Anti-idiotype antibody for Anti-CD19 CAR expression detection

Abstract

The invention relates to the technical field of antibodies, in particular to an anti-idiotype antibody specifically bound with a CAR antigen binding site, and the antibody has the advantages of high affinity, high specificity and no non-specific background, and is more suitable for evaluating the CAR transfection positive rate, the change rule of the number of CAR positive T cells in a subject after administration and the like.

Description

[0001] This application claims priority to Chinese patent application No. 202410002320.4 filed with the Chinese Patent Office on January 2, 2024, entitled “Anti-idiotypic antibodies for detection of Anti-CD19 CAR expression”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the field of antibody technology and the field of immunity in the field of biotechnology, and specifically to an anti-unique antibody for Anti-CD19 CAR expression detection and its application. Background Art

[0003] CAR-T cell therapy is a cancer immunotherapy based on in vitro modification of T cells to express receptor fragments on their surface that specifically recognize tumor surface antigens. The modified T cells are transfused into the patient's body and directly target cancer cells in the body and exert immune killing effects without the help of antigen presenting cells (APCs). For CAR-T cells, the active ingredient that exerts tumor killing effects is CAR-positive T cells. The packaging specifications and clinical dosages of CAR-T cell products are expressed in terms of the number of CAR-T positive cells. Therefore, the CAR transfection positive rate is a must-check item in the development process of CAR-T cells.

[0004] At present, flow cytometry is usually used to detect the positive rate of CAR transfection. There are detection methods for different structural regions of CAR, including those for CAR antigen binding sites, such as CD19 antigen, or anti-Fab antibodies or Protein L proteins for light chains or hinge regions. Among them, the CAR positive rate detection method targeting the antigen binding site is widely used because of its better specificity.

[0005] However, existing CAR detection tools generally have problems such as low sensitivity and high non-specific background. Anti-Fab antibodies and Protein L are universal CAR positive detection reagents, but they cannot stain individual CARs on dual-target CAR cells separately; Protein L specifically binds to antibodies containing κ light chains, and only binds to human κI, III, IV (not binding to VκII subtypes and λ light chain antibodies) and mouse κI light chain subtypes, and is not applicable to other subtypes, and has different affinities for each subtype. At the same time, strict washing is required before and after Protein L staining, otherwise it will cross-react with non-CAR IgG-like proteins and cause serious non-specificity; when using antigens to detect the positive rate of CAR transfection, the activity and concentration of the antigen directly determine the accuracy of the test. In addition, for CAR cell detection in patients, due to the blocking effect of cytotoxicity and free antigens on CAR-scFv, the antigen protein is very unsuitable for in vivo CAR cell detection. In view of this, this application is proposed. SUMMARY OF THE INVENTION

[0007] In response to the above technical problems, this application has developed a series of anti-idiotypic antibodies that specifically bind to CAR antigen binding sites through intensive research. These antibodies can specifically evaluate the CAR transfection positivity rate and the change pattern of the number of CAR-positive T cells in the patient's body after administration. Therefore, this application includes at least the following purposes:

[0008] The first object of the present application is to provide an antibody or an antigen-binding fragment thereof that specifically binds to the CAR antigen binding site;

[0009] The second object of the present application is to provide a polynucleotide encoding the above-mentioned antibody or antigen-binding fragment thereof, a corresponding vector, a cell, or a kit containing the same;

[0010] The third objective of the present application is to provide an application of the above-mentioned antibody or antigen-binding fragment, including application in detecting Anti-CD19 CAR expression, application in evaluating Anti-CD19 CAR transfection positivity rate, and application in evaluating the change pattern of the number of Anti-CD19 CAR positive T cells in the subject after administration, etc.

[0011] The fourth objective of the present application is to provide a detection method using the above-mentioned antibody or antigen-binding fragment.

[0012] In order to achieve the above objectives, this application adopts the following specific technical solutions.

[0013] The first aspect of the present application provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region that specifically binds to a CAR antigen binding site, comprising 3 CDRs in the heavy chain variable region amino acid sequence shown in any one of SEQ ID NO.4, 12, 20, and 28 and 3 CDRs in the light chain variable region amino acid sequence shown in any one of SEQ ID NO..8, 16, 24, and 32; or a variant having a single or multiple CDRs with no more than 2 amino acid changes in each CDR region with the above-mentioned light and heavy chain CDR regions.

[0014] Furthermore, the residue ranges of the CDRs are shown in the following table:

[0015]

[0016] Further, when the antibody HCDRs are encoded according to the IMGT coding rules, the antibody or antigen-binding fragment comprises the following CDR sequences:

[0017] i. The amino acid sequence of the HCDR1 is shown in any one of SEQ ID NO.1, 9, 17, and 25;

[0018] ii. the amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO.2, 10, 18, and 26;

[0019] iii. the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO.3, 11, 19, and 27;

[0020] iv. The amino acid sequence of the LCDR1 is shown in any one of SEQ ID NO.5, 13, 21, and 29;

[0021] v. the amino acid sequence of the LCDR2 is shown in any one of SEQ ID NO.6, 14, 22, and 30;

[0022] vi. The amino acid sequence of the LCDR3 is shown in any one of SEQ ID NO.7, 15, 23, and 31;

[0023] Alternatively, a variant having a single or multiple CDRs with no more than 2 amino acid changes in each CDR region compared to the 6 CDR regions of i-vi above.

[0024] Further, the combination of HCDR1 / HCDR2 / HCDR3 may include one or more groups selected from the following:

[0025] (1) HCDR1 / HCDR2 / HCDR3:

[0026] SEQ ID NO.1 / SEQ ID NO.2 / SEQ ID NO.3,

[0027] SEQ ID NO.9 / SEQ ID NO.10 / SEQ ID NO.11,

[0028] SEQ ID NO.17 / SEQ ID NO.18 / SEQ ID NO.19,

[0029] SEQ ID NO.25 / SEQ ID NO.26 / SEQ ID NO.27,

[0030] (2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids;

[0031] The combination of LCDR1 / LCDR2 / LCDR3 comprises one or more groups selected from the following:

[0032] (1) LCDR1 / LCDR2 / LCDR3 are:

[0033] SEQ ID NO.5 / SEQ ID NO.6 / SEQ ID NO.7,

[0034] SEQ ID NO.13 / SEQ ID NO.14 / SEQ ID NO.15,

[0035] SEQ ID NO.21 / SEQ ID NO.22 / SEQ ID NO.23,

[0036] SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31,

[0037] (2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the CAR antigen-binding site of the present application further includes a heavy chain variable region VH sequence and a light chain variable region VL sequence, wherein the heavy chain variable region VH sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group including the following sequences: SEQ ID NO.4, 12, 20, 28; the light chain variable region VL sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group including the following sequences: SEQ ID NO.8, 16, 24, 32.

[0039] Furthermore, the antibody or antigen-binding fragment may further comprise a coupling portion connected to the polypeptide, wherein the coupling portion is selected from one or more of radionuclides, toxins, cytokines, enzymes, fluorescein, carrier proteins, lipids, and biotin, wherein the polypeptide or antibody and the coupling portion may be selectively connected via a linker, and preferably the linker is a peptide or polypeptide.

[0040] Further, the antibody or antigen-binding fragment can be selected from monoclonal antibodies, polyclonal antibodies, antisera, chimeric antibodies, humanized antibodies and human antibodies; more preferably, the antibody is selected from multispecific antibodies, single-chain Fv (scFv), single-chain antibodies, anti-idiotypic (anti-Id) antibodies, diabodies, miniantibodies, nanoantibodies, single domain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fv (sdFv) and intracellular antibodies.

[0041] In the second aspect of the present application, a nucleic acid encoding the antibody or antigen-binding fragment thereof of the first aspect is also provided.

[0042] The third aspect of the present application provides a vector (preferably a recombinant vector) comprising the nucleic acid of the second aspect, and optionally a regulatory sequence;

[0043] Furthermore, the vector may be a cloning vector or an expression vector, without limitation;

[0044] Furthermore, the regulatory sequence can be selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof, without limitation.

[0045] The fourth aspect of the present application provides a cell (preferably a host cell) comprising the nucleic acid of the second aspect or the vector of the third aspect.

[0046] Furthermore, the cells include but are not limited to mammalian cells such as yeast cells, Chinese hamster ovary cells, human embryonic kidney cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0047] The sixth aspect of the present application provides a kit, characterized in that it comprises one or more of any of the above-mentioned antibodies or antigen-binding fragments, polynucleotides, recombinant vectors and host cells, and is contained in a suitable container.

[0048] The seventh aspect of the present application provides a method for preparing the antibody or antigen-binding fragment thereof that can bind to the CAR antigen binding site of the first aspect, the method comprising expressing the vector of the third aspect in a host cell culture to produce the antibody; and recovering the antibody molecule from the cell culture.

[0049] The eighth aspect of the present application provides a use of the antibody or antigen-binding fragment thereof comprising the first aspect in any of the following aspects:

[0050] a) Application in detecting Anti-CD19 CAR expression;

[0051] b) Application in evaluating the positive rate of Anti-CD19 CAR transfection;

[0052] c) Application in evaluating the changing pattern of the number of Anti-CD19 CAR positive T cells in the subject after administration, or in the preparation of a reagent for evaluating the changing pattern of the number of Anti-CD19 CAR positive T cells in the subject after administration. It can be understood that a) and b) can be in vitro detection, the antibody or antigen binding fragment of the present application is a CAR detection tool, and the sample in practice can be from the body or in vitro. When used for in vitro sample detection, its purpose can be a simple CAR detection or evaluation purpose, thus including non-disease diagnosis applications.

[0053] The ninth aspect of the present application provides a detection method, which comprises the step of detecting a sample to be tested using any of the above-mentioned antibodies or antigen-binding fragments, or the above-mentioned kit.

[0054] Further preferably, the detection method is used to evaluate whether the sample contains Anti-CD19 CAR or the expression level of Anti-CD19 CAR, etc., without limitation, which can be reasonably expected in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Partial results of FACS binding screening of supernatants of positive clones.

[0056] Figure 2 Partial results of FACS blocking screening of supernatants of positive clones.

[0057] Figure 3 Some results of subclone blocking FACS detection.

[0058] Figure 4 Some results of subclone blocking FACS detection.

[0059] Figure 5 The SDS-PAGE identification results of the anti-FMC63 scFv antibody of the present application are shown.

[0060] Figure 6 The results of ELISA binding analysis of the anti-FMC63 scFv antibody of the present application are shown.

[0061] Figure 7 The results of competition ELISA analysis of the anti-FMC63 scFv antibody of the present application are shown.

[0062] Figure 8 The results of SPR analysis of the anti-FMC63 scFv antibody of the present application are shown.

[0063] Fig. 9 The results of FACS binding analysis of the anti-FMC63 scFv antibody of the present application are shown. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present application discloses a separation antibody or an antigen-binding fragment thereof. Those skilled in the art can refer to the contents of this article to realize its application. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The preparation method and application of this application have been described through preferred embodiments. The relevant personnel can obviously modify or appropriately change and combine the preparation method and application of this article without departing from the content, spirit and scope of this application to realize and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the field to which this application belongs.

[0066] The following terms or definitions are provided only to help understand the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0067] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0068] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0069] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" is used, this includes a plural of that noun.

[0070] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0071] 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more of the above values. Also included are any larger numbers or fractions therebetween.

[0072] Conversely, the term "no more than" includes every value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0073] The terms "plurality", "at least two", "two or more", "at least a second", etc., should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also includes any larger number or fraction therebetween.

[0074] Definition of terms:

[0075] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly and in a specific manner bind to the corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by a disulfide bond. Each heavy chain comprises a heavy chain variable region (abbreviated as VH here) and a heavy chain constant region. The heavy chain constant region comprises three domains CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL here) and a light chain constant region. The light chain constant region comprises a domain CL. The VH region and the VL region can be further subdivided into hypervariable regions referred to as complementary determining regions (CDRs), which are interspersed with more conservative regions referred to as framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, including different cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. "Antibody" includes but is not limited to monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies for antibodies of the present disclosure). Antibodies can belong to any isotype / category (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0076] Antibodies contain globular regions of heavy or light chain polypeptides referred to as "domains". Domains may contain peptide loops, typically 3 to 4 loops, which are stabilized, for example, by beta-sheets and / or intrachain disulfide bonds. Domains are often referred to as "constant" or "variable" based on the relative lack of sequence variation within domains of members of different classes in the case of "constant" domains, or the significant variation within domains of members of different classes in the case of "variable" domains. Antibody or polypeptide "domains" are often referred to interchangeably in the art as antibody or polypeptide "regions".

[0077] According to the amino acid sequence of the constant region of the heavy chain of the antibody, antibodies can be divided into five categories: IgA, IgD, IgE, IgG and IgM, and several isotypes in these categories can be further divided into subclasses, such as IgG1, IgG2, IgG3 and IgA1 and IgA2. The heavy chain constant domains corresponding to different types of immunoglobulins are called α, δ, ε, γ and μ, respectively. According to the difference in the constant region (CL) of the light chain of the antibody, κ and λ can be divided. In the full-length light chain and heavy chain, the variable region and the constant region are usually connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

[0078] The term "monoclonal antibody" refers to a preparation of an antibody molecule having a single amino acid composition, and does not refer to the method of its preparation. Monoclonal antibodies or immunologically active fragments thereof can be produced by hybridoma technology, recombinant technology, phage display technology, synthetic technology, or other production technologies known in the art. The methods for preparing monoclonal antibodies in this application include preparation by hybridoma cell culture in vitro or preparation by DNA recombination technology. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Each monoclonal antibody is directed against a single determinant cluster on the antigen.

[0079] The term "antigen" is an entity (eg, a protein entity or a peptide) to which an immunoglobulin or antibody (or antigen-binding fragment thereof) specifically binds.

[0080] The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with the portion when the portion is present in an intact antibody. Fragments can be obtained by chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means.

[0081] The term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms 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 complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative 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-folded configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)). The constant region is not directly involved 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.

[0082] "Complementarity determining domain" or "complementarity determining region" ("CDR") refers interchangeably to the hypervariable regions of VL and VH. CDR is the target protein binding site of the antibody chain that carries the specificity of such a target protein. There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each human VL or VH, totaling about 15-20% of the variable domain. CRD can be referred to by its region and sequence. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDR is structurally complementary to the epitope of the target protein and is therefore directly responsible for binding specificity. The remaining extensions of VL or VH (the so-called framework region) show less variation in the amino acid sequence (Kuby, "Immunology", 4th edition, Chapter 4 WH Freeman & Co., New York, 2000).

[0083] In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health, 10005, USA);

[0084] Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0085] However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. For example, the residue ranges of the CDR regions numbered using Kabat, Chothia, etc. under different assignment systems are shown in the following table.

[0086] CDR residue ranges defined by different assignment systems

[0087]

[0088] Therefore, when referring to antibodies defined by specific CDR sequences defined in this application, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose declared CDR boundaries are different from the specific CDR boundaries defined in this application due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0089] The CDR of the antibody of the present application can be manually evaluated and determined according to any scheme or combination thereof in the art. Unless otherwise specified, in the present application, the term "CDR" or "CDR sequence" covers CDR sequences determined in any of the above ways.

[0090] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above.

[0091] The term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include, but are not limited to: single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F(ab') fragments (i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains); F(ab)2 fragments (i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementary determining regions (CDRs) or other epitope-binding fragments of antibodies. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be connected using recombinant methods by a synthetic linker that enables the two domains to become a single protein chain, in which the VL region and the VH region are used to form a monovalent molecule (referred to as a single-chain Fv ("scFv"); see, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proceedings of the National Academy of Sciences of the United States of America 85:5879-5883, 1988). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment". These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as intact antibodies.

[0092] Antigen binding fragments can also be incorporated into single domain antibodies, large antibodies, minibodies, nanobodies, intrabodies, diabodies, tribodies, tetrabodies, v-NARs and bi-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monoclonal antibodies). Antigen binding fragments can be incorporated into single chain molecules including a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions together with complementary light chain polypeptides (Zapata et al., Protein Eng. 8: 1057-1062, 1995; and U.S. Pat. No. 5,641,870).

[0093] The term "chimeric antibody" refers to a portion of the heavy chain and / or light chain (generally referring to the variable region) derived from a specific species or belonging to a specific antibody class or subclass, and the corresponding sequences in the antibody are identical or homologous, while the rest of the chain (generally referring to the constant region) is derived from an antibody of another species or belongs to another antibody class or subclass, and the corresponding sequences in the fragments of these antibodies are identical or homologous, as long as they exhibit the desired biological activity. The chimeric antibodies involved in this application, such as the heavy chain / light chain variable region from a mouse antibody, are grafted to the constant region of the heavy chain / light chain of a human antibody through antibody engineering technology, which exhibits similar biological activity.

[0094] The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the sequence of a non-human antibody, and all or substantially all of the FRs correspond to the sequence of a human antibody.

[0095] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0096] The term "competition" herein, when referring to the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, means competition between antigen binding proteins, which is determined by the following assay: in the assay, the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen.

[0097] As used herein, the term "variant" refers to a heavy chain variable region or light chain variable region that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, wherein the modified antigen-binding protein comprising a heavy chain or light chain variant substantially retains the biological characteristics of the antigen-binding protein before modification. In one embodiment, the antigen-binding protein containing the variant heavy chain variable region or light chain variable region sequence retains 70%, 80%, 90%, 100% biological characteristics of the antigen-binding protein before modification. It should be understood that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region. The antigen-binding proteins of the present disclosure include heavy chain variable region amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequences described herein. The antigen-binding proteins disclosed herein include light chain variable region amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequences described herein. The homology percentage can be over the entire heavy chain variable region and / or the entire light chain variable region, or the percentage homology can be limited to the framework region, and the sequence corresponding to the CDR has 100% identity with the CDR disclosed herein in the heavy chain variable region and / or the light chain variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified by at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, wherein the modified antigen-binding proteins containing CDR variants substantially retain the biological characteristics of the antigen-binding proteins before modification. In one embodiment, the antigen-binding proteins containing variant CDRs retain 60%, 70%, 80%, 90%, 100% biological characteristics of the antigen-binding proteins before modification. It will be appreciated that each CDR that can be modified can be modified alone or in combination with another CDR. In one embodiment, the modification is a substitution, particularly a conservative substitution.

[0098] The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked by transformation. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0099] The term "host cell" refers to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell, and is capable of expressing the exogenous nucleic acid in the cell or cell membrane or releasing it to the outside of the cell.

[0100] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Except when indicated, the terms "patient" or "subject" are used interchangeably herein.

[0101] The term "kit" is used to refer to a combination of reagents and other materials that aid in sample analysis. In some embodiments, the immunoassay kits described herein include suitable antigens, binding agents comprising detectable moieties, and detection reagents. Systems for amplifying the signals generated by the detectable moieties may also or may not be included in the kit. In addition, in other embodiments, the kit includes, but is not limited to, components such as devices for sample collection, sample tubes, racks, trays, shelves, plates, instructions for kit users, solutions or other chemical reagents, and samples and / or control samples for standardization and normalization.

[0102] 1. Anti-unique antibodies or antigen-binding fragments for Anti-CD19 CAR of the present application

[0103] The terms "anti-unique antibody against Anti-CD19 CAR", "antibody against Anti-CD19 CAR", "antibody that specifically binds to the antigen binding site of Anti-CD19 CAR", and "antigen binding fragment against Anti-CD19 CAR" are used interchangeably herein and refer to such an antibody or antigen binding fragment of the present application, which can bind to Anti-CD19 CAR with sufficient affinity and specificity, so that the antibody can be used for the detection of the CAR. In some embodiments, the antibody or antigen binding fragment of the present application can specifically bind to the CAR antigen binding site. According to ELISA and SPR validation data, the antibody or antigen binding fragment can specifically bind to the antigen recognition site of Anti-CD19 CAR derived from FMC63 and has the characteristics of high affinity. The antibody can detect the expression of Anti-CD19 CAR derived from FMC63 by flow cytometry, showing the characteristics of high sensitivity and no non-specific background.

[0104] The antibodies and antigen-binding fragments of the present application specifically bind to Anti-CD19 CAR with high affinity. In some embodiments, according to the different analysis methods for CDR in the above definition, the antibodies or antigen-binding fragments of the present application are such a sequence:

[0105] It may comprise three CDRs in the heavy chain variable region amino acid sequence shown in any one of SEQ ID NO.4, 12, 20, and 28, and three CDRs in the light chain variable region amino acid sequence shown in any one of SEQ ID NO.8, 16, 24, and 32; or a variant having a single or multiple CDRs with no more than two amino acid changes in each CDR region compared to the above light and heavy chain CDR regions.

[0106] In some embodiments, when the antibody HCDRs are encoded according to the IMGT coding rules, the antibody or antigen-binding fragment comprises the following CDR sequences:

[0107] i. The amino acid sequence of the HCDR1 is shown in any one of SEQ ID NO.1, 9, 17, and 25;

[0108] ii. the amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO.2, 10, 18, and 26;

[0109] iii. the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO.3, 11, 19, and 27;

[0110] iv. The amino acid sequence of the LCDR1 is shown in any one of SEQ ID NO.5, 13, 21, and 29;

[0111] v. the amino acid sequence of the LCDR2 is shown in any one of SEQ ID NO.6, 14, 22, and 30;

[0112] vi. The amino acid sequence of the LCDR3 is shown in any one of SEQ ID NO.7, 15, 23, and 31;

[0113] Alternatively, a variant having a single or multiple CDRs with no more than 2 amino acid changes in each CDR region compared to the 6 CDR regions of i-vi above.

[0114] In some embodiments, the combination of HCDR1 / HCDR2 / HCDR3 may comprise one or more groups selected from the following:

[0115] (1) HCDR1 / HCDR2 / HCDR3:

[0116] SEQ ID NO.1 / SEQ ID NO.2 / SEQ ID NO.3,

[0117] SEQ ID NO.9 / SEQ ID NO.10 / SEQ ID NO.11,

[0118] SEQ ID NO.17 / SEQ ID NO.18 / SEQ ID NO.19,

[0119] SEQ ID NO.25 / SEQ ID NO.26 / SEQ ID NO.27;

[0120] (2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids;

[0121] The combination of LCDR1 / LCDR2 / LCDR3 comprises one or more groups selected from the following:

[0122] (1) LCDR1 / LCDR2 / LCDR3 are:

[0123] SEQ ID NO.5 / SEQ ID NO.6 / SEQ ID NO.7,

[0124] SEQ ID NO.13 / SEQ ID NO.14 / SEQ ID NO.15,

[0125] SEQ ID NO.21 / SEQ ID NO.22 / SEQ ID NO.23,

[0126] SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31;

[0127] (2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids.

[0128] In some specific embodiments, the binding antibody or antigen-binding fragment thereof of the present application further includes a heavy chain variable region VH sequence and a light chain variable region VL sequence. The heavy chain variable region VH sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group including the following sequences: SEQ ID NO.4, 12, 20, 28; the light chain variable region VL sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group including the following sequences: SEQ ID NO.8, 16, 24, 32; preferably, the amino acid changes do not occur in the CDR region.

[0129] In some more specific embodiments, some specific antibody sequences of the present application are shown in the following table (specific VH and VL sequences of antibodies or antigen-binding fragments thereof).

[0130]

[0131]

[0132] In some embodiments, the antibody or antigen-binding fragment of the present application further comprises an Fc region, and the Fc region is derived from IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0133] In some embodiments, the amino acid changes in the above amino acid homology include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions refer to the substitution of an amino acid by another amino acid in the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid. Exemplary substitutions are shown in the following table (amino acid substitutions).

[0134]

[0135] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (eg, in the FRs). More preferably, the amino acid changes described herein occur in the Fc region.

[0136] In some embodiments, the present application encodes antibody CDRs according to the IMGT coding rules, and the CDRs sequences of specific numbered antibodies are shown in the following table:

[0137]

[0138] In some embodiments, the antibody or antigen-binding fragment may further comprise a coupling portion connected to the polypeptide, wherein the coupling portion is selected from one or more of radionuclides, toxins, cytokines, enzymes, fluoresceins, carrier proteins, lipids, and biotin, wherein the polypeptide or antibody and the coupling portion may be selectively connected via a linker, preferably the linker is a peptide or polypeptide.

[0139] In some embodiments, the antibody or antigen-binding fragment can be selected from a monoclonal antibody, a polyclonal antibody, an antiserum, a chimeric antibody, a humanized antibody and a human antibody; more preferably, the antibody is selected from a multispecific antibody, a single-chain Fv (scFv), a single-chain antibody, an anti-idiotypic (anti-Id) antibody, a diabody, a minibody, a nanobody, a single domain antibody, a Fab fragment, a F(ab') fragment, a disulfide-linked bispecific Fv (sdFv) and an intrabody.

[0140] In some embodiments, the antibody or its antigen-binding fragment described in the present application can be produced by recombinant expression. The above-mentioned nucleic acid encoding the light chain and heavy chain variable region optionally connected to the constant region can be inserted into an expression vector. The vector itself comprising the nucleic acid encoding the antibody described herein is an aspect of the present application. The light chain and the heavy chain can be cloned into the same or different expression vectors. The nucleic acid encoding the antibody chain described herein can be operably connected to one or more control sequences in the expression vector to ensure the expression of the antibody chain. The expression control sequence includes but is not limited to a promoter (e.g., a naturally associated or heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Such a vector can be incorporated into a suitable host, whereby the host is maintained under conditions suitable for high-level expression of nucleotide sequences and collection and purification of antibodies.

[0141] 2. The nucleic acid of the present application, the vector containing the same and the host cell

[0142] The application provides nucleic acids encoding any of the above antibodies or their antigen-binding fragments or any of their chains. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is a recombinant vector, such as an expression vector or a cloning vector. In one embodiment, a cell comprising the nucleic acid or the vector is provided, such as a host cell. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or their antigen-binding fragments. In another embodiment, the host cell is prokaryotic.

[0143] The nucleic acid involved in the present application is a nucleic acid encoding an antibody or its antigen-binding fragment or its VH or VL domain; it can be understood that any nucleic acid that can encode the above-mentioned antibody or its antigen-binding fragment or its VH or VL domain is within the scope of the present application.

[0144] The vector of the present application comprising one or more nucleic acids encoding the antibodies described herein may be a cloning vector or an expression vector, which is not limited herein.

[0145] In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC).

[0146] In one embodiment, the vector comprises an optional regulatory sequence; in some embodiments, the regulatory sequence can be selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof, without limitation.

[0147] The host cell comprising the expression vector of the present application, such as host cell is yeast cell, mammalian cell or other cells suitable for preparing antibody or its antigen-binding fragment. In some embodiments, suitable host cell includes prokaryotic microorganism, such as Escherichia coli. Host cell can also be eukaryotic microorganism such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells etc. Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2 / 0, etc. For a review of mammalian host cell lines suitable for producing proteins, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a 293 cell.

[0148] The vector comprising the polynucleotide sequence of interest (for example, heavy and light chain coding sequences and expression control sequences) described herein can be transferred to a host cell by a well-known method, which is different according to the type of the cell host. For example, calcium chloride transfection is generally used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics or virus-based transfection can be used for other cell hosts. (usually referring to Green and Sambrook, Molecular Cloning: Laboratory Manual (Molecular Cloning: A Laboratory Manual) (Cold Spring Harbor Press, 4th edition, 2012). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection (usually referring to Sambrook et al., the same). In order to produce transgenic animals, transgenic microinjection can be carried out into fertilized oocytes, or it can be integrated into the genome of embryonic stem cells, and the nuclei of these cells are transferred into enucleated oocytes.

[0149] 3. Preparation, production and purification of the antibodies or antigen-binding fragments of the present application

[0150] The method for preparing the anti-unique antibody or antigen-binding fragment thereof of Anti-CD19 CAR in the present application may include expressing the vector described herein in a host cell culture to produce the antibody, and recovering the antibody from the cell culture.

[0151] In some embodiments, the method may include transferring a vector comprising one or more nucleic acids encoding an antibody or its antigen-binding fragment or its antibody chain as described above into a host cell as described herein, culturing the host cell culture under conditions that allow expression of the nucleic acid, and recovering the expressed corresponding antibody or its antigen-binding fragment. Any suitable method known in the art may be used.

[0152] The present application provides a method for an anti-unique antibody or antigen-binding fragment of Anti-CD19 CAR, wherein the method includes culturing a host cell comprising a nucleic acid encoding the antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing a nucleic acid encoding the antibody or antigen-binding fragment, and optionally separating the antibody or antigen-binding fragment. In a certain embodiment, the method further includes recovering and purifying the corresponding antibody or antigen-binding fragment from the host cell (or host cell culture medium).

[0153] In some embodiments, the antibodies or antigen-binding fragments prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these are obvious to those skilled in the art. The purity of the antibodies of the present application can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0154] 4. Detection method and application of this application

[0155] The antibodies or antigen-binding fragments provided in the present application can be used to detect the presence or content of Anti-CD19 CAR in a biological sample, and then used for detection or evaluation purposes.

[0156] In some embodiments, the anti-anti-CD19 CAR antibody or its antigen-binding fragment provided in the present application can be conveniently used in the kit, and the Anti-CD19 CAR in the in vivo or in vitro sample can be detected by the antibody or its antigen-binding fragment provided in the present application with specificity, high sensitivity and no non-specific background. Therefore, the antibody or its antigen-binding fragment of the present application is particularly suitable for evaluating the positive rate of CAR transfection and the change pattern of the number of CAR-positive T cells in the subject after administration.

[0157] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, and SPR, etc. In some embodiments, the protein antibody or antigen-binding fragment thereof of the present application may be coupled to a detectable marker such as luciferase or biotinidase, and used in direct or indirect immunoassays such as FACS, SPR, IHC, and ELISA in liquid or solid phase.

[0158] The technical solution of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application. Example

[0159] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0160] Example 1 Preparation, screening and sequencing of monoclonal antibodies

[0161] 1) Preparation of immunogen: An expression plasmid expressing FMC63 scFv, His Tag was constructed by gene synthesis, and then the expression plasmid was transfected into HEK293 cells using Invitrogen Lipofectamine 2000 transfection reagent. The culture supernatant was harvested 48 hours later, and then FMC63 scFv, His Tag was purified by affinity chromatography, and then the purified FMC63 scFv was coupled to KLH carrier protein (purchased from Sigma, Cat. No. H7017).

[0162] 2) Mouse immunization: FMC63 scFv coupled to KLH carrier protein was used as an immunogen, and 10 Balb / c mice were immunized with FMC63 scFv-KLH using conventional immunization. The conventional immunization plan is shown in Table 1. Seven days after each immunization, the serum of the immunized animals was tested by ELISA to determine the level of immune response. After the end of conventional immunization, if the immunized animals can reach the level of immune response to the immunogen (OD value>1.0, titer reaches 1:8,000), cell fusion can be performed.

[0163] Table 1 Immunological table

[0164] step schedule Dosage and route Adjuvant Blood collection before immunization Day 4 First immunization Day 0 50 μg / mouse, subcutaneous Freund's complete adjuvant The second epidemic Day 14 25 micrograms / mouse, subcutaneous Freund's incomplete adjuvant Blood test 1 Day 21 The third epidemic Day 28 25 micrograms / mouse, subcutaneous Freund's incomplete adjuvant Blood test 2 Day 35 Free Day 50±7 25 μg / pc, intraperitoneal Cell fusion Four days after the final exemption

[0165] 3) Cell fusion and plating

[0166] The cells were fused twice by electrofusion, and all cells of each fusion were plated into a 96-well plate.

[0167] 4) Screening analysis

[0168] Initial screening: The supernatant of the fused cells was screened by ELISA, and the supernatant that was positive for anti-scFv was selected. FMC63scFv and several other proteins (as negative controls and isotype controls) were coated separately, and the pre-immune serum and antisera after three immunizations were tested separately (Table 2). The titer is the highest dilution ratio when the signal / blank ratio value is greater than or equal to 2.1, and NC is the pre-immune serum, which is used as a negative control (Table 3).

[0169] Table 2 ELISA initial screening experimental plan

[0170]

[0171]

[0172] Table 3 Results of ELISA method screening supernatant of fusion cells

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] Confirmation screening: When screening mother clones, all positive mother clone cell supernatants were screened by indirect ELISA, and total human IgG and irrelevant his proteins were used for counter-screening. The indirect ELISA screening scheme for positive mother clones is shown in Table 4. The ELISA screening results of pre-immune serum and anti-serum after three immunizations are shown in Table 5. The titer is the highest dilution ratio when the signal / blank ratio value is greater than or equal to 2.1, and NC is the pre-immune serum, which is used as a negative control.

[0179] Table 4 Indirect ELISA screening scheme for positive mother clones

[0180]

[0181] Table 5 ELISA screening results of pre-immune serum and anti-serum after three immunizations

[0182]

[0183]

[0184]

[0185] FACS binding screening: Screening experiments to express FMC63 scFv HEK293, 1x10 5 cells are positive cell lines, conventional HEK293, 1x10 5 cells is a negative cell line, and the primary antibodies are: a, 1:50 dilution of pre-immune serum; b, 3ug / ml mouse IgG (isotype control); c, PBS as a negative control; d, 1:50 dilution of tertiary immunization antiserum. The secondary antibody is Alexa 647AffiniPure Goat Anti-Mouse IgG, Fcγfragmentspecific (min X Hu, Bov, Hrs Sr Prot) (Jackson, 115-605-071), 1ug / ml. FACS binding screening results are shown in Figure 1 The black curve is negative cells + a / b / c / d + secondary antibody; the gray curve is positive cells + a / b / c / d + secondary antibody.

[0186] FACS blocking screening: The cell line used in the screening experiment was HEK293 expressing FMC63 scFv, 1x105 cells. The primary antibodies were: a, 1:50 dilution of pre-immune serum; b, 3ug / ml mouse IgG (isotype control); c, PBS (negative control); d, 1:50 dilution of antiserum after three immunizations; e, 1ug / ml human CD19 recombinant protein ligand with human Fc tag. The secondary antibody was Goat Anti-Human IgG, Fc fragment specific [AlexaFluor647] (Jackson, 109-605-098), 1ug / ml. FACS blocking screening results are shown in Figure 2 The black curve is scFv HEK293+c+e+secondary antibody, and the gray curve is scFv HEK293+a / b / c / d+e+secondary antibody.

[0187] 5) Cloning expansion and cryopreservation

[0188] Transfer the positive mother clone cells to a 24-well plate for expansion culture, up to 10 strains. Collect 2 ml of supernatant from each expanded clone for indirect ELISA and blocking FACS detection. Freeze these specific positive clone cells to avoid clone loss. The indirect ELISA detection scheme is shown in Table 6, and the test results are shown in Table 7.

[0189] Table 6 Indirect ELISA screening scheme for supernatant of expanded culture of positive mother clone cells

[0190]

[0191] Table 7 Indirect ELISA screening results of positive mother clone cell expansion culture supernatant

[0192]

[0193]

[0194] The cell line used for blocking FACS detection is HEK293 expressing FMC63 scFv, 1x10 5 cells. The primary antibodies were: a, 1:50 diluted fusion serum (positive control); b, 3ug / ml mouse IgG (isotype control); c, PBS (negative control); d, 1ug / ml human CD19 recombinant protein ligand with human Fc tag; e, supernatant of positive mother clone cell expansion culture. The secondary antibody was Alexa 647AffiniPure Goat Anti-Human IgG (H+L) (min X Bov,Hrs,Ms Sr Prot) (Jackson, 1μg / ml).

[0195] 6) Subcloning

[0196] The positive mother clones were subcloned by limiting dilution method to ensure that these positive mother clones were derived from a single mother clone cell, and subclone screening was performed by indirect ELISA or blocking FACS method. The indirect ELISA detection scheme is shown in Table 8, and the detection results are shown in Table 9.

[0197] Table 8 Subclone indirect ELISA screening scheme

[0198]

[0199] Table 9 Results of subclone indirect ELISA screening scheme

[0200]

[0201]

[0202]

[0203] The cell line used for blocking FACS detection is HEK293 expressing FMC63 scFv, 1x10 5 cells. The primary antibodies were: a, 1:50 diluted fusion serum (positive control); b, 3ug / ml mouse IgG (isotype control); c, PBS (negative control); d, 1ug / ml human CD19 recombinant protein ligand with human Fc tag; e, subclone supernatant. The secondary antibody was Alexa 647AffiniPure Goat Anti-Human IgG (H+L) (min X Bov, Hrs, Ms Sr Prot) (Jackson, 1 μg / ml). Blocking FACS detection results are shown in Figure 3 and Figure 4 The black curve is scFv HEK293+c+d+secondary antibody, and the gray curve is scFv HEK293+a / b / c / e+d+secondary antibody.

[0204] Based on the confirmation of antigen recognition, two stable subclone cell lines were selected from each mother clone for cryopreservation. Before cryopreservation, 5 ml of supernatant was collected from each subclone, and all subclones were subtyped and stored.

[0205] 7) Antibody production and purification

[0206] The hybridoma cells were expanded and cultured, and the antibodies were purified by protein A / G affinity chromatography and stored in phosphate buffered saline (PBS) by dialysis.

[0207] 8) Hybridoma cell antibody gene sequencing

[0208] Based on the above screening data and results, the present application obtained 4 superior subclone strains: 24C1E4D8, 26A10C3E6, 23D4G9E9 and 16D5E8E7, which exhibited more comprehensive affinity and blocking activities and could be used for subsequent detection purposes. Further, the total RNA of the corresponding hybridoma cells was extracted, the RNA was reverse transcribed into cDNA by RT-PCR reaction, the antibody light chain and heavy chain sequences were cloned, the light chain and heavy chain sequences were constructed into T vectors, and the antibody gene sequences were obtained by DNA sequencing analysis. The specific sequence results are shown in Table 10.

[0209] Table 10 Amino acid sequences of dominant antibodies (CDR determined by IMGT rules)

[0210]

[0211]

[0212] Example 2. Analysis and identification of monoclonal antibodies and functional analysis

[0213] The superior clones screened above were further subjected to antibody identification and functional analysis, and some of the analysis results are exemplarily shown below.

[0214] The results of ELISA testing performed using a subclass kit showed that the subtype of antibodies 24C1E4D8, 26A10C3E6, 23D4G9E9, and 16D5E8E7 was IgG1,κ.

[0215] SDS-PAGE identification results ( Figure 5 ) showed that, taking 16D5E8E7 antibody as an example, the molecular weights of the two bands in reduced electrophoresis were 27 kDa and 50 kDa respectively, and the purity of the antibody was greater than 99%.

[0216] ELISA binding data ( Figure 6 ) and competition ELISA data ( Figure 7 ) showed that the 16D5E8E7 clone antibody could very specifically recognize the antigen binding site of Anti-CD19 (FMC63) CAR.

[0217] SPR analysis data ( Figure 8 ) shows that the seven fitting lines from top to bottom represent the change of affinity and dissociation between FMC63 scFv and 16D5E8E7 clone antibody at concentrations of 125nM, 62.5nM, 31.25nM, 15.625nM, 7.813nM, 3.906nM and 1.953nM respectively. This result shows that the affinity of the antibody binding to FMC63 scFv is as high as 1.1nM.

[0218] FACS binding data ( Fig. 9 ) showed that the 16D5E8E7 clone antibody could specifically bind to the Anti-CD19 (FMC63) CAR expressed on the cell surface, while there was no non-specific binding signal with 293 cells that were not transfected with CAR and PBMC cells that were not transfected with CAR, showing the advantages of good high specificity and no non-specific background.

[0219] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and illustration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a CAR antigen-binding site, characterized in that: Comprising 3 CDRs in the heavy chain variable region amino acid sequence shown in any one of SEQ ID NO.4, 12, 20, 28 and 3 CDRs in the light chain variable region amino acid sequence shown in any one of SEQ ID NO.8, 16, 24, 32; or variants having a single or multiple CDRs with no more than 2 amino acid changes in each CDR region with the above light and heavy chain CDR regions; Preferably, the residue ranges of the CDR are shown in the following table:

2. The antibody or antigen-binding fragment of claim 1, wherein: When the antibody HCDRs are encoded according to the IMGT coding rules, the antibody or antigen-binding fragment comprises the following CDR sequences: i. The amino acid sequence of the HCDR1 is shown in any one of SEQ ID NO.1, 9, 17, and 25; ii. the amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO.2, 10, 18, and 26; iii. the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO.3, 11, 19, and 27; iv. The amino acid sequence of the LCDR1 is shown in any one of SEQ ID NO.5, 13, 21, and 29; v. the amino acid sequence of the LCDR2 is shown in any one of SEQ ID NO.6, 14, 22, and 30; vi. The amino acid sequence of the LCDR3 is shown in any one of SEQ ID NO.7, 15, 23, and 31; Alternatively, a variant having a single or multiple CDRs with no more than 2 amino acid changes in each CDR region compared to the 6 CDR regions of i-vi above.

3. The antibody or antigen-binding fragment of claim 2, wherein: The combination of HCDR1 / HCDR2 / HCDR3 comprises one or more groups selected from the following: (1) HCDR1 / HCDR2 / HCDR3: SEQ ID NO.1 / SEQ ID NO.2 / SEQ ID NO.3; SEQ ID NO.9 / SEQ ID NO.10 / SEQ ID NO.11; SEQ ID NO.17 / SEQ ID NO.18 / SEQ ID NO.19; SEQ ID NO.25 / SEQ ID NO.26 / SEQ ID NO.27; (2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids; The combination of LCDR1 / LCDR2 / LCDR3 comprises one or more groups selected from the following: (1) LCDR1 / LCDR2 / LCDR3 are: SEQ ID NO.5 / SEQ ID NO.6 / SEQ ID NO.7; SEQ ID NO.13 / SEQ ID NO.14 / SEQ ID NO.15; SEQ ID NO.21 / SEQ ID NO.22 / SEQ ID NO.23; SEQ ID NO.29 / SEQ ID NO.30 / SEQ ID NO.31; (2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the sequence of which is selected from the following: a) the amino acid sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 4, 12, 20, 28, or has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity with a sequence in the group; b) the amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 8, 16, 24, 32 or has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity with a sequence in the group.

5. An isolated polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding fragment according to any one of claims 1-4.

6. A vector comprising the polynucleotide of claim 5, and optionally a regulatory sequence; Preferably, the vector is a cloning vector or an expression vector; More preferably, the regulatory sequence is selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof.

7. A cell, characterized in that Comprising the polynucleotide according to claim 5 or the vector according to claim 6; Preferably, the cell is a prokaryotic cell or a eukaryotic cell; More preferably, the cells include but are not limited to yeast cells, Chinese hamster ovary cells, and human embryonic kidney cells.

8. A kit, characterized in that: The method comprises one or more of the antibody or antigen-binding fragment, polynucleotide, recombinant vector and host cell according to any one of claims 1 to 4, and is contained in a suitable container.

9. Any of the following uses of the antibody or antigen-binding fragment of any one of claims 1 to 4: Application in detecting Anti-CD19 CAR expression; Application in evaluating the positive rate of Anti-CD19 CAR transfection; Application in evaluating the changing pattern of the number of Anti-CD19 CAR positive T cells in a subject after administration, or in preparing a reagent for evaluating the changing pattern of the number of Anti-CD19 CAR positive T cells in a subject after administration.

10. A detection method, characterized in that: The step of detecting the sample to be tested using the antibody or antigen-binding fragment described in any one of claims 1 to 4.

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