMA CAR-positive cells is provided, comprising the steps of:

[0110] (I) providing the detection reagent according to the eighth aspect of the present invention;

[0111] (II) detecting the cell population to be detected using the detection reagent to obtain qualitative or quantitative detection results of BCMA CAR-positive cells.

[0112] In another preferred embodiment, the method comprises the steps of:

[0113] (1) Take 1×10 5 -1×106 Wash the cells to be tested 1-3 times with a buffer (e.g., FACS) and resuspend in 50-200 μL of buffer;

[0114] (2) incubating with 50-200 μL of a buffer (e.g., FACS) containing the first antibody (the antibody described in the first aspect of the present invention) at 2-15°C for 10-60 minutes, preferably at 4±2°C for 15-45 minutes;

[0115] (3) Wash cells 1-3 times with pre-chilled buffer (e.g., FACS buffer);

[0116] (4) Resuspend the cells in 50-200 μL of a buffer (e.g., FACS) containing a secondary antibody with a detectable marker (e.g., a fluorescent group) and incubate at 2-15°C for 10-60 minutes, preferably at 4±2°C for 15-45 minutes;

[0117] (5) Wash cells 1-3 times with pre-chilled buffer (e.g., FACS buffer);

[0118] (6) Resuspend the cells in a certain amount (e.g., 100-500 μL) of buffer (e.g., FACS) and perform detection on a flow cytometer.

[0119] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0120] In another preferred embodiment, the method is an in vitro detection method.

[0121] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0122] In another preferred embodiment, the method is a quality control method.

[0123] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 shows some results of flow cytometry detection of phage monoclonal supernatant.

[0125] Figure 2 shows the purity of rabbit monoclonal antibody rA3 detected by SDS-PAGE.

[0126] FIG3 shows the specificity of rA3 antibody binding to C11D5.3 protein detected by ELISA.

[0127] FIG4 shows the results of detecting BCMA CAR-positive cells using rabbit monoclonal antibody rA3 in immunofluorescence assay.

[0128] Figure 5 shows that rabbit monoclonal antibody rA3 has high specificity and accuracy in detecting BCMA CAR-Jurkat cells.

[0129] Figure 6 shows that rabbit monoclonal antibody rA3 has high sensitivity in detecting BCMA CAR-Jurkat cells.

[0130] Figure 7 shows that rabbit monoclonal antibody rA3 detects BCMA CAR-Jurkat cells with high accuracy.

[0131] Figure 8 shows that rabbit monoclonal antibody rA3 detects BCMA CAR-Jurkat cells with high precision. DETAILED DESCRIPTION

[0132] After extensive and in-depth research, the inventors unexpectedly obtained a monoclonal antibody (rA3) that can specifically bind to BCMA CAR through a large number of experimental screenings. The experimental results show that the monoclonal antibody against BCMA CAR has high specificity and strong affinity. The present invention also provides a detection reagent with high sensitivity, good accuracy and good specificity for detecting CAR-positive cells (such as CAR-T cells). The detection method based on the detection reagent of the present invention can not only directly target the extracellular antigen binding region on CAR-positive cells, but also has a detection sensitivity much higher than that of various currently available detection reagents. In addition, the detection reagent of the present invention has a very high degree of differentiation between positive CAR cells and negative CAR cells, and the detection results are more accurate.

[0133] On this basis, the present invention was completed.

[0134] the term

[0135] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0136] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0137] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a class of substantially homogeneous populations, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which are typically different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they can be synthesized by recombinant expression in eukaryotic cells and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population and should not be construed as requiring any special method to produce the antibody.

[0138] The present invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity, such as single-chain antibodies; Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains.

[0139] As used herein, the terms "heavy chain variable region" and "V H ” can be used interchangeably.

[0140] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0141] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes the following three complementarity determining regions CDR:

[0142] H-CDR1, whose amino acid sequence is GIDLNSYV (SEQ ID NO: 4);

[0143] H-CDR2, whose amino acid sequence is ISGGGTS (SEQ ID NO: 5);

[0144] H-CDR3, the amino acid sequence of which is ARLAYDGGDYLDI (SEQ ID NO: 6).

[0145] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:

[0146] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the aforementioned heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of rabbit, mouse or human origin, preferably rabbit origin.

[0147] As used herein, the terms "light chain variable region" and "V L ” can be used interchangeably.

[0148] In a preferred embodiment of the present invention, the light chain variable region of the antibody according to the present invention has a complementarity determining region CDR selected from the following group:

[0149] L-CDR1, whose amino acid sequence is QNVANNKN (SEQ ID NO: 7);

[0150] L-CDR2, whose amino acid sequence is SAS;

[0151] L-CDR3, the amino acid sequence of which is AGDYSGSIIS (SEQ ID NO: 8).

[0152] In another preferred embodiment, the amino acid sequence of the light chain variable region is:

[0153] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and light chain constant region, and the light chain constant region can be of rabbit, mouse or human origin, preferably rabbit origin.

[0154] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain and a light chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the variable region of the heavy chain and light chain of the antibody of the present invention, preferably at least 95% homologous.

[0155] As used herein, the term "CAR" refers to a chimeric antigen receptor, including an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens.

[0156] The present invention provides a rabbit monoclonal antibody that specifically binds to the extracellular antigen binding domain of a BCMA CAR. The BCMA CAR is a BCMA CAR constructed based on the scFv of the anti-BCMA monoclonal antibody C11D5.3, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0157] Detection reagents and kits

[0158] The present invention also provides a detection reagent for detecting BCMA CAR-positive cells, the detection reagent comprising a first detection reagent, the detection reagent being the antibody of the first aspect of the present invention as the first antibody. The first antibody may be an unmodified monoclonal antibody or a monoclonal antibody coupled to a fluorescent or luminescent marker. Furthermore, the detection reagent further comprises a second detection reagent, the second detection reagent specifically binding to the first antibody or a marker coupled to the first antibody. In another preferred embodiment, the second antibody carries a detectable label, such as a fluorescent or luminescent marker.

[0159] In a preferred embodiment of the present invention, the detection reagent comprises: a first antibody, which is an unmodified monoclonal antibody; and a second antibody, which is carrying a detectable label. Preferably, the detectable label is a fluorescent label, such as Cy3, Alexa Fluor 647, etc.

[0160] The detection reagent can also be prepared as a kit for detecting BCMA CAR-positive cells, the kit (direct labeling method):

[0161] (t1) a first container, and a detectably labeled monoclonal antibody located in the first container, wherein the monoclonal antibody specifically binds to the extracellular antigen-binding domain of BCMA CAR.

[0162] (t0) Instructions.

[0163] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0164] In another preferred embodiment, the monoclonal antibody is a fluorescently labeled monoclonal antibody.

[0165] In another preferred embodiment, the kit (secondary antibody method) comprises:

[0166] (t1) a first container, and a first antibody in the first container, wherein the first antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the extracellular antigen-binding domain of BCMA CAR; and

[0167] (t2) a second container, and a second antibody specific for the monoclonal antibody located in the second container; and

[0168] (t0) Instructions.

[0169] In another preferred embodiment, the second antibody is an anti-rabbit IgG antibody.

[0170] In another preferred embodiment, the second antibody is selected from the following group: goat anti-rabbit IgG antibody, donkey anti-rabbit IgG antibody.

[0171] In another preferred embodiment, the second antibody carries a detectable label.

[0172] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0173] In another preferred embodiment, the second antibody is a fluorescently labeled secondary antibody.

[0174] In another preferred embodiment, the detection kit further comprises one or more additional detection reagents selected from the following group:

[0175] (t3) a third container, and a sealing agent located within the third container; and

[0176] (t4) a fourth container, and a positive control (such as BCMA CAR scFv recombinant protein) located in the fourth container; and

[0177] (t5) a fifth container, and a negative control reagent located in the fifth container.

[0178] In another preferred embodiment, the additional detection reagents are located in the same or different containers.

[0179] In another preferred embodiment, the instructions describe a method for detecting BCMA CAR-positive cells.

[0180] Detection method

[0181] The present invention also provides a method for detecting CAR-positive cells based on the antibody described in the first aspect of the present invention. Since the monoclonal antibody of the present invention has high affinity and high specificity for the antigen binding region of the matching or corresponding CAR, the monoclonal antibody of the present invention can be used to efficiently, sensitively and accurately detect CAR-positive cells.

[0182] Representative examples of CAR-positive cells that can be detected by the methods of the present invention include (but are not limited to): T cells, NK cells, NKT cells, CIK cells, and other different cells. For example, taking the rabbit monoclonal antibody rA3 against BCMA scFv of the present invention as an example, it can be used to detect corresponding BCMA CD19 CAR-positive cells (including T, NK, NKT or CIK cells).

[0183] The detection method of the present invention can be used in scientific research, cell drug development, cell drug quality control, cell drug clinical treatment monitoring, clinical patient auxiliary diagnosis and other aspects.

[0184] The main advantages of the present invention include:

[0185] (a) The rabbit monoclonal antibody rA3 of the present invention has a detection sensitivity of 1:2048000 when measuring BCMA CAR-positive cells in a flow cytometric assay. Therefore, subsequent detection kits for BCMA CAR-positive cells developed based on the monoclonal antibody of the present invention will have the characteristics of high detection sensitivity and good specificity.

[0186] (b) The detection reagent and method of the present invention have a very high degree of differentiation between CAR-positive cells and CAR-negative cells, and the detection results are more accurate.

[0187] (c) Compared with existing detection reagents such as anti-human IgG (Fab')2, Protein L, and BCMA / Fc, the rabbit monoclonal antibody of the present invention can detect BCMA CAR-positive cells more specifically, sensitively, and accurately, meeting the needs of CAR-T drug production quality control, as well as clinical treatment monitoring and auxiliary diagnosis.

[0188] (d) The production cost of the rabbit monoclonal antibody of the present invention is significantly reduced, and thus it is more advantageous for use in CAR-T drug quality control, clinical treatment monitoring, and auxiliary diagnosis.

[0189] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0190] Example 1

[0191] Animal immunization and positive clone screening

[0192] In May 2019, Celgene and Bluebird Bio jointly developed bb2121, a CAR-T cell therapy for multiple myeloma. The scFv sequence used in the CAR structure is C11D5.3. The antibodies prepared in the examples of the present invention all target this scFv sequence.

[0193] The sequence information of C11D5.3 is:

[0194] C11D5.3-hFc protein, a single-chain form of the scFv-hFc structure, was prepared and used to immunize rabbits at weeks 0, 3, 5, and 7. After immunization, spleen tissue was extracted and RNA was reverse-transcribed into cDNA. The antibody variable region sequences were amplified and ligated into the phage vector pCANTAB-5E. The pCANTAB-5E plasmid library containing the antibody variable regions was then transformed into Escherichia coli TG1 using a Bio-Rad electroporator to construct a phage antibody library.

[0195] With the assistance of helper phage M13K07, a phage library co-expressing the corresponding antibody scFv structure on the capsid protein was prepared and purified from the phage antibody library. The resulting phage library was co-incubated with BCMA CAR-Jurkat cells with a positive rate of about 10%-30%. Then, flow cytometry was used to screen the BCMA CAR-C11D5.3-specific phage first-round screening library from the constructed phage antibody library. The previous operation was then repeated to obtain the phage second-round screening library and the phage third-round screening library. Finally, monoclonal colonies were picked from the second or third-round phage screening library, and the corresponding monoclonal phage was prepared using helper phage M13K07. The BCMA CAR-C11D5.3-specific monoclonal phage was screened using flow cytometry.

[0196] After testing 8*94 phage monoclones, three positive phage clones, A3, B1, and E7, were finally obtained (as shown in FIG1 ).

[0197] Example 2

[0198] Determination and analysis of monoclonal antibody sequences

[0199] In order to identify the antibody sequence in the positive monoclonal antibody phage, the plasmid in the corresponding glycerol bacteria was extracted, sequenced using pCANTAB-R1 / R2 primers, and the location of the complementary determining region (CDR) was determined using the IgBLAST tool.

[0200] After sequencing, the amino acid sequence of the antibody heavy chain variable region in clone A3 was obtained as follows:

[0201] The amino acid sequence of the light chain variable region is:

[0202] The six CDR regions of the antibody are underlined: (GIDLNSYV (SEQ ID NO: 4), ISGGGTS (SEQ ID NO: 4), ARLAYDGGDYLDI (SEQ ID NO: 6), QNVANNKN (SEQ ID NO: 7), SAS, AGDYSGSIIS (SEQ ID NO: 8)).

[0203] Example 3

[0204] Monoclonal antibody preparation

[0205] The variable regions of the antibody heavy and light chains in the above plasmids were amplified and ligated into the pcDNA3.4 vector containing the rabbit anti-heavy and light chain constant regions. Rabbit full-antibody expression plasmids were constructed and the corresponding rabbit monoclonal antibodies rA3, rB1, and rE7 were expressed using the ExpiCHO-S (Invitrogen, USA) cell line. Before purification, a small amount of culture supernatant was taken to test the antibody specificity and expression. The results showed that the monoclonal antibody rE7 corresponding to the E7 phage was not specific for BCMA CAR-C11D5.3. This may be because the antibody heavy and light chains screened by phage display technology are not in a naturally paired state. When converted to IgG format, the antibody may not express or recognize the original antigen epitope.

[0206] Then the obtained rA3 and rB1 cell culture supernatants were collected using HiTrap TMPurification of the rabbit antibody using an rProtein A FF affinity chromatography column (GE Healthcare, USA) revealed that the expression level of the rB1 antibody was too low, yielding less than 500 μg of antibody per 25 ml of cell culture supernatant. Optimization of expression conditions was required before subsequent functional testing. Furthermore, the expression level of the rA3 antibody reached 7 mg per 25 ml of cell culture supernatant, approximately 14 times the expression level of the rB1 antibody.

[0207] An SDS-PAGE gel was prepared and electrophoresis was performed to detect the purity of the purified rA3 rabbit monoclonal antibody. As shown in FIG2 , the purity of the purified rA3 rabbit monoclonal antibody was greater than 95%.

[0208] Example 4

[0209] Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding specificity of rabbit monoclonal antibodies.

[0210] To determine the specificity of mAb binding, microELISA plates (Nunc, USA) were coated with 50 ng / well of C11D5.3-hFc protein, control mouse IgG, and control human IgG at 4°C overnight. Plates were then blocked with 5% nonfat dry milk in PBS-Tween 20 (PBST). After washing with PBST, 100 μL / well of 10 ng / μL purified rabbit mAb rA3 and control rabbit IgG were added, followed by incubation at 37°C for 2 hours. After washing, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000 dilution; Sigma-Aldrich, USA) was added and incubated at 37°C for 1 hour. After color development, absorbance at 450 nm was monitored using a microplate reader.

[0211] As shown in Figure 3, the ELISA results show that rabbit monoclonal antibody rA3 can bind to the C11D5.3 protein and does not bind to the control human or mouse IgG. This fully demonstrates that rA3 can specifically recognize the C11D5.3-hFc protein and has no cross-reactivity with other mouse or human IgG.

[0212] Example 5

[0213] Immunofluorescence assay (IFA) to determine the binding of rabbit monoclonal antibodies to BCMA CAR-positive cells

[0214] BCMA CAR-Jurkat cells were washed with 1× PBS and fixed in 4% paraformaldehyde for 20 minutes at room temperature. The cells were washed with 1× PBS and then incubated with blocking buffer (PBS containing 10% bovine serum) for 30 minutes at room temperature. After washing with 1× PBS, the cells were incubated with rabbit monoclonal antibody rA3 for 1 hour at room temperature. After washing with 1× PBS, they were incubated with goat anti-rabbit IgG (H+L)-Cy3 secondary antibody for 30 minutes. Cell smears were then observed and photographed using a confocal microscope.

[0215] As shown in Figure 4, rA3 clearly stains BCMA CAR-positive T cells. The red signal on the membrane indicates the localization of BCMA CAR on the cell membrane. BCMA CAR-negative T cells show no red fluorescence signal. Blue fluorescence represents the cell nucleus.

[0216] Example 6

[0217] Specificity, accuracy, sensitivity and precision of rabbit monoclonal antibodies detected by flow cytometry

[0218] (1) Rabbit monoclonal antibody rA3 binds to BCMA CAR-Jurkat cells

[0219] Jurkat cells were infected with BCMA CAR lentivirus, CD19 CAR lentivirus, or CD20 CAR lentivirus to obtain the corresponding CAR-Jurkat cells. 2C6-Jurkat cells are CD20 CAR+ cells, and DUAL CAR-Jurkat cells are CD19 CAR+CD20 CAR+Jurkat cells. rA3 and isotype control (Rabbit IgG) antibodies were diluted into 50 μL of pre-chilled FACS buffer. 2×10 5 Suspend each cell in 50 μL of FACS buffer. Then, add 50 μL of the antibody solution to the 50 μL cell suspension and incubate the mixture on ice for 30 minutes. Wash the cells twice with cold FACS buffer. Add the corresponding secondary antibody (Goat anti-Rabbit IgG (H+L), Alexa Fluor 647) and incubate the mixture on ice for 30 minutes. Wash the cells twice with cold FACS buffer. Resuspend the cells in 200 μL of FACS buffer and analyze by flow cytometry.

[0220] As shown in Figure 5, rA3 specifically recognizes the BCMA CAR structure on the cell surface, but not the CD19 CAR structure or the CD20 CAR structure (Figure 5A). The only difference between the three is the scFv portion. Therefore, rA3 specifically recognizes BCMA CAR-positive cells containing the C11D5.3 scFv sequence. In addition, rA3 can clearly distinguish between BCMA CAR-positive and -negative cell populations, with non-overlapping signal peaks (Figure 5B), demonstrating high detection accuracy.

[0221] Because rA3 has high specificity and accuracy and can clearly distinguish BCMA CAR-positive and negative cell populations, it can conveniently and accurately count BCMA CAR-positive cells, which is very important for BCMA CAR-T drug production quality control and clinical treatment-assisted diagnosis.

[0222] (2) Analysis of rA3 detection sensitivity

[0223] BCMA CAR-Jurkat cells and Jurkat negative cells were mixed, washed with FACS buffer and diluted to approximately 2 × 10 cells per sample. 5 cells / test. Rabbit monoclonal antibody rA3 was diluted to a concentration of 1 mg / ml and then serially diluted two-fold in FACS buffer starting at a dilution ratio of 1:1000. The cells were then resuspended with the diluted antibody and the mixture was incubated on ice for 30 minutes. The cells were then washed twice with cold FACS buffer. The corresponding secondary antibody (Goat anti-Rabbit IgG (H+L), Alexa Fluor 647) was added and the mixture was incubated on ice for 30 minutes. The cells were then washed twice with cold FACS buffer. The cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry.

[0224] As shown in Figure 6, even at a dilution ratio of 1:2,048,000, rabbit monoclonal antibody rA3 clearly separated negative and positive cells, demonstrating the high sensitivity of rA3 in flow cytometry detection of BCMA CAR-positive cells. This allows for convenient and accurate enumeration of low-abundance BCMA CAR-positive cells, which is crucial for in vivo BCMA CAR-T cell detection, clinical treatment monitoring, and auxiliary diagnosis.

[0225] (3) Analysis of rA3 detection accuracy

[0226] BCMA CAR-Jurkat cells and Jurkat cells were taken separately, washed, counted, and the cell density was adjusted to 1×10 6 cells / ml. Take 1×10 5CAR-Jurkat cells and negative Jurkat cells were mixed to create a 50% BCMA CAR-Jurkat sample. Unstained cells served as a negative control; rA3 antibody staining was used as a test control. After staining, the cells were washed twice and then stained with the corresponding secondary antibody (Goat anti-Rabbit IgG (H+L), Alexa Fluor 647). The cells were then washed three times and analyzed.

[0227] As shown in FIG7 , the positive result of the test group was 49.0%, ranging from 45.0% to 55.0%. It can be seen that the rabbit monoclonal antibody rA3 has a very high accuracy in flow cytometry detection of BCMA CAR-Jurkat cells.

[0228] (4) Analysis of rA3 detection precision

[0229] CAR-Jurkat positive cells and Jurkat negative cells were collected, washed, counted, and the cell density was adjusted to 1.0×10 6 cells / mL. CAR-Jurkat and Jurkat cells were mixed as test cells. The precision test experiment was repeated 12 times. Each sample was about 2×10 5 Stain with rA3-AF647 antibody, incubate at room temperature in the dark for 30 minutes, wash twice, and then analyze.

[0230] As shown in Figure 8 , the detection results of the 12 replicate wells were similar, indicating that rabbit monoclonal antibody rA3 has extremely high precision in flow cytometry detection of BCMA CAR-Jurkat cells.

[0231] Example 7

[0232] Determination of the Binding Affinity of Rabbit Monoclonal Antibodies by Biofilm Interferometry (BLI)

[0233] Before BLI assay, purified C11D5.3-hFc protein was labeled with EZ-Link™ 64 Sulfo-NHS-LC-LC-Biotin Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and then stained with Zeba TM Excess unreacted biotin was removed by centrifugation through a desalting column (Thermo 66 Fisher Scientific).

[0234] In order to measure the binding affinity of antibody rA3 to C11D5.3-hFc, BLI assay was performed on an Octet RED96 machine (Pall Forté Bio, the United States) according to the manufacturer's instructions. Briefly, biotinylated C11D5.3-hFc protein was fixed on a streptavidin-coated biosensor (Pall Forté Bio) until saturation. The biosensor bound to the antigen (C11D5.3-hFc protein) was placed in a well containing a series of diluted monoclonal antibody samples to allow antigen-antibody binding, and then immersed in dissociation buffer (0.01M PBS supplemented with 0.1% bovine serum albumin and 0.02% Tween 20) to dissociate. The equilibrium dissociation constant (KD) was calculated using Octet data analysis software (Pall Forté Bio).

[0235] The results showed that rA3 bound to C11D5.3-hFc with high affinity.

[0236] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An antibody or antigen-binding fragment thereof that specifically targets BCMA CAR, characterized in that: The heavy chain variable region of the antibody comprises the following three complementarity determining regions (CDRs): H-CDR1 as shown in SEQ ID NO: 4, H-CDR2 as shown in SEQ ID NO: 5, and H-CDR3 as shown in SEQ ID NO: 6; and The light chain variable region of the antibody comprises the following three complementarity determining regions (CDRs): L-CDR1 as shown in SEQ ID NO: 7, The amino acid sequence of L-CDR2 is SAS, and L-CDR3 as shown in SEQ ID NO:

8.

2. The antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:

2.

3. The antibody according to claim 1, wherein The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

3.

4. The antibody according to claim 1, wherein The antibodies include: monoclonal antibodies, single-chain antibodies (scFv), Fab or (Fab')2 fragments, and double-chain antibodies.

5. The antibody according to claim 1, wherein The antibody is a rabbit-derived monoclonal antibody.

6. The antibody according to any one of claims 1 to 5, wherein The antibody specifically binds to the scFv in BCMA CAR, and the scFv in BCMA CAR is C11D5.3, whose amino acid sequence is shown in SEQ ID NO:

1.

7. The antibody according to claim 6, wherein The equilibrium dissociation constant K of the antibody and the scFv in BCMA CAR D ≤1nM, more preferably, K D ≤100pM, optimally, K D ≤50pM.

8. A recombinant protein comprising: (i) the antibody or antigen-binding fragment thereof according to claim 1; and (ii) optionally a tag sequence to facilitate expression and / or purification.

9. A polynucleotide encoding a polypeptide selected from the group consisting of: the antibody or antigen-binding fragment thereof according to claim 1; or the recombinant protein according to claim 8.

10. An immunoconjugate comprising: (a) the antibody of claim 1; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a magnetic bead, and agarose.

11. A detection reagent for detecting BCMA CAR-positive cells, the detection reagent comprising: The first detection reagent is the antibody according to claim 1 as the first antibody.

12. The detection reagent according to claim 11, wherein The detection reagent further includes a second detection reagent, which specifically binds to the first antibody or a label coupled to the first antibody. The detection reagent according to claim 12 , wherein the second detection reagent is a second antibody specific for the first antibody.

14. A kit for detecting BCMA CAR-positive cells, comprising: (t1) a first container containing the antibody according to claim 1, wherein the antibody specifically binds to the extracellular antigen-binding domain of BCMA CAR, and optionally (t0) instructions.

15. Use of the antibody according to claim 1 for preparing a detection reagent or kit for detecting BCMA CAR-positive cells.

16. A method for detecting BCMA CAR-positive cells, comprising the steps of: (1) providing the detection reagent according to claim 11; (II) detecting the cell population to be detected using the detection reagent to obtain qualitative or quantitative detection results of BCMA CAR-positive cells.

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