Anti-CD19 CAR expression detection anti-idiotype antibody

Abstract

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

Description

[0001] This application claims priority to the Chinese patent application No. 202410002320.4, filed on January 2, 2024, entitled "Anti-idiotype antibody for Anti-CD19 CAR expression detection", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of antibodies and the field of immunity in the field of biotechnology, in particular to an Anti-idiotype antibody for Anti-CD19 CAR expression detection and application thereof. BACKGROUND

[0003] CAR-T cell therapy is a cancer immunotherapy based on in vitro modification of T cells to express receptor fragments on the surface of the T cells that specifically recognize tumor surface antigens. The modified T cells are input into the patient's body, do not need the help of antigen presenting cells (APC), and directly target cancer cells in the body and play an immune killing role. For CAR-T cells, the effective component that plays a tumor killing role is CAR-positive T cells. The packaging specifications and clinical use dose 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] Currently, the CAR transfection positive rate is usually detected by flow cytometry, and there are detection methods for different structural regions of CAR, including anti-Fab antibodies or Protein L proteins for the antigen binding site of CAR, such as CD19 antigen, or for the light chain or hinge region. Among them, the CAR positive rate detection method for the antigen binding site is widely used because it has better specificity.

[0005] However, the existing CAR detection tools generally have low sensitivity, high non-specific background and other problems. Anti-Fab antibodies and Protein L belong to general CAR positive detection reagents, but they cannot separately stain individual CARs on double-target CAR cells; Protein L specifically binds to antibodies containing kappa light chains, only binds to human kappa I, III, IV (does not bind to V kappa II subtype and lambda light chain antibodies) and mouse kappa I light chain subtype, and is not suitable for other subtypes, and the affinity for each subtype is different. At the same time, strict washing is required before and after using Protein L staining, otherwise it will cross-react with non-CAR IgG-like proteins and cause serious non-specificity; when using antigen to detect CAR transfection positive rate, the activity and use concentration of the antigen directly determine the accuracy of the detection. In addition, for the detection of CAR cells in the patient's body, due to the effects of cell digestion and free antigen blocking CAR-scFv, antigen proteins are not suitable for in vivo CAR cell detection. In view of this, the present application is proposed. SUMMARY

[0006] In order to solve the above technical problems, the present application provides a series of anti-idiotypic antibodies which specifically bind to the antigen binding site of CAR. These antibodies can be used to evaluate the transfection positive rate of CAR and the change of the number of CAR positive T cells in the body of a patient after administration. Therefore, the present application at least includes the following purposes:

[0007] The first purpose of the present application is to provide an antibody or antigen binding fragment thereof which specifically binds to the antigen binding site of CAR.

[0008] The second purpose of the present application is to provide a polynucleotide encoding the above antibody or antigen binding fragment thereof, a corresponding vector, a cell, or a kit comprising the same.

[0009] The third purpose of the present application is to provide an application of the above antibody or antigen binding fragment, including an application in detecting Anti-CD19 CAR expression, an application in evaluating Anti-CD19 CAR transfection positive rate, and an application in evaluating the change of the number of Anti-CD19 CAR positive T cells in the body of a subject after administration.

[0010] The fourth purpose of the present application is to provide a detection method using the above antibody or antigen binding fragment.

[0011] In order to achieve the above purposes, the present application adopts the following specific technical solutions.

[0012] The first aspect of the present application provides an antibody or antigen binding fragment thereof which specifically binds to the antigen binding site of CAR, comprising a heavy chain variable region and a light chain variable region, comprising 3 CDRs in the heavy chain variable region amino acid sequence of any one of SEQ ID NO. 4, 12, 20, 28 and 3 CDRs in the light chain variable region amino acid sequence of any one of SEQ ID NO. 8, 16, 24, 32; or a variant having no more than 2 amino acid changes per CDR region.

[0013] Further, the residue range of the CDR is shown in the following table:

[0014]

[0015] Further, when the antibody HCDRs are encoded according to the IMGT encoding rule, the antibody or antigen binding fragment comprises the following CDR sequences:

[0016] i. the amino acid sequence of the HCDR1 is any one of SEQ ID NO. 1, 9, 17, 25;

[0017] ii. the amino acid sequence of the HCDR2 is as set out in any one of SEQ ID NO. 2, 10, 18, 26;

[0018] iii. the amino acid sequence of the HCDR3 is as set out in any one of SEQ ID NO. 3, 11, 19, 27;

[0019] iv. the amino acid sequence of the LCDR1 is as set out in any one of SEQ ID NO. 5, 13, 21, 29;

[0020] v. the amino acid sequence of the LCDR2 is as set out in any one of SEQ ID NO. 6, 14, 22, 30;

[0021] vi. the amino acid sequence of the LCDR3 is as set out in any one of SEQ ID NO. 7, 15, 23, 31;

[0022] or a variant of any one of i-vi above having a single or multiple amino acid changes in the six CDR regions, not more than 2 amino acid changes in each CDR region.

[0023] Further, the combination of HCDR1 / HCDR2 / HCDR3 can comprise one or more groups selected from:

[0024] (1) HCDR1 / HCDR2 / HCDR3:

[0025] SEQ ID NO. 1 / SEQ ID NO. 2 / SEQ ID NO. 3,

[0026] SEQ ID NO. 9 / SEQ ID NO. 10 / SEQ ID NO. 11,

[0027] SEQ ID NO. 17 / SEQ ID NO. 18 / SEQ ID NO. 19,

[0028] SEQ ID NO. 25 / SEQ ID NO. 26 / SEQ ID NO. 27,

[0029] (2) or the HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids;

[0030] The combination of LCDR1 / LCDR2 / LCDR3 comprises one or more groups selected from:

[0031] (1) LCDR1 / LCDR2 / LCDR3 is:

[0032] SEQ ID NO. 5 / SEQ ID NO. 6 / SEQ ID NO. 7,

[0033] SEQ ID NO. 13 / SEQ ID NO. 14 / SEQ ID NO. 15,

[0034] SEQ ID NO. 21 / SEQ ID NO. 22 / SEQ ID NO. 23,

[0035] SEQ ID NO. 29 / SEQ ID NO. 30 / SEQ ID NO. 31,

[0036] (2) or HCDR of (1) above containing one or more amino acid substitutions, deletions or insertions of no more than 2 amino acids.

[0037] In some aspects, the antibody or antigen-binding fragment thereof of the present application that specifically binds to the CAR antigen binding site further comprises a heavy chain variable region (VH) sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 4, 12, 20, 28; and a light chain variable region (VL) sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 8, 16, 24, 32.

[0038] Further, the antibody or antigen-binding fragment can further comprise a conjugated moiety linked to the polypeptide, the conjugated moiety selected from the group consisting of a radionuclide, a toxin, a cytokine, an enzyme, a fluorophore, a carrier protein, a lipid, and biotin, wherein the polypeptide or antibody is selectively linked to the conjugated moiety via a linker, preferably the linker is a peptide or polypeptide.

[0039] Further, the antibody or antigen-binding fragment can be selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antisera, a chimeric antibody, a humanized antibody, and a human antibody; more preferably, the antibody is selected from the group consisting of 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.

[0040] The second aspect of the present application also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof of the first aspect.

[0041] 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;

[0042] Further, the vector can be a cloning vector or an expression vector, without limitation;

[0043] Further, 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.

[0044] 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.

[0045] Further, the cell includes but is not limited to a yeast cell, a Chinese hamster ovary cell, a human embryonic kidney cell, and other mammalian cells or other cells suitable for producing an antibody or an antigen-binding fragment thereof.

[0046] The sixth aspect of the present application provides a kit, characterized in that it comprises one or more of the above-mentioned antibody or antigen-binding fragment, polynucleotide, recombinant vector, and host cell, and is contained in a suitable container.

[0047] The seventh aspect of the present application provides a method for producing the antibody or antigen-binding fragment thereof of the first aspect that can bind to the antigen-binding site of the CAR, 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.

[0048] The eighth aspect of the present application provides a use of the antibody or antigen-binding fragment thereof of the first aspect in any of the following aspects:

[0049] a) in detecting Anti-CD19 CAR expression;

[0050] b) in evaluating Anti-CD19 CAR transfection positivity rate;

[0051] c) in evaluating the change pattern of the number of Anti-CD19 CAR positive T cells in the body of a subject after administration, or in preparing a reagent for evaluating the change pattern of the number of Anti-CD19 CAR positive T cells in the body of a subject after administration. It can be understood that a) and b) can be in vitro detection, and the antibody or antigen-binding fragment of the present application is a CAR detection tool. The sample in practice can be from the body or in vitro. When used for in vitro sample detection, its purpose can be simple CAR detection or evaluation, thus including non-disease diagnosis applications.

[0052] The ninth aspect of the present application provides a detection method, which comprises the step of detecting a sample to be tested by using any of the antibodies or antigen binding fragments described above, or the kit described above.

[0053] Further preferably, the detection method is used to evaluate whether the sample contains Anti-CD19 CAR or the expression amount of Anti-CD19 CAR, without limitation, which can be reasonably expected in the art. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 Partial results of FACS binding screening of supernatant of positive clones.

[0055] Figure 2 Partial results of FACS blocking screening of supernatant of positive clones.

[0056] Figure 3 Partial results of FACS blocking screening of subclones.

[0057] Figure 4 Partial results of FACS blocking screening of subclones.

[0058] Figure 5 SDS-PAGE identification results of the antibodies of Anti-FMC63 scFv of the present application.

[0059] Figure 6 ELISA binding analysis results of the antibodies of Anti-FMC63 scFv of the present application.

[0060] Figure 7 Competitive ELISA analysis results of the antibodies of Anti-FMC63 scFv of the present application.

[0061] Figure 8 SPR analysis results of the antibodies of Anti-FMC63 scFv of the present application.

[0062] Figure 9 FACS binding analysis results of the antibodies of Anti-FMC63 scFv of the present application. DETAILED DESCRIPTION

[0063] The present application discloses an isolated antibody or antigen binding fragment thereof, and those skilled in the art can refer to the content herein to realize its application. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all considered to be included in the present application. The preparation method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation method and application herein without departing from the content, spirit and scope of the present application to realize and apply the technology of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art.

[0064] The following terms or definitions are provided merely to aid in the understanding of the present application. These definitions should not be construed to limit the scope or applicability of the claims.

[0065] Unless otherwise defined, all technical and scientific terms used in the present application are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Although it is believed that the following terms are well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate the understanding of the present application.

[0066] As used in the present application, the terms "comprise", "contain", "have", "include" or "involve" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term "consist of" is considered to be a preferred embodiment of the term "comprise". If in the following a group is defined to comprise at least a certain number of embodiments, this is also to be understood as disclosing a group which preferably consists only of these embodiments.

[0067] The indefinite article "a" or "an" or the definite article "the" used in the description of singular forms, such as "one" or "said", include the plural forms of the noun.

[0068] The terms "about", "approximately" in the present application mean an interval of accuracy which can still guarantee the technical effect referred to the feature as understood by those skilled in the art. The term generally means ±10%, preferably ±5% deviation from the indicated numerical value.

[0069] The terms "or more," "at least," "more than," and the like, e.g., "at least one," are understood to include but not be limited to at least 1, 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 than the stated value. Any larger number or fraction in between is also included.

[0070] 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, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fraction in between is also included.

[0071] The terms "a plurality," "at least two," "two or more," "at least a second," and the like, should be understood to include but not be limited to 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. Any larger number or fraction thereof is also included.

[0072] Definitions of terms:

[0073] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that is capable of non-covalently, reversibly and specifically binding to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer that includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) 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 herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. "Antibodies" include, but are not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies, including, for example, anti-Id antibodies to antibodies of the present disclosure. Antibodies can belong to any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).

[0074] Antibodies comprise the globular regions of a heavy or light chain polypeptide termed "domains". Domains can comprise peptide loops, typically 3 to 4, stabilized, for example, by beta sheets and / or intrachain disulfide bonds. Domains are typically referred to as "constant" or "variable", based on relative lack of sequence variation within the domain of different class members, in the case of "constant" domains, or significant variation, in the case of "variable" domains. Antibody or polypeptide "domains" are often referred to interchangeably in the art as antibody or polypeptide "regions".

[0075] Antibodies can be assigned to five major classes: IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequences of their constant regions. Several of these classes can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, and IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The light chains can be of two types, kappa and lambda, based on the amino acid sequences of their constant regions. Within full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids and, in the case of the heavy chain, a "D" region of about 10 or more amino acids.

[0076] The term "monoclonal antibody" refers to a preparation of antibody molecules of single amino acid composition, and is not intended to be limited with respect to the manner in which they are made. Monoclonal antibodies or immunologically active fragments thereof can be produced by hybridoma technology, recombinant technology, phage display technology, synthetic technology, and other production techniques known in the art. The methods of making monoclonal antibodies referred to herein include in vitro culturing of hybridoma cells or production by DNA recombination technology. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each monoclonal antibody is directed against a single determinant on the antigen.

[0077] The term "antigen" is an entity (e.g., a protein entity or a peptide) to which an immunoglobulin or antibody (or antigen-binding fragment thereof) specifically binds.

[0078] The term "fragment" refers to a portion or part of an antibody or antibody chain, which comprises fewer amino acid residues than the whole 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 the whole antibody, The fragment can be obtained by chemical or enzymatic treatment of the whole or complete antibody or antibody chain. The fragment can also be obtained by recombinant means.

[0079] The term "variable" indicates certain portions of the variable regions of antibodies that differ in sequence among antibodies and are responsible for binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, joined by three CDRs, which are generally termed CDR1, CDR2 and CDR3, in sequence from the N-terminus. The CDRs in each chain are closely associated with each other and with the CDRs from the other chain via the FR regions, and collectively contribute to the formation of the antigen binding site of an antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions of the antibodies are not directly involved in binding of the antibodies to antigen, but exhibit diverse effector functions, such as participation in antibody-dependent cellular cytotoxicity.

[0080] "Complementarity determining region" or "complementarity determining region" ("CDR") interchangeably refers to the hypervariable regions of VL and VH. CDRs are the target protein binding sites of the antibody chains that carry the specificity of such target proteins. There are three CDRs in each human VL or VH (CDR1-3, numbered sequentially from the N-terminus), which account for about 15-20% of the variable domain. CDRs can be referred to by their region and order. For example, "VH CDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDRs are structurally complementary to the epitope of a target protein and are therefore directly responsible for binding specificity. The remaining stretches of VL or VH (so-called framework regions) exhibit less variation in amino acid sequence (Kuby, Immunology, 4th Ed., Chapter 4 W.H. Freeman & Co., New York, 2000).

[0081] 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 or a combination of many known antibody CDR assignment systems, including, for example: Chothia (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)) based on antibody sequence variability; and Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, Department of Health and Human Services, National

[0082] 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.

[0083] However, it should be noted that the boundaries of the CDRs (Continuous Derivatives) of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may vary. For example, the residue ranges of CDR regions numbered using Kabat, Chothia, etc., under different assignment systems are shown in the table below.

[0084] CDR residue ranges defined by different assignment systems

[0085]

[0086] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this application, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this application due to the application of different schemes (e.g., different assignment system rules or combinations).

[0087] CDRs of antibodies of the present application can be artificially assessed to determine boundaries according to any of the schemes in the art or combinations thereof. Unless otherwise indicated, in the present application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the manners described above.

[0088] 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 chains 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, univalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide bonded Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen binding fragments of any of the above.

[0089] The term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilization / de-stabilization, spatial distribution) an epitope of an antigen. Examples of binding fragments include, but are not limited to, a single-chain Fv (scFv), a disulfide linked Fv (sdFv), a Fab fragment, a F(ab') fragment (i.e., a monovalent fragment consisting of the VL, VH, CL, and CHI domains); a F(ab)2 fragment (i.e., a bivalent fragment including two Fab fragments agnostic at the hinge region by a disulfide bridge); an Fd fragment consisting of the VH and CHI domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., Nature 341 :544-546, 1989) that consists of a VH domain; and an isolated complementarity determining region (CDR) or other epitope- binding fragments of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv ("scFv")); see e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 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 with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0090] Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFv (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. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). Antigen-binding fragments can be incorporated into single chain molecules that include a pair of tandem Fv segments (VH-CH1-VH-CH1) called a diabody (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Patent No. 5,641,870.

[0091] The term "chimeric antibody" refers to an antibody in which a portion (generally the variable region) of the heavy and / or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (generally the constant region) is identical with or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. In the present application, the chimeric antibody refers to the heavy chain / light chain variable region from the murine antibody grafted to the constant region of the heavy chain / light chain of the human antibody by antibody engineering technology, which exhibits similar biological activity.

[0092] The term "humanized antibody" refers to a chimeric antibody that contains 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, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.

[0093] The term "affinity" refers to the strength of the interaction between antibody and antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at a number of sites; the more interactions, the stronger the affinity.

[0094] The term "compete" as used herein, when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, means that the antigen binding proteins compete with each other as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) the specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen.

[0095] As used herein, the term "variant" refers to a heavy chain variable region or a light chain variable region that has been modified by at least one, e.g., 1, 2, or 3, amino acid substitution, deletion, or addition, wherein the modified antigen binding protein comprising the variant heavy chain or light chain variable region substantially retains the biological characteristics of the antigen binding protein prior to modification. In one embodiment, an antigen binding protein containing a variant heavy chain variable region or light chain variable region sequence retains 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to modification. It will be appreciated that each heavy chain variable region or light chain variable region can be modified, either alone or in combination with another heavy chain variable region or light chain variable region. The antigen binding proteins of the disclosure include a heavy chain variable region amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a heavy chain variable region amino acid sequence described herein. The antigen binding proteins of the disclosure include a light chain variable region amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a light chain variable region amino acid sequence described herein. The percentage homology 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 regions, while the sequences corresponding to the CDRs have 100% identity to the CDRs disclosed herein within 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, e.g., 1, 2, or 3, amino acid substitution, deletion, or addition, wherein the modified antigen binding protein comprising the CDR variant substantially retains the biological characteristics of the antigen binding protein prior to modification. In one embodiment, an antigen binding protein containing a variant CDR retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to 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.

[0096] The term "vector," as used herein, refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated by transformation. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has 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."

[0097] The term "host cell" refers to a cell into which foreign nucleic acid has been introduced, including the progeny of such a cell. The host cell is capable of expressing the foreign nucleic acid within the cell or cell membrane or releasing it extracellularly.

[0098] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, unless otherwise indicated.

[0099] The term "kit" is used to refer to a combination of reagents and other materials that aid in the analysis of a sample. In some embodiments, the immunoassay kits described herein include suitable antigens, binding agents comprising a detectable moiety, and detection reagents. Systems for amplifying the signal generated by the detectable moiety can or can not be included in the kit. In addition, in other embodiments, the kits include, but are not limited to, components such as devices for sample collection, sample tubes, racks, trays, shelves, dishes, plates, instructions for the user of the kit, solutions or other chemical reagents, and samples for standardization, normalization, and / or control samples.

[0100] 1. Anti-idiotype antibodies or antigen-binding fragments against Anti-CD19 CAR of the application

[0101] The terms "anti-idiotype antibodies against Anti-CD19 CAR," "antibodies against Anti-CD19 CAR," "antibodies that specifically bind to Anti-CD19 CAR antigen-binding site," "antigen-binding fragments against Anti-CD19 CAR" are used interchangeably herein to refer to antibodies or antigen-binding fragments of the application that are capable of binding Anti-CD19 CAR with sufficient affinity and specificity so as to be useful in the detection of the CAR. In some embodiments, the antibodies or antigen-binding fragments of the application that are capable of specifically binding to the CAR antigen-binding site, according to ELISA and SPR validation data, are capable of specifically binding to the antigen recognition site of the FMC63-derived Anti-CD19 CAR and have high affinity properties. The antibodies are capable of detecting the expression of the FMC63-derived Anti-CD19 CAR by flow cytometry, exhibiting high sensitivity, non-specific background properties.

[0102] The antibodies and antigen-binding fragments of the application specifically bind to Anti-CD19 CAR with high affinity. In some embodiments, the antibodies or antigen-binding fragments of the application are a class of sequences according to the different analysis methods for CDRs in the above definition:

[0103] which can comprise 3 CDRs in the heavy chain variable region amino acid sequence of any one of SEQ ID NO. 4, 12, 20, 28, and 3 CDRs in the light chain variable region amino acid sequence of any one of SEQ ID NO. 8, 16, 24, 32; or variants thereof having single or multiple amino acid changes in the CDRs not more than 2 amino acids per CDR.

[0104] In some embodiments, the antibody or antigen binding fragment comprises the following CDR sequences when the antibody HCDRs are encoded according to IMGT numbering:

[0105] i. the amino acid sequence of the HCDR1 is as set forth in any one of SEQ ID NO. 1, 9, 17, 25;

[0106] ii. the amino acid sequence of the HCDR2 is as set forth in any one of SEQ ID NO. 2, 10, 18, 26;

[0107] iii. the amino acid sequence of the HCDR3 is as set forth in any one of SEQ ID NO. 3, 11, 19, 27;

[0108] iv. the amino acid sequence of the LCDR1 is as set forth in any one of SEQ ID NO. 5, 13, 21, 29;

[0109] v. the amino acid sequence of the LCDR2 is as set forth in any one of SEQ ID NO. 6, 14, 22, 30;

[0110] vi. the amino acid sequence of the LCDR3 is as set forth in any one of SEQ ID NO. 7, 15, 23, 31;

[0111] or variants thereof having single or multiple amino acid changes in the 6 CDRs of i-vi not more than 2 amino acids per CDR.

[0112] In some embodiments, the combination of HCDR1 / HCDR2 / HCDR3 can comprise one or more groups selected from:

[0113] (1) HCDR1 / HCDR2 / HCDR3:

[0114] SEQ ID NO. 1 / SEQ ID NO. 2 / SEQ ID NO. 3,

[0115] SEQ ID NO. 9 / SEQ ID NO. 10 / SEQ ID NO. 11,

[0116] SEQ ID NO. 17 / SEQ ID NO. 18 / SEQ ID NO. 19,

[0117] SEQ ID NO. 25 / SEQ ID NO. 26 / SEQ ID NO. 27;

[0118] (2) the HCDRs of (1) above that contain one or more amino acid substitutions, deletions, or insertions of no more than 2 amino acids.

[0119] the combination of LCDR1 / LCDR2 / LCDR3 comprises one or more groups selected from:

[0120] (1) the LCDR1 / LCDR2 / LCDR3 are:

[0121] SEQ ID NO. 5 / SEQ ID NO. 6 / SEQ ID NO. 7,

[0122] SEQ ID NO. 13 / SEQ ID NO. 14 / SEQ ID NO. 15,

[0123] SEQ ID NO. 21 / SEQ ID NO. 22 / SEQ ID NO. 23,

[0124] SEQ ID NO. 29 / SEQ ID NO. 30 / SEQ ID NO. 31;

[0125] (2) the HCDRs of (1) above that contain one or more amino acid substitutions, deletions, or insertions of no more than 2 amino acids.

[0126] In some specific embodiments, the antibody or antigen-binding fragment thereof that can bind of the present application further comprises 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 to an amino acid sequence selected from the group consisting of SEQ ID NO. 4, 12, 20, 28; and 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 to an amino acid sequence selected from the group consisting of SEQ ID NO. 8, 16, 24, 32; preferably, the amino acid changes occur to the CDR regions.

[0127] In some more specific embodiments, certain antibody sequences of the application are shown in the following table (specific VH and VL sequences of antibodies or antigen binding fragments thereof).

[0128]

[0129]

[0130] In some embodiments, the antibody or antigen binding fragment of the application further comprises an Fc region, which is from an IgG, such as IgGl, IgG2, IgG3, or IgG4.

[0131] In some embodiments, the amino acid changes in the above-described amino acid homologies comprise substitutions, insertions, or deletions of amino acids. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions are ones in which the amino acid is replaced with one from the same class, e.g., an acidic amino acid is replaced with another acidic amino acid, a basic amino acid is replaced with another basic amino acid, or a neutral amino acid is replaced with another neutral amino acid. Exemplary substitutions are shown in the following table (amino acid substitutions).

[0132]

[0133] In preferred embodiments, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in the Fc region.

[0134] In some embodiments, the CDRs of the antibodies are encoded according to the IMGT numbering scheme, and the CDR sequences of certain numbered antibodies are shown in the following table

[0135]

[0136] In some embodiments, the antibody or antigen binding fragment can further comprise a conjugation moiety linked to the polypeptide, the conjugation moiety selected from one or more of a radionuclide, a toxin, a cytokine, an enzyme, a fluorescein, a carrier protein, a lipid, and biotin, wherein the polypeptide or antibody and the conjugation moiety are optionally linked by a linker, preferably the linker is a peptide or polypeptide.

[0137] In some embodiments, the antibody or antigen binding fragment can be selected from a monoclonal antibody, a polyclonal antibody, an anti-serum, 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.

[0138] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be produced by recombinant expression. The above-described nucleic acids encoding the light and heavy chain variable regions, optionally linked to constant regions, can be inserted into expression vectors. Vectors comprising nucleic acids encoding the antibodies described herein are aspects of the present application per se. The light and heavy chains can be cloned into the same or different expression vectors. The nucleic acids encoding the antibody chains described herein can be operably linked to one or more control sequences in the expression vector to ensure expression of the antibody chains. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Such vectors can be incorporated into the appropriate host, which maintains the vector under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the antibodies.

[0139] 2. Nucleic acids of the present application, vectors comprising the same, and host cells

[0140] The present application provides nucleic acids encoding any of the above antibodies or antigen-binding fragments thereof, or any of the chains thereof. 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, such as a host cell, comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for making an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

[0141] The nucleic acids contemplated by the present application are nucleic acids encoding an antibody or antigen-binding fragment thereof, or a VH or VL domain thereof; it is understood that any nucleic acid capable of encoding an antibody or antigen-binding fragment thereof, or a VH or VL domain thereof, as described above, is within the scope of the present application.

[0142] The present application contemplates vectors comprising one or more of the above-described nucleic acids encoding an antibody described herein, which vectors can be cloning vectors or expression vectors, without limitation.

[0143] In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs), and the like.

[0144] In one embodiment, the vector comprises optional regulatory sequences; in some embodiments, the regulatory sequences can be selected from, without limitation, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof.

[0145] Host cells of the application comprising the expression vectors, such as host cells are yeast cells, mammalian cells, or other cells suitable for production of antibodies or antigen-binding fragments thereof. In some embodiments, suitable host cells include prokaryotic microorganisms, such as E. coli. Host cells can also be eukaryotic microorganisms, such as filamentous fungi or yeasts, or various vertebrate cells, e.g., insect cells, etc. Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be used. Examples of useful mammalian host cell lines include the COS (SV40-transformed human embryonic kidney) 293 or 293F cells, 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog 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, NS0 cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2 / 0, and the like. A review of suitable mammalian host cell lines for producing proteins is found, for example, in Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a 293 cell.

[0146] Vectors described herein comprising polynucleotide sequences of interest (e.g., heavy and light chain-encoding sequences and expression control sequences) can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. (See generally, Green and Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 4th ed., 2012). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (See generally, Sambrook et al., supra). To generate transgenic animals, transgenes can be microinjected into fertilized oocytes, or they can be integrated into the genome of embryonic stem cells, and the nuclei of these cells transferred into enucleated oocytes.

[0147] 3、Preparation, production, and purification of antibodies or antigen-binding fragments of the application

[0148] The method of producing an Anti-CD19 CAR anti-idiotypic antibody or antigen-binding fragment thereof in the present application can comprise expressing a vector described herein in a host cell culture to produce the antibody, and recovering the antibody from the cell culture.

[0149] In some embodiments, the method can comprise transferring a vector comprising one or more nucleic acids encoding an antibody or antigen-binding fragment thereof or antibody chain thereof as described above into a host cell as described herein, culturing the host cell culture under conditions that allow expression of the nucleic acids, and recovering the expressed corresponding antibody or antigen-binding fragment thereof. Any suitable method known in the art can be employed.

[0150] The present application provides a method of producing an Anti-CD19 CAR anti-idiotypic antibody or antigen-binding fragment, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the antibody or antigen-binding fragment, and optionally isolating the antibody or antigen-binding fragment. In a certain embodiment, the method further comprises recovering the purified corresponding antibody or antigen-binding fragment from the host cell (or host cell culture medium).

[0151] In some embodiments, the antibodies or antigen-binding fragments produced as described herein can be purified by known art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent 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, and the like.

[0152] 4, Detection methods and uses of the present application

[0153] The antibodies or antigen-binding fragments provided by the present application can be used to detect the presence or amount of Anti-CD19 CAR in a biological sample, and thus for detection or evaluation purposes.

[0154] In some embodiments, the antibodies or antigen-binding fragments against Anti-CD19 CAR provided by the present application can be conveniently used in a kit, and Anti-CD19 CAR in a sample in vivo or in vitro can be specifically, sensitively and without non-specific background detected by the antibodies or antigen-binding fragments provided by the present application. Thus, the antibodies or antigen-binding fragments of the present application are particularly suitable for evaluating CAR transfection positive rate and the change rule of the number of CAR positive T cells in the body of a subject after administration, etc.

[0155] The term "detecting" as used herein includes quantitative or qualitative detection. Exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, and SPR, etc. In some embodiments, the protein antibodies or antigen-binding fragments thereof of the present application can be conjugated with luciferase, biotinase, etc. detectable labels for direct or indirect immunoassay in liquid or solid phase, such as FACS, SPR, IHC, ELISA, etc.

[0156] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. Embodiments

[0157] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0158] Preparation, screening and sequencing of monoclonal antibodies

[0159] 1) Preparation of immunogen: An expression plasmid expressing FMC63 scFv, His Tag was constructed by gene synthesis method, and then the expression plasmid was transfected into HEK293 cells using Invitrogen Lipofectamine 2000 transfection reagent. After 48 hours, the culture supernatant was harvested, and then FMC63 scFv, His Tag was purified by affinity chromatography. Subsequently, the purified FMC63 scFv was coupled with KLH carrier protein (purchased from Sigma, Cat. No. H7017).

[0160] 2) Mouse immunization: FMC63 scFv coupled KLH carrier protein was used as immunogen, and 10 Balb / c mice were immunized with FMC63 scFv-KLH. The conventional immunization plan is shown in Table 1. Seven days after each immunization, the serum of the immunized animals was detected by ELISA method to determine the level of immune response. After the completion of the conventional immunization, if the immunized animals can reach the immune response level (OD value > 1.0, titer reaches 1:8,000) against the immunogen, cell fusion can be performed.

[0161] Table 1 Immunization table

[0162] Step Schedule Dose and Route Adjuvant Pre-immune bleeds Day -4 First immunization Day 0 50 μg / animal, s.c. Freund's complete adjuvant Second immunization Day 14 25 μg / animal, s.c. Freund's incomplete adjuvant Bleeds for testing 1 Day 21 Third immunization Day 28 25 μg / animal, s.c. Freund's incomplete adjuvant Bleeds for testing 2 Day 35 Final immunization Day 50 ± 7 25 μg / animal, i.p. Cell fusion Four days after final immunization

[0163] 3) Cell fusion and plating

[0164] Two cell fusions were performed using the electrofusion method. All cells from each fusion were plated into 96-well plates.

[0165] 4) Screening analysis

[0166] Primary screening: Supernatants from the fusion cells were screened by ELISA. Supernatants that were positive against the scFv were selected. FMC63 scFv and several other proteins (as negative and isotype controls) were coated separately, and pre-immune serum and three post-immune antisera were tested (Table 2). Titers were the highest dilution ratios where the signal / blank ratio was greater than or equal to 2.1. NC is the pre-immune serum, which was used as a negative control (Table 3).

[0167] Table 2 ELISA primary screening experimental scheme

[0168]

[0169]

[0170] Table 3 ELISA screening of supernatants from the fusion cells

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] Confirmation screening: When performing the parental clone screening, all positive parental clone cell supernatants were screened by indirect ELISA, with total human IgG and irrelevant his protein as counter screens. The positive parental clone indirect ELISA screening scheme is shown in Table 4. The ELISA results for the pre-immune serum and three post-immune antisera screening are shown in Table 5. Titers were the highest dilution ratios where the signal / blank ratio was greater than or equal to 2.1. NC is the pre-immune serum, which was used as a negative control.

[0177] Table 4 Positive parental clone indirect ELISA screening scheme

[0178]

[0179] Table 5 ELISA screening results for the pre-immune serum and three post-immune antisera

[0180]

[0181]

[0182]

[0183] FACS binding screening: Screening experiment with FMC63 scFv HEK293, 1x10 5 cells as positive cell line and regular HEK293, 1x10 5 cells as negative cell line. Primary antibodies were: a, pre-immune serum diluted 1:50; b, mouse IgG (isotype control) at 3ug / ml; c, PBS as negative control; d, post-immune serum diluted 1:50. Secondary antibody was Alexa 647 AffiniPure Goat Anti-Mouse IgG, Fc gamma fragments specific (min X Hu, Bov, Hrs Sr Prot) (Jackson, 115-605-071) at 1ug / ml. FACS binding screening results are shown in Figure 1 . Black curve: negative cells + a / b / c / d + secondary antibody; grey curve: positive cells + a / b / c / d + secondary antibody.

[0184] FACS blocking screening: Screening experiment with FMC63 scFv HEK293, 1x10 Figure 2 cells. Primary antibodies were: a, pre-immune serum diluted 1:50; b, mouse IgG (isotype control) at 3ug / ml; c, PBS (negative control); d, post-immune serum diluted 1:50; e, human CD19 recombinant protein ligand with human Fc tag at 1ug / ml. Secondary antibody was Goat Anti-Human IgG, Fc fragment specific [Alexa Fluor 647] (Jackson, 109-605-098) at 1ug / ml. FACS blocking screening results are shown in . Black curve: scFv HEK293 + c + e + secondary antibody; grey curve: scFv HEK293 + a / b / c / d + e + secondary antibody.

[0185] 5) Clone expansion and cryopreservation

[0186] Positive maternal cell lines were transferred to 24-well plates for expansion culture, up to a maximum of 10 lines. Two mL of supernatant was collected from each expanded culture clone for indirect ELISA and blocking FACS detection. These specific positive cell lines were cryopreserved to avoid clone loss. Indirect ELISA

[0187] The ELISA testing protocol is shown in Table 6, and the test results are shown in Table 7.

[0188] Table 6. Indirect ELISA screening protocol for expanded culture supernatant of positive maternal clones.

[0189]

[0190] Table 7 Results of indirect ELISA screening using expanded culture supernatant of positive maternal clones

[0191]

[0192]

[0193] The cell line used to block FACS detection was FMC63-expressing scFv HEK293, 1x10 5 Cells. Primary antibodies were: a) 1:50 diluted fusion serum (positive control); b) 3 μg / ml mouse IgG (isotype control); c) PBS (negative control); d) 1 μg / ml human CD19 recombinant protein ligand with a human Fc tag; e) supernatant from expanded culture of positive primordial clones. Secondary antibody was Alexa. 647AffiniPure Goat Anti-Human IgG (H+L) (min X Bov,Hrs,Ms Sr Prot) (Jackson, 1μg / ml).

[0194] 6) Subcloning

[0195] Subcloning of positive maternal clones was performed using a limiting dilution method to ensure that these positive maternal clones originated from individual maternal clone cells. Subclonal screening was conducted using indirect ELISA or blocking FACS methods. The indirect ELISA assay protocol is shown in Table 8, and the assay results are shown in Table 9.

[0196] Table 8 Subclonal Indirect ELISA Screening Scheme

[0197]

[0198] Table 9 Results of the subclonal indirect ELISA screening protocol

[0199]

[0200]

[0201]

[0202] The cell line for blocking FACS detection is FMC63 scFv HEK293, 1x10 5 The primary antibodies are: a, 1:50 dilution of fusion serum (positive control); b, 3 ug / ml of mouse IgG (isotype control); c, PBS (negative control); d, 1 ug / ml of human CD19 recombinant protein ligand with human Fc tag; e, subclone supernatant. The secondary antibody is Alexa 647 AffiniPure Goat Anti-Human IgG (H+L) (min X Bov, Hrs, Ms Sr Prot) (Jackson, 1 ug / ml). The results of the blocking FACS detection are shown in Figure 3 and Figure 4 The black curve is scFv HEK293 + d + secondary antibody, and the gray curve is scFv HEK293 + a / b / c / e + d + secondary antibody.

[0203] On the basis of antigen recognition confirmation, 2 stable subclone cell lines were selected from each parent clone for cryopreservation. Before cryopreservation, 5 ml of supernatant was collected from each subclone, and subtype identification and preservation were performed on all subclones.

[0204] 7) Antibody production and purification

[0205] Hybridoma cells were cultured in large quantities, and protein A / G affinity chromatography was used to purify the antibodies. Dialysis was used to store the purified antibodies in phosphate buffered saline (PBS).

[0206] 8) Sequencing of hybridoma cell antibody genes

[0207] Based on the above screening data and results, 4 dominant subclone strains were obtained: 24C1E4D8, 26A10C3E6, 23D4G9E9, and 16D5E8E7. These antibodies showed more comprehensive affinity and blocking activities and could be used for subsequent detection purposes. Further, total RNA was extracted from the corresponding hybridoma cells, and RNA was reverse transcribed into cDNA through RT-PCR reaction. The light chain and heavy chain sequences were cloned into T vectors, and DNA sequencing analysis was performed to obtain the antibody gene sequences. The specific sequence results are shown in Table 10.

[0208] Table 10 Amino acid sequences of dominant antibodies (IMGT rules determine CDR)

[0209]

[0210]

[0211] Example 2. Analytical characterization and functional analysis of monoclonal antibodies

[0212] The above selected each dominant clone further carried out antibody identification and functional analysis, part of the analysis results are shown as follows.

[0213] ELISA detection results by subclass kit showed that the subtypes of antibodies 24C1E4D8, 26A10C3E6, 23D4G9E9 and 16D5E8E7 were IgG1, K.

[0214] SDS-PAGE identification results( Figure 5 ) showed that, for example, the 16D5E8E7 antibody, its reduced electrophoresis two bands were 27 kDa and 50 kDa in size, and the purity of the antibody was greater than 99%.

[0215] ELISA binding data( Figure 6 ) and competitive ELISA data( Figure 7 ) showed that the 16D5E8E7 clone number antibody could very specifically recognize the antigen binding site of Anti-CD19 (FMC63) CAR.

[0216] SPR analysis data( Figure 8 ) showed that the 7 fitting lines from top to bottom represented the affinity and dissociation between FMC63 scFv and 16D5E8E7 clone number antibody with time at concentrations of 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.813 nM, 3.906 nM and 1.953 nM. The results showed that the affinity of the antibody to FMC63 scFv was as high as 1.1 nM.

[0217] FACS binding data( Figure 9 ) showed that the 16D5E8E7 clone number antibody could specifically bind to Anti-CD19 (FMC63) CAR expressed on the cell surface, and had no non-specific binding signal with 293 cells not transfected with CAR and PBMC cells transfected with CAR, showing good high specificity and no non-specific background advantage.

[0218] The foregoing description of specific exemplary embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and various modifications and variations are possible in light of the above teachings. It is intended that the embodiments be limited only by the claims as interpreted in their broadest terms.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a CAR antigen-binding site, characterized in that, It contains three CDRs from the heavy chain variable region amino acid sequence shown in SEQ ID NO. 28 and three CDRs from the light chain variable region amino acid sequence shown in SEQ ID NO.

32.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that, When antibody HCDRs are encoded according to the IMGT encoding rules, the CDR sequence of the antibody or antigen-binding fragment is as follows: i. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27, respectively; ii. The amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.29, SEQ ID NO.30, and SEQ ID NO.31, respectively.

3. An isolated polynucleotide, characterized in that, The polynucleotide encodes the antibody or antigen-binding fragment as described in any one of claims 1-2.

4. A carrier, characterized in that, It contains the polynucleotide as described in claim 3.

5. The carrier according to claim 4, characterized in that, The vector is a cloning vector or an expression vector.

6. The carrier according to claim 4, characterized in that, The vector further includes a regulatory sequence selected from leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, transcription terminators, or any combination thereof.

7. A host cell, characterized in that, It comprises the polynucleotide of claim 3 or the vector of any one of claims 4-6.

8. The host cell according to claim 7, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

9. The host cell according to claim 7, characterized in that, The host cells were selected from yeast cells, Chinese hamster ovary cells, and human embryonic kidney cells.

10. A reagent kit, characterized in that, It comprises an antibody or antigen-binding fragment as described in any one of claims 1-2, a polynucleotide as described in claim 3, a vector as described in any one of claims 4-6, and a host cell as described in any one of claims 7-9, and is contained in a suitable container.

11. Any of the following applications of the antibody or antigen-binding fragment according to any one of claims 1-2: Application in the preparation of reagents for detecting Anti-CD19 CAR expression; Application in the preparation of kits for assessing the positive rate of Anti-CD19 CAR transfection; Application in the preparation of reagents for assessing the changes in the number of Anti-CD19 CAR-positive T cells in subjects after drug administration.

12. A detection method, characterized in that, The steps of detecting a test sample using the antibody or antigen-binding fragment according to any one of claims 1-2, wherein the detection method is not a diagnostic method for a disease.

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