Monoclonal antibody for detecting anti-CD19 Car expression level and application thereof in activating CD19 CAR-T cells

Abstract

Antibodies to the CD19 CAR molecule and methods of using the antibodies are provided. The antibody can be specifically combined with CD19 CAR-FMC63, can specifically, sensitively and accurately detect CD19 CAR positive cells, can be used for detecting the self-activation function of CAR-T cells, and meets the requirements of CAR-T drug production quality control, clinical treatment monitoring and auxiliary diagnosis.

Description

Monoclonal antibodies for detecting anti-CD19 CAR expression levels and their application in activating CD19 CAR-T cells Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-FMC63 monoclonal antibody, a kit and applications for CD19 CAR-T cell detection. Background Art

[0002] Chimeric antigen receptor (CAR-T) cell therapy is an emerging immunotherapy that has revolutionized the treatment of certain cancers, particularly relapsed and refractory (r / r) lymphoma, offering a new, potentially curative treatment option for terminally ill patients. While the therapeutic efficacy of CAR-T cells is impressive, the high cost of treatment is undeniable due to the quality control and specialized manufacturing processes involved. The seven FDA-approved CAR-T cell products are priced at over US$300,000, and the two CAR-T cell products available in China, Yikaida and Beinoda, are priced at over RMB 1.2 million.

[0003] CAR-T cells need to be activated and expanded during the production process, and the proportion and number of cells transduced with CAR must meet certain requirements. In addition, due to the transduction and amplification process involved in CAR-T cell production, the preparation cycle takes about 2 weeks, which further limits the company's production capacity and increases the cost of product production to a certain extent.

[0004] There are now many ways to stimulate, activate and expand CAR-T cells. Due to the special form of CAR-T cells, the activation method needs to consider safety factors in addition to cost and effect, and the existing activation methods generally have their own shortcomings. The first is monoclonal antibodies and interleukins. The common method is to add OKT3 (anti-CD3 monoclonal antibody) and interleukin 2 (IL-2). Because of their poor effect and easy to cause cell death, they are rarely used; the second is artificial antigen-presenting cells. In recent clinical studies, CAR-T cells are activated by inactive antigen-presenting cells, such as K562 cell line antigens (TAA) that co-express the required stimulatory molecules and tumor-associated molecules. After irradiation, the dead cells meet the current good manufacturing practices (cGMP), and they do not express human leukocyte antigens A and B, and selectively stimulate CAR-T-TAA-specific cells. Although antigen-presenting cells have good activation performance and can only Activate CAR-T cells without activating non-CAR-T cells. However, since CAR-T cells need to be infused back into the body, activation through antigen-presenting cells will undoubtedly bring safety risks and make the entire process and quality control more complicated. The third and currently commonly used method is cell-sized anti-CD3 / CD28 antibody-coated magnetic beads. Studies have found that the effect of using anti-CD3 / CD28 antibody-coated magnetic beads as artificial antigen-presenting particles for activation is stronger than that of using anti-CD3 antibodies and IL-2 for activation, and the activation causes less T cell consumption, so the effect is more lasting. However, this type of activation can only activate T cells indiscriminately, but cannot specifically activate CAR-T cells.

[0005] In summary, scFv-specific antibodies that can specifically activate CAR-T cells and do not pose any safety risks have enormous innovative application value and market prospects.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a detection and quality control method for CAR-T cell detection technology in view of the limitations of the existing technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] In the first part, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, wherein the polypeptide sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of:

[0010] a. SEQ ID Nos: 3, 4, 5, 7, 8, 9; or

[0011] b. SEQ ID Nos: 3, 4, 5, 11, 12, 13; or

[0012] c.SEQ ID Nos: 15, 16, 17, 19, 20, 21;

[0013] wherein the antibody or antigen-binding fragment thereof specifically binds to CD19 CAR-FMC63; and

[0014] Any of the above polypeptide sequences also includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-5 (or 1, 2, 3) amino acids and capable of retaining FMC63 binding affinity.

[0015] In a preferred embodiment, the heavy chain of an antibody of the present invention further comprises a heavy chain constant region and / or the light chain further comprises a light chain constant region.

[0016] In a preferred embodiment, the number of amino acids added, deleted, modified and / or substituted in the three HCDRs and three LCDRs of the antibody of the present invention is 1-5 (eg, 1-3, preferably 1-2, more preferably 1).

[0017] In a preferred embodiment, the heavy chain variable region of an antibody of the present invention further comprises a human or humanized framework, and / or the light chain variable region of an antibody further comprises a human or humanized framework.

[0018] In preferred embodiments, the antibodies of the present invention are diabodies or single-chain antibodies.

[0019] In preferred embodiments, the antibodies of the present invention are full-length antibody proteins or antigen-binding fragments.

[0020] In a preferred embodiment, the polypeptide sequence of the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention is selected from the group consisting of:

[0021] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 2 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 6; or

[0022] b. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 2 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; or

[0023] c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 14 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18.

[0024] In preferred embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least (≥) 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 or 14; or a light chain variable region having a polypeptide sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 6, 10 or 18.

[0025] In a preferred embodiment, the V of the monoclonal antibody H Chain and V L The chains are respectively the same as SEQ ID NO: 2 (V H ) and SEQ ID NO: 6 (V L ) have at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence identity with the amino acid sequence of the present invention.

[0026] In a preferred embodiment, the V of the monoclonal antibody H Chain and V L The chains are respectively the same as SEQ ID NO: 2 (V H ) and SEQ ID NO: 10 (V L ) have at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence identity with the amino acid sequence of the present invention.

[0027] In a preferred embodiment, the V of the monoclonal antibody H Chain and V L The chains are respectively the same as SEQ ID NO: 14 (V H ) and SEQ ID NO: 18 (V L ) have at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence identity with the amino acid sequence of the present invention.

[0028] In preferred embodiments, the monoclonal antibody is IgA, IgD, IgE, IgG, or IgM.

[0029] In a preferred embodiment, the monoclonal antibody is selected from: (i) a single-chain antibody, a single-chain variable region fragment (scFv), a monovalent antibody lacking a hinge region, or a minibody; (ii) a Fab, Fab', or F(ab')2 fragment; (iii) an intact antibody; or (iv) an antibody comprising a human IgG Fc domain.

[0030] In a preferred embodiment, the monoclonal antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 and synthetic IgG.

[0031] In a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric.

[0032] In preferred embodiments, the isolated monoclonal antibodies or antigen-binding fragments thereof of the present invention are of murine or rabbit origin.

[0033] In preferred embodiments, the isolated monoclonal antibodies or antigen-binding fragments thereof of the present invention are human or humanized.

[0034] In a preferred embodiment, the derivative antibodies of the present invention include two types: those that cannot activate CAR-T cells and those that can activate CAR-T cells, and can be selected according to experimental requirements.

[0035] In the second part, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment of the first part of the present invention.

[0036] In the third part, the present invention provides a vector comprising a polynucleotide encoding the monoclonal antibody or antigen-binding fragment of the first part of the present invention.

[0037] In preferred embodiments, the vector comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a lentivirus, a retrovirus, or other vector.

[0038] In the fourth part, the present invention provides an engineered host cell, the genome of which contains the nucleic acid of the second part or the vector of the third part.

[0039] In the fifth part, the present invention provides a use of an active ingredient, comprising:

[0040] (1) for preparing detection reagents or kits; and / or

[0041] (2) Used to specifically activate CAR-T cells; and / or

[0042] (2) Used to detect the self-activation function of CAR-T cells;

[0043] Wherein, the active ingredient is selected from the isolated monoclonal antibody or antigen-binding fragment thereof in the first part.

[0044] In a preferred embodiment, the detection reagent is a test strip or a test disc.

[0045] In a preferred embodiment, the detection reagent or kit is used for:

[0046] (1) detecting the target CD19 CAR-T cells, the nucleic acid (particularly DNA or RNA) of the second part, the vector of the third part, or the engineered host cells of the fourth part in the sample; and / or

[0047] (2) Detecting the target CAR-T cell's self-activation function; and / or

[0048] (3) Detect the expression level of CD19 CAR molecules on CAR-T cells.

[0049] In the sixth part, the present invention provides a method for in vitro detection (including diagnostic or non-diagnostic detection) of FMC63 in a sample, comprising the steps of:

[0050] (1) contacting a sample with the antibody of the first part of the present invention in vitro;

[0051] (2) Detecting whether an antigen-antibody complex is formed. The formation of the complex indicates the presence of FMC63 in the sample.

[0052] In the seventh part, the present invention provides a method for specifically activating CAR-T cells, comprising the following steps:

[0053] (1) contacting a CAR-T cell sample with the antibody of the first part of the present invention and incubating at 37° C.; wherein, an antibody that cannot activate CAR-T cells is used as a control;

[0054] (2) Detect the INFgamma content in the supernatant to confirm the activation ability.

[0055] In a preferred embodiment, the above-mentioned CAR-T cells are CD19 CAR-T cells.

[0056] In the eighth part, the present invention provides a kit comprising:

[0057] (1) a first container containing the antibody of the present invention as a first antibody; and

[0058] (2) A second container containing a second antibody directed against the first antibody of the present invention.

[0059] In a preferred embodiment, the first antibody comprises antibodies that can activate CAR-T cells and antibodies that cannot activate CAR-T cells.

[0060] In the ninth part, the present invention provides a method for preparing a recombinant polypeptide, comprising:

[0061] (1) culturing the engineered host cell of the fourth aspect of the present invention under conditions suitable for expression; and

[0062] (2) Isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the monoclonal antibody or antigen-binding fragment thereof of the first portion.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) When the anti-FMC63 monoclonal antibody of the present invention detects FMC63 scFv in flow cytometry, the detection sensitivity reaches 1:2048000, and the discrimination between CAR-positive cells and CAR-negative cells is very high. Therefore, the detection method and reagent of CAR-positive cells developed based on the monoclonal antibody of the present invention have high detection sensitivity, good specificity, and more accurate results.

[0065] (2) The anti-FMC63 monoclonal antibody of the present invention has a high affinity for FMC63 and can detect CD19 CAR-positive cells more specifically, sensitively, and accurately, meeting the needs of CAR-T drug production quality control, clinical treatment monitoring, and auxiliary diagnosis.

[0066] (3) The anti-FMC63 monoclonal antibodies of the present invention include two types: one that cannot activate CAR-T cells and the other that can activate CAR-T cells. They can be used to detect the self-activation function of CAR-T cells and meet the requirements of CAR-T drug production quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 shows the results of SDS-PAGE electrophoresis detection of the purified anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3.

[0068] FIG2 shows the ELISA results of the specific detection of anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 binding to FMC63 protein.

[0069] Figure 3, including Figures 3A and 3B, shows flow cytometric analysis of the binding specificity of anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 to CD19 CAR-Jurkat cells. Figure 3A shows that r1D6 exhibited a positive signal detection rate of 72.8% when detecting CD19 CAR-Jurkat cells with a positivity rate of approximately 70%, a positive signal detection rate of 50.8% when detecting DUAL CAR-Jurkat cells with a positivity rate of approximately 50%, and a positive signal detection rate of almost 0% when detecting BCMA CAR-Jurkat cells, CD20 CAR-Jurkat cells, and all-negative Jurkat cells with a positivity rate of approximately 50%. Figure 3B is a peak plot of Figure 3A.

[0070] FIG4 shows the flow cytometric test results of the anti-FMC63 monoclonal antibodies r1D6, rC1, rC3, and R19M for detecting the binding accuracy of CD19 CAR-Jurkat cells.

[0071] Figure 5, including Figures 5A, 5B, and 5C, shows flow cytometric analysis of the binding sensitivity of anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 to CD19 CAR-Jurkat cells. Figure 5A shows the sensitivity analysis of r1D6; Figure 5B shows the sensitivity analysis of rC1; and Figure 5C shows the sensitivity analysis of rC3.

[0072] FIG6 shows the flow cytometric test results of the binding accuracy of anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 to CD19 CAR-Jurkat cells.

[0073] FIG7 shows the flow cytometric test results of the binding precision of anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 to CD19 CAR-Jurkat cells.

[0074] Figure 8 shows the test results of the anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 on the functional activation of CD19 CAR-T cells. It can be seen that the rabbit monoclonal antibody r1D6 does not activate CD19 CAR-T cells, while rC1, rC3 and the known R19M and OKT3 monoclonal antibodies all activate CD19 CAR-T cells.

[0075] FIG9 shows the test results of anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 for detecting CD19 CAR-positive cells in immunofluorescence assays.

[0076] Figure 10 shows the equilibrium dissociation constants K of anti-FMC63 monoclonal antibodies r1D6, rC1, and rC3 binding to FMC63-hFc protein. D value. DETAILED DESCRIPTION

[0077] Through in-depth research and extensive screening, the inventors unexpectedly obtained a group of anti-FMC63 antibodies with a completely new amino acid sequence. These antibodies have extremely high affinity for FMC63 and include two types: those that cannot activate CAR-T cells and those that can activate CAR-T cells. These antibodies can be used to detect the autoactivation function of CAR-T cells. The present invention was completed on this basis.

[0078] definition:

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. In other cases, some terms used herein will have their meanings clarified in the specification.

[0080] It should be noted that, as used in the specification and claims of the present invention, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0081] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified by the term "about" in all cases. Thus, numerical values ​​generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Numerical ranges herein explicitly include all possible subranges, all individual values ​​within the range, and integers and fractions within the range.

[0082] Unless otherwise indicated, the term "at least" in a series of elements should be understood to include every element in the series. Those skilled in the art will recognize or be able to ascertain using only routine experimentation the specific embodiments of the invention described herein or many equivalents. The present invention encompasses such equivalents.

[0083] As used herein, the terms "comprises," "includes," "has," or any other similar words will be understood to include the stated integers or combinations of integers, but not to exclude any other integers or combinations of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, preparation, method, article, or apparatus need not be limited to only the elements on the wording, but may include other elements not expressly listed or inherent in the wording. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive "or" rather than an exclusive "or." For example, condition A or B satisfies any of the following conditions: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), or both A and B are true (or exist).

[0084] As used herein, the term "and / or" in conjunction with multiple listed elements should be understood to encompass both individual elements and combinations of elements. For example, where two elements are linked by "and / or," the first applicable option is the first element without the second; the second applicable option is the second element without the first; and the third applicable option is both the first and second elements. Any of these options, or situations where more than one of these options applies, should be considered within the above meaning and meet the criteria for use of the term "and / or" herein.

[0085] The term "consisting of" or other similar words used in this specification and claims means that any integer or combination of integers is included, but additional elements or combinations of integers cannot be added to the specified method, structure or combination.

[0086] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-FMC63 antibodies and polynucleotides encoding them, CD19 polypeptides and CD19 polynucleotides encoding them) refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence using a sequence alignment algorithm or by visual inspection.

[0087] Sequence alignment typically uses one sequence as a reference sequence to which test sequences are compared. When using a sequence alignment algorithm, the test and reference sequences are input into a computer, subsequence coordinates are set, if necessary, and the algorithm program parameters are set. The sequence alignment algorithm then calculates the percent sequence identity of the test sequences relative to the reference sequence based on the set program parameters.

[0088] Optimal sequence alignment methods for comparison include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995)). Supplement)(Ausubel)).

[0089] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (NCBI). The algorithm first identifies high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy a positive threshold score T when aligned with a word of the same length in a database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood hits act as seeds for initiating searches for longer HSPs containing them. The hits are then extended in both directions along each sequence until the cumulative alignment score increases.

[0090] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; >0) and N (penalty score for mismatching residues; <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of a fragment hit in each direction is stopped when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a fragment length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses by default a fragment length (W) of 3, an expectation (E) of 10, and the BLOSEIM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. ETSA 89:10915 (1989)).

[0091] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat T. Acad. Sci. ETSA 90:5873-5787 (1993)). One similarity calculation provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the smallest sum probability in a comparison of a test nucleic acid to a reference sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0092] As described below, a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, whereby the polypeptide is typically substantially identical to the second polypeptide, e.g., the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0093] As used herein, the term "polynucleotide" is defined as a chain of nucleotides, and nucleic acids are polymers of nucleotides, so nucleic acid and polynucleotide are used interchangeably. It is well known to those skilled in the art that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides," which can be hydrolyzed into nucleosides. Polynucleotides as referred to herein include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant methods, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes, using conventional cloning techniques and PCR, and by synthetic means.

[0094] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that constitute a protein or peptide sequence. The above terms used herein include long and short chains, short chains are also commonly referred to in the art as peptides, oligopeptides, and oligomers; while for longer chains, they are generally referred to in the art as proteins, which include many types, for example, "polypeptide" includes biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like.

[0095] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment comprising at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to an antigen of interest. In a particularly preferred embodiment herein, the antigen is FMC63. The antigen-binding fragment of the antibody described herein may comprise a V or V fragment of an antibody that binds to FMC63. H and / or VL One, two, three, four, five or all six CDRs in the sequence. The antigen-binding fragments of the FMC63-specific antibodies described herein are capable of binding to FMC63. In certain embodiments, the antigen-binding fragments specifically bind to and / or inhibit or modulate the biological activity of CAR-T cells.

[0096] The term "antigen" refers to a molecule or portion of a protein molecule that can be bound by a selective binding agent (such as an antibody) and can also be administered to an animal to produce antibodies that can bind to the antigen epitope. An antigen may have one or more epitopes.

[0097] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, epitope determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes the target antigen in a complex mixture of proteins and / or macromolecules. According to certain embodiments, an antibody is said to specifically bind an antigen when the equilibrium dissociation constant for antibody-antigen binding is less than or equal to 10 -6 M, or less than or equal to 10 -7 M, or less than or equal to 10 -8 When M, it can be said that the antibody specifically binds to the antigen. In some embodiments, the equilibrium dissociation constant can be less than or equal to 10 -9 M, or less than or equal to 10 -10 M.

[0098] The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector suitable for transforming a host cell and carrying a nucleic acid sequence that directs and / or controls the expression of an inserted heterologous nucleic acid sequence, including, but not limited to, transcription, translation, and RNA splicing, if introns are present.

[0099] Antibody

[0100] The present invention generally relates to isolated anti-FMC63 antibodies, nucleic acids encoding the antibodies and expression vectors, recombinant cells containing the vectors, and compositions containing the antibodies. Methods for preparing the antibodies and methods for using the antibodies to detect the quality of CAR-T cells are also provided. The antibodies of the present invention have one or more desired functional properties, including but not limited to high-affinity binding to FMC63, high specificity for FMC63, and the ability to activate or not activate CAR-T cell activity.

[0101] Generally, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind FMC63.

[0102] The term "antibody" as used herein is used in a broad sense and includes immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, composite antibodies and chimeric antibodies, as well as monoclonal or polyclonal antibody fragments. Generally, antibodies are proteins or peptide chains that exhibit binding specificity to specific antigens, and their structures are well known. According to the amino acid sequence of the heavy chain constant domain, immunoglobulins can be divided into five major categories (i.e., IgA, IgD, IgE, IgG, and IgM), and IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibodies of the present invention can be any of the five major categories or corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of their constant domains, the antibody light chains of vertebrates can be classified into two distinct types, i.e., κ and λ. Therefore, the antibodies of the present invention can comprise κ or λ light chain constant domains. According to a specific embodiment, the antibodies of the present invention include heavy chain and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light chain constant domains, the antibodies also include an antigen-binding region composed of a light chain variable region and a heavy chain variable region. Each heavy chain variable region includes three domains (i.e., complementary determining regions 1-3; CDR1, CDR2 and CDR3). The light chain variable region domains are also referred to as LCDR1, LCDR2 and LCDR3, and the heavy chain variable region domains are also referred to as HCDR1, HCDR2 and HCDR3.

[0103] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to FMC63 is substantially free of antibodies that do not bind to FMC63), and the isolated antibody is substantially free of other cellular material and / or chemicals.

[0104] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, meaning that the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the present invention can be prepared by hybridoma methods, phage display technology, single lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse or rat, having a genome comprising human heavy chain transgenes and light chain transgenes.

[0105] The term "antigen binding fragment" as used herein refers to an antibody fragment, such as Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv1), single-chain antibody molecule (scFv), single domain antibody (sdab), camelized single domain antibody (camelized single domain antibody), nanobody, domain antibody, bivalent domain antibody or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. The antigen binding fragment is capable of binding to the same antigen bound by the parent antibody or parent antibody fragment. According to specific embodiments, the antigen binding fragment comprises a light chain variable region, a light chain constant region, and the Fd segment of the heavy chain. According to other specific embodiments, the antigen binding fragment comprises Fab and F(ab').

[0106] The term "single-chain antibody" as used herein refers to a single-chain antibody conventional in the art, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to 20 amino acids. The term "single-domain antibody" as used herein refers to a single-domain antibody conventional in the art, which comprises a heavy chain variable region and a heavy chain constant region, or only a heavy chain variable region.

[0107] The term "human antibody" as used herein refers to an antibody produced by humans or an antibody having an amino acid sequence corresponding to an antibody produced by humans prepared using any technique known in the art. The definition of a human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide.

[0108] The term "humanized antibody" as used herein refers to a non-human antibody that has been modified to increase sequences homologous to human antibodies, thereby retaining the antigen-binding properties of the antibody while reducing its antigenicity in the human body.

[0109] The term "chimeric antibody" as used herein refers to an antibody whose immunoglobulin amino acid sequence is derived from two or more species. The variable regions of the light and heavy chains typically correspond to the variable regions of an antibody derived from one mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and avidity, while the constant regions correspond to antibody sequences derived from another mammal (e.g., human) to avoid eliciting an immune response in that species.

[0110] As used herein, an antibody that "specifically binds to FMC63" refers to an antibody that binds to FMC63 with a specific binding affinity of 1×10 -7 M or smaller, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10M or smaller, 1×10 -10 M or smaller K D The term "K D ” refers to the dissociation constant, which is derived from K d and K a The ratio (K d / K a ) and expressed as molar concentration (M). The present application can use the method in the art to determine the antibody K D For example, the K D can be determined by using surface plasmon resonance, for example by using a biosensor system ( systems), or by using biofilm interferometry (BLI), such as the Octet RED96 system.

[0111] Antibody K D The smaller the value, the higher the affinity of the antibody binding to the target antigen.

[0112] As used herein, the term "specific binding" for an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species, but this cross-species reactivity itself does not change the specific classification of the antibody. In another embodiment, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen, but this cross-reactivity itself does not change the specific classification of the antibody. In some cases, the term "specific binding" or "specific binding" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, indicating that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than the general structure of the protein. If the antibody is specific for epitope "A," then in a reaction containing a marker "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of marker A bound to the antibody.

[0113] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include heavy chain and light chain CDR sets, which are inserted between heavy chain and light chain framework regions (FR) sets, and the set provides support for CDR and defines the spatial relationship of CDR relative to each other. As used herein, the term "CDR set" refers to three hypervariable regions of heavy chain or light chain variable region, starting from the N-terminus of heavy chain or light chain, and these regions are expressed as "CDR1", "CDR2" and "CDR3", respectively. Therefore, the antigen binding site includes six CDRs, including CDR sets from each of the heavy chain and light chain variable region. The polypeptide comprising a single CDR (such as CDR1, CDR2 or CDR3) is referred to as a "molecular recognition unit" in this article. Crystallographic analysis of many antigen-antibody complexes shows that the amino acid residues of CDR form extensive contacts with the combined antigen, and the heavy chain CDR3 is the most widely contacted with the antigen. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen binding site.

[0114] As used herein, the term "FR set" refers to the four amino acid sequences flanking the CDRs that make up the variable region of a heavy or light chain. Some FR residues may contact the bound antigen; however, the FRs are primarily responsible for folding the variable region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Certain amino acid residues and certain structural features are extremely conserved within the FRs. In this regard, all variable region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the variable region folds into the binding site, the CDRs appear as prominent circular motifs that form the antigen-binding surface. It is generally believed that, regardless of the exact CDR amino acid sequence, conserved structural regions within the FRs influence the folding of the CDR loops into certain "canonical" structures. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction between the antibody heavy and light chains.

[0115] The structure and location of immunoglobulin variable regions can be found in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4 th Edition, US Department of Health and Human Services, 1987, and its updates, now available on the Internet (immuno.bme.nwu.edu).

[0116] The term "antibody heavy chain" as used herein refers to the larger of the two types of polypeptide chains present in all antibody molecules in their native structure. The heavy chains of any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM, which are also designated as α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function, with humans expressing the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0117] The term "antibody light chain" as used herein refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their native structure, kappa and lambda light chains being the two major antibody light chain isotypes.

[0118] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a phage as described herein. The term should also be construed to refer to an antibody produced by synthesizing a DNA molecule encoding the antibody, and wherein the DNA molecule expresses the antibody protein, or specifies the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using techniques for synthesizing DNA or amino acid sequences that are customary and well known in the art.

[0119] In the present invention, the antibodies of the present invention also include conservative mutants thereof, meaning polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids having the same or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative mutant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0120] Table A

[0121] The present invention relates to isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof. It will be appreciated by those skilled in the art that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore, it will be appreciated by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibodies or antigen-binding fragments thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0122] Polynucleotides, vectors, host cells and preparation methods

[0123] The present invention also provides polynucleotides encoding the antibodies of the present invention.

[0124] The present invention also provides a vector comprising an isolated nucleic acid encoding a monoclonal antibody of the present invention or its antigen-binding fragment. Based on the disclosure of the present application, any vector known to those skilled in the art, such as a plasmid, cosmid, phage vector or viral vector, can be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements for establishing the conventional functions of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selective marker and an origin of replication, and the promoter may be a constitutive, inducible or repressible promoter. Many expression vectors capable of delivering nucleic acid to cells are known in the art and can be applied herein to produce antibodies or their antigen-binding fragments in cells. Conventional cloning techniques or artificial gene synthesis can be used to produce recombinant expression vectors based on the embodiments of the present invention, and these technologies are well known to those skilled in the art.

[0125] The present invention also provides the host cell of the isolated nucleic acid comprising encoding monoclonal antibody of the present invention or its Fab. Based on the application disclosure, any host cell well known by persons skilled in the art can be used for recombinant expression antibody of the present invention or its Fab. In some embodiments, the host cell is Escherichia coli TG1 or BL21 cell (for expression such as scFv or Fab antibody), CHO-DG44 or CHO-K1 cell or HEK293 cell (for expression such as full-length IgG antibody). According to specific embodiment, the recombinant expression vector is transformed into the host cell by conventional method such as chemical transfection, heat shock or electroporation, and it is stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0126] The present invention also provides a method for producing a monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising culturing cells containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions that produce the monoclonal antibody or antigen-binding fragment thereof of the present invention, and recovering the antibody or antigen-binding fragment thereof from the cells or cell culture (e.g., supernatant). The expressed antibody or antigen-binding fragment thereof can be recovered from the cells and purified according to conventional techniques known in the art and as described herein.

[0127] As will be understood by those skilled in the art, a polynucleotide may include genomic sequences, extragenomic and plasmid-encoded sequences, as well as smaller engineered gene fragments that express or can be used to express proteins, polypeptides, peptides, etc., which can be naturally isolated or modified and synthesized by those skilled in the art.

[0128] Those skilled in the art will also recognize that polynucleotide can be single-stranded (coding strand or antisense strand) or double-stranded, and can be DNA (genome, cDNA or synthetic DNA) or RNA molecule, RNA molecule can include and comprise intron and correspond to the HnRNA molecule of DNA molecule in a one-to-one manner, and the mRNA molecule that does not comprise intron.Extra coding or non-coding sequence can but not necessarily be present in the polynucleotide of the application, and polynucleotide can but not necessarily be connected to other molecules and / or auxiliary materials.Polynucleotide can comprise native sequence, also can comprise the variant of coding such sequence or the sequence of derivative.

[0129] Typically, polynucleotide variants may contain one or more substitutions, additions, deletions and / or insertions. Preferably, the binding affinity of the antibody encoded by the variant polynucleotide is not substantially reduced relative to the antibody encoded by the polynucleotide sequence specifically listed in this application.

[0130] The polynucleotides described herein, or fragments thereof, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme cutting sites, multiple cloning sites, other coding segments, etc., and thus their total length may vary greatly. Thus, nucleic acid segments of virtually any length can be used, preferably, their total length is limited by the ease of preparation and use in the intended recombinant DNA protocol, for example, exemplary polynucleotide segments having a total length of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs (including all intermediate lengths) are contemplated as useful.

[0131] Site-directed mutagenesis allows for the generation of mutants by using a specific oligonucleotide sequence encoding the desired mutant DNA sequence, along with a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on either side of the deleted junction being crossed. Mutations can be employed in a selected polynucleotide sequence to improve, modify, reduce, modify, or otherwise alter the properties of the polynucleotide itself, and / or to alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0132] Detection Applications and Kits

[0133] The antibody or antigen-binding fragment thereof according to the present invention can be used in detection, for example, for sample detection to provide quality control information.

[0134] The present invention also provides a kit comprising the antibody (or fragment thereof) of the present invention; in a preferred embodiment of the present invention, the kit further comprises a container, instructions, a buffer, and the like.

[0135] In a preferred embodiment, the antibodies of the present invention may be immobilized on a test plate.

[0136] The kit of the present invention may also include tools and / or reagents known in the art for ELISA. Depending on the requirements, the kit of the present invention may also include tubes, well plates, instructions for use, etc. for mixing the components.

[0137] In one embodiment, the number of CD19 CAR-T cells is confirmed by detecting the presence of FMC63 in the sample.

[0138] In another embodiment, the self-activation ability of CAR-T cells is detected by adding antibodies that can activate CAR-T cells and antibodies that cannot activate CAR-T cells to the sample.

[0139] The following examples further illustrate the present invention. 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 procedures in the examples, unless otherwise specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer-recommended conditions. Unless otherwise indicated, percentages and other numerical values ​​in the examples are by weight. Cell lines were commercially available or purchased from ATCC, and all plasmids were commercially available.

[0140] Example 1: Animal immunization and positive clone screening

[0141] The antigen specifically recognized by each antibody in this patent is CD19 CAR-FMC63, which has the amino acid sequence of SEQ ID No. 1. The specific sequence information is: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

[0142] FMC63-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.

[0143] 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 CD19 CAR-Jurkat cells with a positive rate of approximately 10%-30%. Flow cytometry was then used to screen the CD19 CAR-FMC63-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 were prepared using helper phage M13K07. The CD19 CAR-FMC63-specific monoclonal phage was screened using flow cytometry.

[0144] In this example, a total of 11×94 phage monoclones were detected, and three positive phage clones, 1D6, C1, and C3, were finally obtained.

[0145] Example 2: Determination and analysis of anti-FMC63 monoclonal antibody sequences

[0146] In order to identify the antibody sequence in the positive monoclonal antibody phage, this example extracted the plasmid from the corresponding TG1 glycerol bacteria, used pCANTAB-R1 / R2 primers (primer sequence: pCANTAB5-R1 (SEQ ID No: 22): CCATGATTACGCCAAGCTTTGGAGCC, pCANTAB5-R2 (SEQ ID No: 23): CGATCTAAAGTTTTGTCGTCTTTCC) for sequence determination, and used the IgBLAST tool to determine the position of the complementary determining region (CDR). After sequencing, the antibody heavy chain variable region nucleic acid sequence of the 1D6 clone was SEQ ID No. 24, the antibody light chain variable region nucleic acid sequence in the 1D6 clone was SEQ ID No. 25, the antibody heavy chain variable region nucleic acid sequence in the C1 clone was SEQ ID No. 26, and the antibody light chain variable region nucleic acid sequence in the C1 clone was SEQ ID No. 27; specifically, V H 、V L The nucleic acid sequences are shown in Table 1 below.

[0147] Table 1: Nucleic acid sequences of antibody clones 1D6 and C1

[0148] The nucleic acid sequences of the antibody heavy chain variable region and antibody light chain variable region of clone C1 are SEQ ID No.2 and SEQ ID No.10 respectively, and the nucleic acid sequences of the antibody heavy chain variable region and antibody light chain variable region of clone C3 are SEQ ID No.14 and SEQ ID No.18 respectively; specifically V H 、V L The amino acid sequences of the CDRs are shown in Table 2 below.

[0149] Table 2: Amino acid sequences of antibody clones 1D6, C1, and C3

[0150] Example 3: Preparation of anti-FMC63 monoclonal antibodies

[0151] The antibody heavy and light chain variable regions from the plasmids described in the previous examples were amplified and ligated into the pcDNA3.4 vector containing the rabbit heavy and light chain constant regions to construct rabbit full-antibody expression plasmids. The corresponding rabbit monoclonal antibodies, r1D6, rC1, and rC3, were expressed using the ExpiCHO-S (Invitrogen, USA) cell line. The resulting supernatants from the r1D6, rC1, and rC3 cell cultures were then purified using HiTrap™ rProtein A FF affinity chromatography columns (GE Healthcare, USA).

[0152] SDS-PAGE gels were prepared and electrophoresis was performed to examine the purity of the purified r1D6, rC1, and rC3 rabbit monoclonal antibodies. As shown in Figure 1, the purity of the purified monoclonal antibodies was greater than 95%. However, in subsequent antibody stability studies, we found that r1D6 and rC1 had better stability.

[0153] Example 4: Anti-FMC63 monoclonal antibody binding specificity test

[0154] To determine the specificity of mAb binding, in this example, micro-ELISA plates (Nunc, USA) were coated with 50 ng / well of FMC63 protein and control mouse IgG at 4°C overnight. The plates were then blocked with 5% nonfat dry milk in PBS-Tween 20 (PBST). Following washing with PBST, 100 μL / well of 10 ng / μL purified rabbit mAbs r1D6, rC1, and rC3, 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.

[0155] As shown in Figure 2, the enzyme-linked immunosorbent assay (ELISA) results showed that rabbit monoclonal antibodies r1D6, rC1, and rC3 could bind to the FMC63 protein and did not bind to the control mouse IgG, which fully demonstrated that rabbit monoclonal antibodies r1D6, rC1, and rC3 could specifically recognize the FMC63 protein and had no cross-reaction with other mouse IgG.

[0156] Example 5: Binding test of anti-FMC63 monoclonal antibody to CD19 CAR-Jurkat cells

[0157] Jurkat cells were infected with CD19 CAR lentivirus, BCMA CAR lentivirus or CD20 CAR lentivirus to obtain the corresponding CAR-Jurkat cells. Among them, DUAL CAR-Jurkat cells are CD19 CAR + -CD20 CAR + -Jurkat cells.

[0158] First, rabbit monoclonal antibodies r1D6, rC1, rC3 and isotype control (Rabbit IgG) were diluted into 50 μL pre-cooled FACS buffer, and 2×10 5 Each cell was suspended in 50 μL of FACS buffer. 50 μL of antibody solution was then added to the 50 μL cell suspension 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 and resuspended in 200 μL of FACS buffer for flow cytometry analysis.

[0159] The results are shown in Figure 3A. Using rabbit monoclonal antibody r1D6 as an example, when detecting CD19 CAR-Jurkat cells with a positivity rate of approximately 70%, r1D6 had a positive signal detection rate of 72.8%. When detecting DUAL CAR-Jurkat cells with a positivity rate of approximately 50%, the antibody had a positive signal detection rate of 50.8%. However, when detecting BCMA CAR-Jurkat cells, CD20 CAR-Jurkat cells, and all-negative Jurkat cells with a positivity rate of approximately 50%, the positive signal detection rate was almost 0%, as there is no CD19 CAR scFv epitope on the cell surface. Figure 3B shows the peak plot of Figure 3A, which provides a more intuitive view of the detection of positive cells. The results indicate that rabbit mAb r1D6 specifically recognizes the CD19 CAR structure on the cell surface, but not the BCMA CAR and CD20 CAR structures. The only difference between the three is the scFv portion of the extracellular CAR structure. Therefore, rabbit mAb r1D6 specifically recognizes CD19 CAR-positive cells containing the FMC63 scFv sequence. Flow cytometry results for rabbit mAbs rC1 and rC3 were similar.

[0160] In addition, as shown in Figure 4 , rabbit monoclonal antibodies r1D6, rC1, and rC3 can clearly distinguish CD19 CAR-positive and -negative cell populations, and the signal peaks of the two do not overlap, indicating high detection accuracy.

[0161] Combining the results in Figures 3 and 4 , we can see that rabbit monoclonal antibodies r1D6, rC1, and rC3 have high specificity and accuracy, and can clearly distinguish between CD19 CAR-positive and negative cell populations. Therefore, CD19 CAR-positive cells can be counted conveniently and accurately, which is very important for CD19 CAR-T drug production quality control and clinical treatment-assisted diagnosis.

[0162] Example 6: Anti-FMC63 monoclonal antibody detection sensitivity analysis

[0163] CD19 CAR-Jurkat cells and Jurkat negative cells were mixed, washed with FACS buffer and diluted to approximately 2 × 10 cells per sample. 5cells / test. Rabbit monoclonal antibodies r1D6, rC1, and rC3 were then diluted to 1 mg / mL and serially diluted two-fold in FACS buffer, starting at a 1:1000 dilution ratio. The cells were resuspended in the diluted antibodies and incubated on ice for 30 minutes. The cells were washed twice with cold FACS buffer, and the corresponding secondary antibody (Goat anti-Rabbit IgG (H+L), Alexa Fluor 647) was added. The mixture was incubated on ice for 30 minutes. The cells were then washed twice with cold FACS buffer and resuspended in 200 μL of FACS buffer for flow cytometry analysis.

[0164] The results are shown in Figure 5. When the dilution ratio of rabbit monoclonal antibodies r1D6 (Figure 5A) and rC1 (Figure 5B) reached 1:2048000, and the dilution ratio of rC3 (Figure 5C) reached 1:1024000, obvious negative-positive cell clusters were still visible. It can be seen that rabbit monoclonal antibodies r1D6, rC1, and rC3 have high sensitivity in flow cytometry detection of CD19 CAR-positive cells. Therefore, low-abundance CD19 CAR-positive cells can be easily and accurately counted, which is very important for detecting CD19 CAR-T cells in vivo, conducting clinical treatment monitoring and auxiliary diagnosis.

[0165] Example 7: Analysis of Detection Accuracy of Anti-FMC63 Monoclonal Antibodies

[0166] CD19 CAR-Jurkat cells and Jurkat cells were taken separately, washed and counted, and the cell density was adjusted to 1×10 6 cells / ml, and then 1×10 5 CAR-Jurkat cells and negative Jurkat cells were mixed to create a CD19 CAR-Jurkat sample with a 50% positive rate. Unstained cells served as a negative control and were stained with rabbit monoclonal antibodies r1D6, rC1, and rC3 as the test group. After staining, the cells were washed twice and stained with the corresponding secondary antibody (Goat anti-Rabbit IgG (H+L), Alexa Fluor 647). The cells were then washed three times and analyzed.

[0167] The results are shown in Figure 6. Using rabbit monoclonal antibody r1D6 as an example, flow cytometry results showed a 50.0% positive rate in the test group, ranging from 45.0% to 55.0%. This indicates that rabbit monoclonal antibody r1D6 demonstrated extremely high accuracy in flow cytometry detection of CD19 CAR-Jurkat cells. Flow cytometry results for the remaining antibodies were similar, with detailed data provided in Table 3.

[0168] Table 3: Accuracy of antibody clones r1D6, rC1, and rC3 in detecting CD19 CAR-Jurkat cells

[0169] Example 8: Precision Analysis of Anti-FMC63 Monoclonal Antibody Detection CD19 CAR-Jurkat positive cells and Jurkat negative cells were collected, washed, and counted. The cell density was adjusted to 1.0×10 6 cells / mL, and then CD19 CAR-Jurkat and Jurkat cells were mixed as test cells. The precision test experiment was repeated 12 times, and each sample was about 2×10 5 cells / test, stain with rA3-AF647 antibody, incubate at room temperature in the dark for 30 minutes, wash twice and then detect on the instrument.

[0170] The results are shown in Figure 7. Using the rabbit monoclonal antibody r1D6 as an example, the results from 12 replicate wells were similar, demonstrating that rabbit monoclonal antibody r1D6 exhibits extremely high precision in flow cytometry detection of CD19 CAR-Jurkat cells. Flow cytometry results for the remaining antibodies are similar, with detailed data provided in Table 4.

[0171] Table 4: Precision of antibody clones r1D6, rC1, and rC3 in detecting CD19 CAR-Jurkat cells

[0172] Example 9: Anti-FMC63 monoclonal antibody cell function activation test. Antibodies such as r1D6, rC1, rC3, R19M (FMC63 specific antibody), and OKT3 (CD3 specific antibody) were diluted to 10 μg / mL and 50 μg / mL, and then 50 μL of the diluted antibody solution was added to each well of a 96-well cell culture plate and incubated at 4°C overnight. The next day, the cells were washed three times with PBS, and CD19 CAR-T cells were taken and diluted to 1×10 6 cells / mL, and then 200 μL of the cell suspension was added to the treated 96-well cell culture plate and incubated in a 37°C cell culture incubator for 3 days. After 3 days, the culture supernatant was taken and the IFNgamma content in the supernatant was detected using an IFNgamma detection kit to analyze the CAR-T cell activation ability of the corresponding antibody.

[0173] As shown in Figure 8, rabbit mAb r1D6 does not activate CD19 CAR-T cells, while rC1, rC3, and the known R19M and OKT3 mAbs all activate CD19 CAR-T cells. Based on known sequence information, r1D6 and rC1 share high sequence similarity, yet exhibit distinct differences in activation of CD19 CAR-T cells. Therefore, these two mAbs can be used as control antibodies in subsequent CD19 CAR-T cell activation experiments, effectively eliminating nonspecific background results caused by experimental manipulation or system changes.

[0174] Example 10: Anti-FMC63 monoclonal antibody testing in CD19 CAR-positive cells

[0175] CD19 CAR-Jurkat cells were washed with 1× PBS and then fixed in 4% paraformaldehyde at room temperature for 20 minutes. The cells were washed with 1× PBS and then incubated with blocking solution (PBS containing 10% bovine serum) at room temperature for 30 minutes. After washing with 1× PBS, rabbit monoclonal antibodies r1D6, rC1, and rC3 were added and incubated at room temperature for 1 hour. After washing with 1× PBS, the cells were incubated with goat anti-rabbit IgG (H+L)-Cy3 secondary antibody for 30 minutes. The cells were smeared and photographed using a confocal microscope.

[0176] As shown in Figure 9, rabbit monoclonal antibodies r1D6, rC1, and rC3 can clearly stain CD19 CAR-positive cells. The red signal on the membrane represents the localization of CD19 CAR on the cell membrane. CD19 CAR-negative cells have no red fluorescence signal, and blue fluorescence represents the cell nucleus. Example 11: Equilibrium dissociation constant test of anti-FMC63 monoclonal antibody protein

[0177] Ligand coupling was performed according to the instructions provided in the amino coupling kit. HBS-EP+10X was used as the running buffer. The Fc1 and Fc2 channels of the chip were activated with 11.5 mg / ml NHS and 75 mg / ml EDC (1:1) for 420 s. Antibodies r1D6 and rC1 were diluted to 5 μg / mL with Acetate pH 5.5 and then injected into the activated channel Fc4 for coupling at a flow rate of 10 μL / min. The coupling level was set to 150 RU. Finally, 1 mmol / L of Methanolamine was injected to block the Fc1 and Fc2 channels for 420 s.

[0178] Next, a multi-cycle kinetic assay was used: the antigen FMC63-hFc (SLP004) was diluted to 5 μg / mL with HBS-EP+buffer. This was then doubled to 78 ng / mL as the highest concentration. The flow rate was set at 30 μL / min. The serially diluted antigen was injected into the flow channel, where it bound to the antibody coupled to the chip. The binding time was 180 s and the dissociation time was 300 s. After dissociation, the antibody was regenerated using 10 mM Glycine at pH 1.5. Different concentrations of antigen were repeatedly injected, followed by binding, dissociation, and regeneration. Data were analyzed using the Biacore T200 evaluation software using a 1:1 binding model.

[0179] As shown in Figure 10, the equilibrium dissociation constants (KD) of rabbit monoclonal antibodies r1D6 and rC1 for binding to FMC63-hFc reached 1.241 pM and 5.295 pM, respectively, demonstrating that both rabbit monoclonal antibodies r1D6 and rC1 bind to FMC63-hFc with high affinity and exhibit high sensitivity. All references cited herein are hereby incorporated by reference into this application as if equivalent. It should also be understood that, after reviewing the present disclosure, those skilled in the art may make various modifications or adjustments to the present disclosure, and such equivalents are intended to fall within the scope of the claims herein.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the polypeptide sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from the group consisting of: a. SEQ ID Nos: 3, 4, 5, 7, 8, 9; or b. SEQ ID Nos: 3, 4, 5, 11, 12, 13; or c.SEQ ID Nos: 15, 16, 17, 19, 20, 21; in, The antibody or antigen-binding fragment thereof specifically binds to the CD19 CAR molecule.

2. The monoclonal antibody according to claim 1, characterized in that The polypeptide sequence of the isolated monoclonal antibody or antigen-binding fragment thereof is selected from the following groups: a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 2 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 6; or b. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 2 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10; or c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 14 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

18.

3. The monoclonal antibody according to claim 1, characterized in that The antibody is an FMC63-specific antibody capable of binding to the CD19 CAR molecule.

4. The antibody according to any one of claims 1 to 3, characterized in that The antibody is selected from the following groups: (i) a single-chain antibody, a single-chain variable region fragment, a monovalent antibody lacking a hinge region, or a minibody; or (ii) a Fab, Fab' or F(ab')2 fragment; or (iii) intact antibodies; or (iv) Antibodies comprising an IgG Fc domain.

5. The antibody according to any one of claims 1 to 3, characterized in that The monoclonal antibody is selected from IgG1, IgG2, IgG3, IgG4 or synthetic IgG.

6. The antibody according to any one of claims 1 to 3, characterized in that The monoclonal antibody or antigen-binding fragment thereof is of murine or rabbit origin.

7. An isolated nucleic acid, characterized in that The nucleic acid encodes the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 3.

8. A carrier, characterized in that The vector comprises a nucleic acid encoding the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 3, or the nucleic acid according to claim 7.

9. An engineered host cell, characterized in that The genome of the engineered host cell comprises the nucleic acid of claim 7 or the vector of claim 8.

10. A use of an active ingredient, comprising: (1) Used for preparing detection reagents or kits; and / or (2) Used to specifically activate CAR-T cells; The active ingredient is selected from the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

11. The use according to claim 10, characterized in that The detection reagent or kit is used for: (1) detecting the expression level of CD19 CAR molecules on CAR-T cells in the sample; and / or (2) Detect the self-activation function of CD19 CAR-T cells in the sample.

12. An in vitro method for detecting FMC63 in a sample, comprising the following steps: (1) contacting a sample in vitro with the antibody according to any one of claims 1 to 3; (2) Detect whether an antigen-antibody complex is formed.

13. A method for specifically activating CAR-T cells, comprising the following steps: (1) contacting a CAR-T cell sample with the antibody according to any one of claims 1 to 3 of the present invention, and incubating and culturing at 37°C; wherein, Antibodies that cannot activate CAR-T cells were used as controls; (2) Detect the INFgamma content in the supernatant to confirm the activation ability.

14. A detection kit comprising: (1) A first container containing the antibody according to any one of claims 1 to 3 as a first antibody; and (2) A second container containing a second antibody directed against the first antibody.

15. The kit according to claim 14, characterized in that The first antibody includes antibodies that can activate CAR-T cells and antibodies that cannot activate CAR-T cells.

16. A method for preparing a recombinant polypeptide, comprising: (1) culturing the engineered host cell according to claim 9 under conditions suitable for expression; and (2) Isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

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