Monoclonal antibody of anti-CD20 antibody and application thereof

Abstract

The invention provides a monoclonal antibody of an anti-CD20 antibody and application of the monoclonal antibody. The monoclonal antibody aiming at the CD20 antibody is high in specificity and strong in affinity. The CD20 CAR-T cell detection reagent provided by the invention can aim at an extracellular antigen binding region of a CD20 CAR-T cell, and has the characteristics of high detection sensitivity, good specificity and the like.

Description

Anti-CD20 monoclonal antibodies and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody for detecting anti-CD20 CAR expression levels and applications thereof. Background Art

[0002] CAR-T cell immunotherapy has become a research hotspot both domestically and internationally due to its remarkable efficacy in treating a variety of malignancies, including leukemia, lymphoma, and multiple myeloma. Patients with chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL) have a poor prognosis, and CD19 and CD20 are promising targets for the treatment of B-cell malignancies. However, despite the significant success of anti-CD19 CAR therapy, with five CD19-targeted CAR-T cell products currently available worldwide and two approved in China in 2021, only 30-35% of DLBCL patients achieve sustained remission for more than two to three years after anti-CD19 CAR T cell therapy. Relapse and nonresponse may be due to decreased CD19 expression after anti-CD19 treatment and low CD19 expression in certain lymphomas.

[0003] To overcome the limitations of CD19 CAR T therapy, bispecific CAR T targeting CD19 and CD20 has been developed and clinical trials have been conducted. It was found that bispecific CAR targeting CD19 and CD20 showed greater efficacy than CAR targeting CD19 alone, prolonging the remission period and reducing the relapse rate.

[0004] During the development, production, and clinical use of these bispecific CAR T-cell products targeting CD19 and CD20, CAR positive expression rate is a mandatory test. Flow cytometry is typically used to measure CAR positive expression rate. Because these are bispecific CAR-Ts, the need for specific detection reagents for both CARs is even more pressing.

[0005] Currently, there are detection methods targeting different structural regions of CARs, including those targeting the CAR antigen binding site, such as the target antigen, or anti-Fab antibodies or Protein L proteins targeting the light chain or hinge region. The latter two lack exclusive specificity and cannot distinguish between two CARs expressed simultaneously. While the CD20 target antigen is specific, the protein itself has multiple transmembrane spans and a short extracellular region, making its preparation difficult and its detection performance low. In addition, these CAR detection tools also generally suffer from low sensitivity and / or high nonspecific background, making it impossible to accurately and conveniently detect CAR expression on the surface of transduced T cells, resulting in quantification and a large degree of potential variability between CAR T cell preparations. In addition, in clinical trials, the expansion and persistence of CAR-T cells need to be monitored after infusion. Due to the complex background, small number of CAR-T cells, and low ratio, there is an even greater need for highly sensitive and specific anti-CD20 CAR idiotypic flow cytometric detection antibodies.

[0006] Summary of the Invention

[0007] The present invention aims to provide a highly sensitive monoclonal antibody that specifically binds to CD20 CAR, and its use in detecting CD20 CAR-positive cells.

[0008] In a first aspect of the present invention, an antibody or antigen-binding fragment thereof that specifically targets CD20 CAR is provided, wherein the heavy chain variable region of the antibody comprises the following three complementarity determining regions (CDRs):

[0009] H-CDR1 as shown in SEQ ID NO: 4,

[0010] H-CDR2 as shown in SEQ ID NO: 5, and

[0011] H-CDR3 as shown in SEQ ID NO: 6; and

[0012] The light chain variable region of the antibody comprises the following three complementarity determining regions (CDRs):

[0013] L-CDR1 as shown in SEQ ID NO: 7,

[0014] The amino acid sequence is L-CDR2 of GAS, and

[0015] L-CDR3 as shown in SEQ ID NO:8.

[0016] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2.

[0017] In another preferred embodiment, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 3.

[0018] 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.

[0019] In another preferred embodiment, the heavy chain constant region and light chain constant region of the antibody are of rabbit origin.

[0020] 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.

[0021] In another preferred embodiment, the antibody is a rabbit-derived monoclonal antibody.

[0022] In another preferred embodiment, the antibody specifically binds to the scFv in CD20 CAR.

[0023] In another preferred embodiment, the scFv in the CD20 CAR is 2C6, and its amino acid sequence is shown in SEQ ID NO: 1.

[0024] In another preferred embodiment, the scFv in the CD20 CAR is 2F2, and its amino acid sequence is shown in SEQ ID NO:9.

[0025] In another preferred embodiment, the equilibrium dissociation constant K between the antibody and the scFv in CD20 CAR is D ≤1nM, more preferably, K D ≤100pM, optimally, K D ≤50pM.

[0026] The second aspect of the present invention provides a recombinant protein, comprising:

[0027] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0028] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0029] In another preferred embodiment, the tag sequence includes a secretory peptide tag.

[0030] In another preferred embodiment, the tag sequence includes a 6*His tag and / or an Fc tag.

[0031] In another preferred embodiment, the recombinant protein is a specific anti-CD20scFv antibody.

[0032] The third aspect 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.

[0033] In another preferred example, the polynucleotide encodes the heavy chain variable region of the antibody, and its sequence is shown in SEQ ID NO: 10.

[0034] In another preferred example, the polynucleotide encodes the light chain variable region of the antibody, and its sequence is shown in SEQ ID NO:11.

[0035] The nucleotide sequence of the heavy chain variable region of the rabbit monoclonal antibody r1F5 of the present invention is:

[0036] The nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody r1F5 of the present invention is:

[0037] The fourth aspect of the present invention provides a vector comprising the polynucleotide according to the third aspect of the present invention.

[0038] 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.

[0039] 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.

[0040] In another preferred embodiment, the cells include: prokaryotic cells or eukaryotic cells.

[0041] The sixth aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0042] (a) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0043] (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.

[0044] 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.

[0045] 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:

[0046] (a) providing CD20 CAR scFv recombinant protein;

[0047] (b) immunizing rabbits with the recombinant protein, taking spleen tissue from the immunized rabbits, extracting RNA therefrom and reverse transcribing it into cDNA;

[0048] (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;

[0049] (d) Screening CD20 CAR scFv-specific positive monoclones from the constructed phage antibody library;

[0050] (e) sequencing the positive monoclonal antibody to obtain the light and heavy chain gene sequences of the monoclonal antibody;

[0051] (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;

[0052] (g) culturing the transfected eukaryotic host cell under conditions suitable for expression, thereby expressing the monoclonal antibody; and

[0053] (h) isolating and / or purifying the monoclonal antibody from the culture system.

[0054] In another preferred embodiment, the expression includes secretory expression (ie, secretion into the cell culture medium).

[0055] 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.

[0056] In another preferred embodiment, the eukaryotic host cell is a CHO cell, specifically an ExpiCHO-S cell.

[0057] In another preferred embodiment, the CD20 CAR scFv recombinant protein is a 2C6-hFc single chain, wherein the amino acid sequence of 2C6 is shown in SEQ ID NO: 1.

[0058] In another preferred embodiment, the method further comprises the optional steps of:

[0059] (h) performing performance tests on the isolated and / or purified monoclonal antibody.

[0060] In another preferred embodiment, the performance measurement comprises one or more tests selected from the following group:

[0061] (1) Specificity detection;

[0062] (2) potency testing;

[0063] (3) Sensitivity testing;

[0064] (4) Precision testing;

[0065] (5) neutralization blocking assay; and

[0066] (6) Binding kinetics detection.

[0067] In an eighth aspect of the present invention, a detection reagent for detecting CD20 CAR-positive cells is provided, the detection reagent comprising:

[0068] The first detection reagent is the antibody described in the first aspect of the present invention as the first antibody.

[0069] 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.

[0070] In another preferred embodiment, the first antibody is a monoclonal antibody coupled to a fluorescent or luminescent marker.

[0071] In another preferred embodiment, the first antibody is an unmodified monoclonal antibody.

[0072] In another preferred embodiment, the second detection reagent is a second antibody specific to the first antibody.

[0073] In another preferred embodiment, the first antibody is a biotinylated monoclonal antibody, and the second detection reagent is streptavidin with a detectable label.

[0074] In another preferred embodiment, the second antibody carries a detectable label, and the detectable label includes a fluorescent or luminescent label.

[0075] In another preferred example, the detection sensitivity of the detection reagent is ≥1 CD20 CAR-positive cell / 1000 cells, more preferably ≥5 CD20 CAR-positive cells / 10000 cells, more preferably ≥1 CD20 CAR-positive cell / 10000 cells, and more preferably ≥2 CD20 CAR-positive cells / 100000 cells.

[0076] In another preferred embodiment, the monoclonal antibody is a rabbit monoclonal antibody.

[0077] In another preferred embodiment, the first antibody and the second antibody are from different mammalian species.

[0078] In another preferred embodiment, the first antibody is from rabbit, and the second antibody is from donkey or sheep.

[0079] In another preferred embodiment, the CAR-positive cells include CAR immune cells and CAR non-immune cells.

[0080] 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.

[0081] In another preferred embodiment, the CAR-positive cells include: HEK-293T cells, Jurkat cells, or a combination thereof.

[0082] In another preferred embodiment, the monoclonal antibody specifically recognizes and binds to the CD20 CAR constructed based on the scFv of the anti-CD20 monoclonal antibody 2C6.

[0083] In another preferred embodiment, the CD20 CAR scFv recombinant protein is a 2C6-hFc single chain, wherein the amino acid sequence of 2C6 is shown in SEQ ID NO: 1.

[0084] A ninth aspect of the present invention provides a kit for detecting CD20 CAR-positive cells, the kit comprising:

[0085] (t1) a first container containing the antibody according to the first aspect of the present invention, wherein the antibody specifically binds to the extracellular antigen-binding domain of CD20 CAR,

[0086] and optionally (t0) instructions.

[0087] In another preferred embodiment, the antibody is a monoclonal antibody, and the monoclonal antibody carries a detectable label.

[0088] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0089] In another preferred embodiment, the antibody is a fluorescently labeled monoclonal antibody.

[0090] 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.

[0091] In another preferred embodiment, the second antibody is an anti-rabbit IgG antibody.

[0092] In another preferred embodiment, the second antibody is selected from the following group: goat anti-rabbit IgG antibody, donkey anti-rabbit IgG antibody.

[0093] In another preferred embodiment, the second antibody carries a detectable label.

[0094] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0095] In another preferred embodiment, the second antibody is a fluorescently labeled secondary antibody.

[0096] In another preferred embodiment, the first container and the second container may be integrated or separated.

[0097] In another preferred embodiment, the detection kit further comprises one or more additional detection reagents selected from the following group:

[0098] (t3) a third container, and a sealing agent located within the third container; and

[0099] (t4) a fourth container, and a positive control (such as CD20 CAR scFv recombinant protein) located in the fourth container; and

[0100] (t5) a fifth container, and a negative control reagent located in the fifth container.

[0101] In another preferred embodiment, the additional detection reagents are located in the same or different containers.

[0102] In another preferred embodiment, the instructions describe a method for detecting CD20 CAR-positive cells.

[0103] In another preferred embodiment, the scFv in the CD20 CAR is 2C6, whose amino acid sequence is shown in SEQ ID NO: 1, or the scFv in the CD20 CAR is 2F2, whose amino acid sequence is shown in SEQ ID NO: 9.

[0104] The tenth aspect of the present invention provides a use of the antibody according to the first aspect of the present invention for preparing a detection reagent or kit for detecting CD20 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 CD20 CAR-positive cells comprise a CD20 CAR constructed based on the scFv of the anti-CD20 monoclonal antibody 2C6, and / or a CD20 CAR constructed based on the scFv of the anti-CD20 monoclonal antibody 2F2.

[0109] In an eleventh aspect of the present invention, a method for detecting CD20 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 CD20 CAR-positive cells.

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

[0113] (1) Take 1×10 5 -1×10 6 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 CD20 CAR-positive cells comprise a CD20 CAR constructed based on the scFv of the anti-CD20 monoclonal antibody 2C6, and / or a CD20 CAR constructed based on the scFv of the anti-CD20 monoclonal antibody 2F2.

[0120] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0121] In another preferred embodiment, the method is an in vitro detection method.

[0122] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0123] In another preferred embodiment, the method is a quality control method.

[0124] 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

[0125] Figure 1 shows some results of flow cytometry detection of phage monoclonal supernatant.

[0126] Figure 2 shows the purity of rabbit monoclonal antibodies r1F5 and r4A8 detected by SDS-PAGE.

[0127] FIG3 shows the specificity of r1F5 and r4A8 antibodies binding to 2C6 protein detected by ELISA.

[0128] Figure 4 shows that rabbit monoclonal antibody r1F5 has high specificity and accuracy in detecting CD20 CAR-Jurkat cells.

[0129] Figure 5 shows the sensitivity analysis of rabbit monoclonal antibodies r1F5 and r4A8 in detecting CD20 CAR-Jurkat cells; wherein A is the sensitivity result of r1F5 in detecting CD20 CAR-Jurkat cells, and B is the sensitivity result of r4A8 in detecting CD20 CAR-Jurkat cells.

[0130] Figure 6 shows that rabbit monoclonal antibody r1F5 detects CD20 CAR-Jurkat cells with high accuracy.

[0131] Figure 7 shows that rabbit monoclonal antibody r1F5 detects CD20 CAR-Jurkat cells with high precision.

[0132] Figure 8 shows that r1F5-PE and R19M-AF647 can co-stain CD19 CAR + CD20 CAR+ -Jurkat-positive cells.

[0133] FIG9 shows that rabbit monoclonal antibody r1F5 can specifically recognize CD20 CAR-Jurkat cells carrying the 2F2 scFv epitope. DETAILED DESCRIPTION

[0134] After extensive and in-depth research and a large number of experimental screenings, the inventors unexpectedly obtained a monoclonal antibody (rA3) that can specifically bind to CD20 CAR. Experimental results show that the monoclonal antibody against CD20 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 far 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.

[0135] On this basis, the present invention was completed.

[0136] the term

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] As used herein, the terms "heavy chain variable region" and "V H ” can be used interchangeably.

[0142] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0143] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes the following three complementarity determining regions CDR:

[0144] H-CDR1, whose amino acid sequence is GFDFTGYW (SEQ ID NO: 4);

[0145] H-CDR2, whose amino acid sequence is IDPVFNNT (SEQ ID NO: 5);

[0146] H-CDR3, the amino acid sequence of which is ARDRGIVVAGVPYNL (SEQ ID NO: 6).

[0147] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:

[0148] PQKQLVESGGGLVQPGGSLKLSCKASGFDFTGYWMSWVRQAPGKGLEWIGYIDPVFNNTYFASWVNGRFTISRENTQNTLYLQLNSLTAADTATYFCARDRGIVVAGVPYNLWGPGTLVTISS (SEQ ID NO: 2).

[0149] 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.

[0150] As used herein, the terms "light chain variable region" and "V L ” can be used interchangeably.

[0151] 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:

[0152] L-CDR1, whose amino acid sequence is QNIYSN (SEQ ID NO: 7);

[0153] L-CDR2, whose amino acid sequence is GAS;

[0154] L-CDR3, the amino acid sequence of which is QSAYYSSSGLT (SEQ ID NO: 8).

[0155] In another preferred embodiment, the amino acid sequence of the light chain variable region is:

[0156] 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.

[0157] 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.

[0158] 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.

[0159] The present invention provides a rabbit monoclonal antibody that specifically binds to the extracellular antigen binding domain of CD20 CAR. The CD20 CAR is constructed based on the scFv of the anti-CD20 monoclonal antibody 2C6, the amino acid sequence of which is shown in SEQ ID NO: 1. In another preferred embodiment of the present invention, the rabbit monoclonal antibody can also specifically recognize CD20 CAR-positive cells expressing CD20 on the cell surface using another monoclonal antibody 2F2 scFv, the amino acid sequence of which is shown in SEQ ID NO: 9.

[0160] Detection reagents and kits

[0161] The present invention also provides a detection reagent for detecting CD20 CAR-positive cells, the detection reagent comprising a first detection reagent, wherein the detection reagent is 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.

[0162] 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.

[0163] The detection reagent can also be prepared as a kit for detecting CD20 CAR-positive cells, wherein the kit (direct labeling method):

[0164] (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 CD20 CAR.

[0165] (t0) Instructions.

[0166] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0167] In another preferred embodiment, the monoclonal antibody is a fluorescently labeled monoclonal antibody.

[0168] In another preferred embodiment, the kit (secondary antibody method) comprises:

[0169] (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 CD20 CAR; and

[0170] (t2) a second container, and a second antibody specific for the monoclonal antibody located in the second container; and

[0171] (t0) Instructions.

[0172] In another preferred embodiment, the second antibody is an anti-rabbit IgG antibody.

[0173] In another preferred embodiment, the second antibody is selected from the following group: goat anti-rabbit IgG antibody, donkey anti-rabbit IgG antibody.

[0174] In another preferred embodiment, the second antibody carries a detectable label.

[0175] In another preferred embodiment, the detectable label includes a fluorescent or luminescent label.

[0176] In another preferred embodiment, the second antibody is a fluorescently labeled secondary antibody.

[0177] In another preferred embodiment, the detection kit further comprises one or more additional detection reagents selected from the following group:

[0178] (t3) a third container, and a sealing agent located within the third container; and

[0179] (t4) a fourth container, and a positive control (such as CD20 CAR scFv recombinant protein) located in the fourth container; and

[0180] (t5) a fifth container, and a negative control reagent located in the fifth container.

[0181] In another preferred embodiment, the additional detection reagents are located in the same or different containers.

[0182] In another preferred embodiment, the instructions describe a method for detecting CD20 CAR-positive cells.

[0183] Detection method

[0184] 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.

[0185] 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 CD20scFv of the present invention as an example, it can be used to detect corresponding CD20 CAR-positive cells (including T, NK, NKT, or CIK cells).

[0186] 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.

[0187] The main advantages of the present invention include:

[0188] (a) The rabbit monoclonal antibody r1F5 of the present invention has a detection sensitivity of 1:1024000 when measuring CD20 CAR-positive cells in a flow cytometric assay. Therefore, subsequent detection kits for CD20 CAR-positive cells developed based on the monoclonal antibody of the present invention will have the characteristics of high detection sensitivity and good specificity.

[0189] (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.

[0190] (c) Compared with existing detection reagents such as anti-human IgG (Fab')2, Protein L, and CD20 / Fc, the rabbit monoclonal antibody of the present invention can detect CD20 CAR-positive cells more specifically, sensitively, and accurately, meeting the needs of CAR-T drug production quality control, clinical treatment monitoring, and auxiliary diagnosis.

[0191] (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.

[0192] 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.

[0193] Example 1

[0194] Animal immunization and positive clone screening

[0195] The antibodies of the present invention are all scFv single-chain antibodies 2C6 (CD20 CAR-2C6) that target CD20 protein.

[0196] The CD20 CAR-2C6 sequence information is:

[0197] 2C6-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 harvested to extract RNA from the cells, and cDNA was synthesized using reverse transcriptase. 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.

[0198] With the assistance of helper phage M13K07, a phage library co-expressing the corresponding antibody scFv construct on the capsid protein was prepared and purified from the phage antibody library. Simultaneously, a previously prepared adenovirus expressing the anti-CD20 single-chain antibody 2C6 was used to infect 293T cells to generate CD20 CAR-293T cells expressing 2C6 on the cell membrane and GFP intracellularly. The resulting phage library was then incubated with CD20 CAR-293T cells with a positive rate of approximately 10%-30%. Flow cytometry was then used to select the first-round selection pool of phage specific for CD20 CAR-2C6 from the constructed phage antibody library. The previous steps were repeated to obtain the second-round selection pool and the third-round selection pool. Finally, monoclonal colonies were picked from the second or third round of phage screening library, and the corresponding monoclonal phages were prepared using helper phage M13K07. The CD20 CAR-2C6-specific monoclonal phages were screened using flow cytometry.

[0199] After testing 20*94 phage monoclones, multiple positive phage clones including C9, E2, 1F5, 4A8, and 4F3 were finally obtained (as shown in Figure 1).

[0200] Example 2

[0201] Determination and analysis of monoclonal antibody sequences

[0202] 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.

[0203] After sequencing,

[0204] The amino acid sequence of the antibody heavy chain variable region in the 1F5 clone is:

[0205] The amino acid sequence of the light chain variable region is:

[0206] The six CDR regions of the antibody are underlined: GFDFTGYW (SEQ ID NO: 4), IDPVFNNT (SEQ ID NO: 5), ARDRGIVVAGVPYNL (SEQ ID NO: 6), QNIYSN (SEQ ID NO: 7), GAS, QSAYYSSSGLT (SEQ ID NO: 8).

[0207] Example 3

[0208] Monoclonal antibody preparation

[0209] The variable regions of the heavy and light chains of the antibodies in the plasmids corresponding to the positive phage clones C9, E2, 1F5, 4A8, and 4F3 described in Example 1 were amplified respectively and ligated to the pcDNA3.4 vector containing the rabbit anti-heavy and light chain constant regions to construct a rabbit full-antibody expression plasmid and express the corresponding rabbit monoclonal antibodies rC9, rE2, r1F5, r4A8, and r4F3 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 rE2 corresponding to the E2 phage did not recognize the 2C6 protein. This may be because the heavy and light chains of the antibodies screened by the phage display technology are not in a naturally paired state, and when transformed into an IgG format, the antibody will not express or recognize the original antigen epitope.

[0210] Then the obtained rC9, r1F5, r4A8, and r4F3 cell culture supernatants were collected using HiTrap TM Rabbit antibodies were purified using an rProtein A FF affinity chromatography column (GE Healthcare, USA). The results revealed that the expression levels of the rC9 and r4F3 antibodies were too low, with less than 200 μg of antibody produced per 25 ml of cell culture supernatant. Optimization of expression conditions was required before subsequent functional testing. Furthermore, in the same 25 ml cell culture system, the expression level of the r1F5 antibody reached 11.1 mg, approximately 55 times the expression levels of the two aforementioned antibodies; and the expression level of the r4A8 antibody reached 7 mg, approximately 35 times the expression levels of the two aforementioned antibodies.

[0211] SDS-PAGE gel was prepared and electrophoresis was performed to detect the purity of the purified r1F5 and r4A8 rabbit monoclonal antibodies. Figure 2 shows the results of SDS-PAGE electrophoresis, which shows that the purity of the purified r1F5 and r4A8 rabbit monoclonal antibodies is greater than 95%.

[0212] Example 4

[0213] Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding specificity of rabbit monoclonal antibodies.

[0214] To determine the specificity of mAb binding, microELISA plates (Nunc, USA) were coated with 50 ng / well of 2C6-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 of purified rabbit mAbs r1F5 and r4A8, as well as control rabbit IgG, were added and incubated 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.

[0215] As shown in Figure 3, the ELISA results show that rabbit monoclonal antibodies r1F5 and r4A8 bind to the 2C6 protein and do not bind to the control human IgG. This demonstrates that r1F5 and r4A8 specifically recognize the 2C6-hFc protein and have no cross-reactivity with other human IgGs. Furthermore, Figure 3 also shows that r1F5 binds to 2C6-hFc more effectively than r4A8, a finding consistent with the results of subsequent sensitivity analysis.

[0216] Example 5

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

[0218] (1) Rabbit monoclonal antibody r1F5 binds to CD20 CAR-Jurkat cells

[0219] Jurkat cells were infected with CD19 CAR lentivirus, CD20 CAR lentivirus or CD20 CAR lentivirus to obtain the corresponding CAR-Jurkat cells. + CD20CAR + -Jurkat cells. Dilute r1F5 and isotype control (Rabbit IgG) antibodies into 50 μL of pre-chilled FACS buffer. 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 4A, when the r1F5 antibody was used to detect DUAL CAR-Jurkat cells with a positivity rate of approximately 40%, the positive signal detection rate was 40.9%. When it was used to detect CD20 CAR-Jurkat cells with a positivity rate of approximately 35%, the positive signal detection rate was 33.7%. However, when it was used to detect BCMA CAR-Jurkat cells, CD19CAR-Jurkat cells with a positivity rate of approximately 50%, and all-negative Jurkat cells, the positive signal detection rate was almost 0% due to the lack of CD20 CAR scFv epitope on the cell surface.

[0221] This indicates that r1F5 specifically recognizes the CD20 CAR structure on the cell surface, but not the CD19 CAR structure or the CD20 CAR structure. The only difference between the three is the scFv portion. Therefore, r1F5 specifically recognizes CD20 CAR-positive cells containing the 2C6 scFv sequence. Furthermore, r1F5 can clearly distinguish between CD20 CAR-positive and -negative cell populations, with non-overlapping signal peaks and high detection accuracy (Figure 4B).

[0222] Because r1F5 has high specificity and accuracy and can clearly distinguish between CD20 CAR-positive and negative cell populations, it can conveniently and accurately count CD20 CAR-positive cells, which is very important for CD20 CAR-T drug production quality control and clinical treatment-assisted diagnosis.

[0223] (2) Analysis of r1F5 Detection Sensitivity

[0224] CD20 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 antibodies r1F5 and r4A8 were diluted to a concentration of 1 mg / ml, and then serially diluted two-fold using FACS buffer starting from a dilution ratio of 1:1000. The cells were then resuspended with the diluted antibodies 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, after which the cells were washed twice with cold FACS buffer. The cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry.

[0225] As shown in Figure 5A, rabbit monoclonal antibody r1F5 exhibited clear differentiation of negative and positive cell clusters even at a dilution ratio of 1:1024000, demonstrating its high sensitivity in flow cytometry detection of CD20 CAR-positive cells. This allows for convenient and accurate enumeration of low-abundance CD20 CAR-positive cells, which is crucial for in vivo CD20 CAR-T cell detection, clinical treatment monitoring, and auxiliary diagnosis. Figure 5B shows that r4A8 only exhibited clear differentiation of cells at a dilution ratio less than 1:256000. This result is consistent with previous ELISA results. Therefore, the sensitivity of rabbit monoclonal antibody r1F5 is significantly superior to that of r4A8.

[0226] (3) Analysis of r1F5 detection accuracy

[0227] CD20 CAR-Jurkat cells and Jurkat cells were taken separately, washed, counted, and the cell density was adjusted to 1×10 6 cells / mL. CD20 CAR-Jurkat cells and negative Jurkat cells were mixed to create a CD20 CAR-Jurkat sample with a 20% positive rate. Unstained cells served as a negative control; r1F5 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.

[0228] As shown in FIG6 , the positive result of the test group was about 22.0%, ranging from 15.0% to 25.0%. It can be seen that the rabbit monoclonal antibody r1F5 has a very high accuracy in flow cytometry detection of CD20 CAR-Jurkat cells.

[0229] (4) Analysis of r1F5 detection precision

[0230] 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 10 times. Each sample was about 2×10 5 cells / test. Stain with r1F5-AF647 antibody, incubate at room temperature in the dark for 30 minutes, wash twice, and then analyze.

[0231] As shown in Figure 7, the detection results of the 10 replicate wells were similar, indicating that the rabbit monoclonal antibody r1F5 has extremely high precision in the flow cytometry detection of CD20 CAR-Jurkat cells.

[0232] Example 6

[0233] Application of r1F5 in flow cytometry

[0234] (1) r1F5 in CD19 CAR + CD20 CAR + -Application of Jurkat cell flow cytometry

[0235] CD19 CAR + CD20 CAR + - Jurkat-positive and Jurkat-negative cells were 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. Each sample was approximately 2×10 5 Co-stain with r1F5-PE and R19M-AF647 (R19M is a CD19 CAR-specific rabbit monoclonal antibody) antibodies, incubate at room temperature in the dark for 30 minutes, wash twice, and then analyze.

[0236] As shown in Figure 8, r1F5-PE and R19M-AF647 can co-stain CD19 CAR + CD20 CAR + -Jurkat-positive cells and had no effect on each other's detection efficiency.

[0237] (2) Application of r1F5 in flow cytometry detection of other CD20 CAR-positive cells

[0238] There are currently two main chimeric antigen receptors targeting CD20 used in cell therapy, with scFv clone numbers 2C6 and 2F2. Later testing revealed that the monoclonal antibody r1F5 against 2C6 can also specifically recognize 2F2. This may be due to the high sequence homology between 2C6 and 2F2. The specific amino acid sequence of 2F2 is:

[0239] CD20 CAR-Jurkat cells without 2C6 scFv epitope and only with 2F2 scFv epitope on cell surface were mixed with Jurkat negative cells to prepare 2F2 scFv-CD20 CAR-Jurkat cells with a positive rate of about 75%. After co-incubation with r1F5 antibody, the cells were detected by flow cytometry.

[0240] As shown in Figure 9, it can be seen that rabbit monoclonal antibody r1F5 can also specifically recognize CD20 CAR-positive cells expressing CD20 on the cell surface of another monoclonal antibody 2F2 scFv.

[0241] Example 7

[0242] Immunofluorescence assay (IFA) was used to detect the binding of rabbit monoclonal antibody r1F5 to CD20 CAR-positive cells.

[0243] CD20 CAR-Jurkat cells were washed with 1× PBS and fixed in 4% paraformaldehyde for 20 minutes at room temperature. The cells were then washed with 1× PBS and 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 r1F5 for 1 hour at room temperature. After washing with 1× PBS, the cells 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.

[0244] The results showed that r1F5 could clearly stain CD20 CAR-positive T cells.

[0245] Example 8

[0246] Biofilm interferometry (BLI) determination of the binding affinity of mAb r1F5 to 2C6-hFc

[0247] Before BLI assay, purified 2C6-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 The biosensor of the present invention is fixed on the biosensor of streptavidin coating until saturation.The biosensor of the present invention is placed in the hole containing a series of dilution monoclonal antibody sample to allow antigen-antibody combination, 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) is calculated using Octet data analysis software (Pall Forté Bio).

[0248] The results showed that the antibody r1F5 bound to 2C6-hFc with high affinity and had high detection sensitivity.

[0249] 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 CD20 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 is L-CDR2 of GAS, 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 CD20 CAR; the scFv in the CD20 CAR is 2C6, and its 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 CD20 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 or antigen-binding fragment thereof according to 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 CD20 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.

13. A kit for detecting CD20 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 CD20 CAR, and optionally (t0) instructions.

14. Use of the antibody according to claim 1 for preparing a detection reagent or kit for detecting CD20 CAR-positive cells.

15. A method for detecting CD20 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 CD20 CAR-positive cells.

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