Antibody specifically binding to GAD or antigen binding fragment thereof, preparation method and application

Abstract

The invention relates to the technical field of biology, in particular to an antibody specifically bound with GAD or an antigen binding fragment thereof, a preparation method and application. The antibody or the antigen binding fragment thereof contains a heavy chain variable region and a light chain variable region, wherein a complementary determining region of the heavy chain variable region has an amino acid sequence consistent with VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region as shown in SEQ ID NO.1; the complementarity determining region of the light chain variable region has an amino acid sequence consistent with the amino acid sequences of VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region as shown in SEQ ID NO.2. The antibody or the antigen binding fragment thereof has good specific binding capacity with glutamate decarboxylase (GAD), and can be used for identifying an anti-glutamate decarboxylase antibody (GADA) or a GAD antigen, and carrying out or assisting in carrying out diagnosis detection on GADA positive diseases.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to antibodies that specifically bind to GAD or their antigen-binding fragments, preparation methods, and applications. Background Technology

[0002] Type 1 diabetes is an autoimmune-related metabolic disease in which the immune system mistakenly attacks and destroys insulin-secreting beta cells in the pancreas, leading to an absolute deficiency of insulin. Type 1 diabetes is often accompanied by various immune dysfunctions during its development and treatment. Detection of diabetes-related autoantibodies, such as ICA (anti-islet cell autoantibody), IAA (anti-insulin autoantibody), GADA (anti-glutamate decarboxylase autoantibody), and IA-2 (anti-islet tumor-associated antigen-2 autoantibody), is helpful in the clinical classification of diabetes, treatment selection, and prognostic assessment.

[0003] Glutamic acid decarboxylase (GAD) is a key enzyme that catalyzes the conversion of glutamate into the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Glutamic acid decarboxylase antibody (GADA), as a specific autoantibody of pancreatic β-cells, is often positive before and in the early stages of type 1 diabetes, but mostly negative in normal individuals and patients with type 2 diabetes. Therefore, GADA is considered an early immunomarker for type 1 diabetes and is widely used for the differential diagnosis and prediction of type 1 diabetes (including LADA) and type 2 diabetes. Compared with other diabetes autoantibodies, GADA has the characteristics of early appearance, long duration, resistance to disappearance, and high sensitivity and specificity, and has important clinical value in diabetes classification, disease progression assessment, and treatment guidance.

[0004] Currently, most common diagnostic kits use anti-GAD antibodies derived from human positive serum. However, the source of such serum is limited, and there are significant differences between different batches, affecting the consistency of detection. In addition, existing technologies also include detection methods that use rabbit or mouse monoclonal antibodies prepared using hybridoma technology, or methods that purify polyclonal antibodies after obtaining positive serum from immunized animals and then conjugate them with human IgG. However, these methods generally suffer from complex operating procedures, long production cycles, and high costs.

[0005] Therefore, developing a high-affinity anti-GAD antibody that is easy to obtain, can be mass-produced, and is suitable for clinical testing kits has important application value and practical significance. Summary of the Invention

[0006] The purpose of this application is to provide an antibody or antigen-binding fragment thereof that specifically binds to GAD, and which exhibits good specific binding ability to GAD. Based on the antibody or antigen-binding fragment provided in this application, another purpose of this application is to provide its applications. Another purpose of this application is to develop antibodies or antigen-binding fragments thereof with good GAD-binding activity using a simpler method, which can be used for the identification of GAD antigens and for the diagnosis or auxiliary diagnosis of diseases positive for anti-GAD antibodies.

[0007] To solve the above technical problems, the following technical solution is adopted:

[0008] Definition of noun:

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0010] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0011] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0012] In this article, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0013] In this article, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0014] In this article, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0015] In this paper, for methods involving multiple steps, unless otherwise explicitly stated otherwise, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times. Their execution order is not necessarily sequential, but can be performed alternately, concurrently, or in turn with other steps or parts of the sub-steps or stages of other steps.

[0016] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another, and does not necessarily require or imply any actual relationship, order, or degree of importance between these entities or actions, such as numbering (Ⅰ), (Ⅱ)...(Ⅶ), etc. In the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., the terms "first," "second," "third," "fourth," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumerable descriptive purpose and should be understood not to constitute a closed limitation on quantity.

[0017] In this article, glutamate decarboxylase (GAD) is a key enzyme that catalyzes the conversion of glutamate into the inhibitory neurotransmitter γ-aminobutyric acid (GABA). It is a pyridoxal phosphate enzyme that can catalyze the irreversible decarboxylation of L-glutamate or its salt at the α-position to form γ-aminobutyric acid.

[0018] In this article, "glutamate decarboxylase antigen" and "GAD antigen" refer to the antigens of glutamate decarboxylase that can be bound by natural anti-glutamate decarboxylase antibodies. These antigens can be complete glutamate decarboxylase or partial components derived from glutamate decarboxylase.

[0019] In this article, "antibodies that specifically bind to GAD or their antigen-binding fragments" refers to antibodies that specifically bind to glutamate decarboxylase or their antigen-binding fragments, which can bind to the aforementioned glutamate decarboxylase antigen. Hereinafter, they are also referred to as "GAD antibodies or their antigen-binding fragments".

[0020] In this document, the technical term "antibody or its antigen-binding fragment" refers to a protein that binds to a specific antigen, broadly encompassing all proteins and protein fragments containing a complementarity-determining region (CDR). The terms "antibody" and "full-length antibody" include both polyclonal and monoclonal antibodies. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. Non-naturally occurring antibodies are also referred to as "recombinant antibodies" in this document. The term "antibody" is used interchangeably with "immunoglobulin."

[0021] In this article, peptides, polypeptides, and proteins are not strictly distinguished and can be used interchangeably in some cases. Generally, peptides refer to polymers composed of amino acids linked by peptide bonds, whether naturally occurring or synthetic.

[0022] In this article, antibodies or their antigen-binding fragments can also be antigen-binding fragments containing part or all of the antibody CDR, lacking at least some amino acids present in the full-length antibody chain but still capable of specifically binding to antigens. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from, but are not limited to, F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody with VH and VL linked by a linker peptide), dsFv (disulfide-stabilized Fv fragments, dsFv)), bispecific antibodies, nanobodies, and the smallest recognition unit of an antibody. In addition to the functional fragments mentioned above, any fragment with an extended half-life is also included.

[0023] In this article, the term "Fab" in antibody refers to a portion of an antibody composed of a single light chain (including variable and constant regions) and a single heavy chain whose variable and first constant regions are linked by disulfide bonds. "Fab' fragment" refers to a Fab fragment containing a portion of the hinge region. "F(ab')2" refers to a Fab' dimer. "Fv fragment" is composed of the variable regions of a single light chain and / or a single heavy chain. "Single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by the direct interconnection of light chain variable regions and heavy chain variable regions or by linkage through peptide linker sequences. "Minimum recognition unit of antibody" refers to a single CDR structure containing only the variable region; although the minimum recognition unit has a small molecular weight and low affinity, it possesses the ability to bind to antigens.

[0024] In this article, the term "variable region" or "variable domain" refers to the amino-terminal domain of an antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable domain can be referred to as "VH," and the light chain variable domain as "VL." Variable domains contain antigen-binding sites. Both the heavy and light chain variable regions consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the backbone region and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well-known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0025] In this document, the term "constant region" or "constant domain" refers to the constant region of a single or combined antibody light chain or antibody heavy chain. The antibody heavy chain has a variable domain (VH) followed by a number of constant domains or regions, such as one or more of the hinge, CH1, CH2, CH3, and CH4. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the antibody heavy chain, and does not form a portion of the Fc region of the antibody. The hinge region includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. The N-terminus of CH2 is typically the CH3 domain, which usually forms the C-terminal portion of the antibody. In some antibody types, such as IgM and IgE, the constant region also includes the CH4 domain. The constant region of an antibody can be derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as the constant regions of their subclasses and mutant forms.

[0026] This application does not limit the method of obtaining GAD antibodies or their antigen-binding fragments. In some optional embodiments, the corresponding antibody can be obtained by linking a polynucleotide encoding the antibody or its antigen-binding fragment to a vector and expressing it in cells. The above-mentioned vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a structure capable of expressing the antibody or its antigen-binding fragment. In other optional embodiments, the antibody or its antigen-binding fragment can also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as automated peptide synthesizers (e.g., automated peptide synthesizers sold by AppliedBioSystems, etc.); the antigen-binding fragment can also optionally be generated by enzymatic cleavage of antigen-binding molecules (including intact antibodies), such as pepsin or papain cleavage; or by chemical cleavage, such as by chemical reduction of disulfide bonds to obtain the above-mentioned antigen-binding fragment.

[0027] In this article, the terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of an antibody or its antigen-binding fragment to a pre-determined epitope on an antigen. Typically, antibodies or their antigen-binding fragments bind at a rate of approximately less than 10... -5 M, for example, approximately less than 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller KD values ​​are required for binding. The KD value of an antibody can be determined using methods well-established in the art. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry analyses.

[0028] In this document, the term "signaling substance" refers to a substance that can provide a detectable signal, which can be directly observed by the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can provide a signal directly, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can provide a signal indirectly through a reaction in which the signaling substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0029] In this document, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a polynucleotide encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0030] In this article, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the cells.

[0031] Vectors are well known to those skilled in the art and include, but are not limited to: plasmids, episome plasmids, microcircular DNA, phage particles, and Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this application contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0032] In this document, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ from primary cells morphologically and / or in genomic DNA. “Transformant” and “transformed cell” include primary test cells and cultures derived therefrom. Cells may be prokaryotic or eukaryotic, with prokaryotic cells including, but not limited to, *Escherichia coli*, *Bacillus*, or *Staphylococcus*. Eukaryotic cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.

[0033] In this document, the terms “purified” or “isolated” associated with peptides or nucleic acids mean that the peptide or nucleic acid is not in its native medium or in its native form. Therefore, the term “isolated” includes peptides or nucleic acids removed from their original environment, such as if they are naturally occurring. For example, isolated peptides typically do not contain at least some proteins or other cellular components that are normally bound to or mixed with or in solution with them. Isolated peptides include naturally produced peptides contained in cell lysates, peptides in purified or partially purified forms, recombinant peptides, peptides expressed or secreted by cells, and peptides in heterologous cells or cultures. As associated with nucleic acids, the terms “isolated” or “purified” indicate, for example, that the nucleic acid is not in its native genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into heterologous cells).

[0034] In this article, "anti-glutamate decarboxylase (GAD) antibody positive disease" refers to a disease that can cause patients to have higher levels of anti-glutamate decarboxylase antibodies than before they have the disease. Exemplary anti-glutamate decarboxylase antibody positive diseases include, but are not limited to, type 1 diabetes.

[0035] In one aspect, a GAD antibody or its antigen-binding fragment is provided, comprising a heavy chain variable region and a light chain variable region.

[0036] The variable region of the heavy chain includes complementarity-determining regions VH-CDR1, VH-CDR2 and VH-CDR3; the variable region of the light chain includes complementarity-determining regions VL-CDR1, VL-CDR2 and VL-CDR3.

[0037] VH-CDR1 has an amino acid sequence identical to that of VH-CDR1 in the heavy chain variable region shown in SEQ ID NO.1; VH-CDR2 has an amino acid sequence identical to that of VH-CDR2 in the heavy chain variable region shown in SEQ ID NO.1; VH-CDR3 has an amino acid sequence identical to that of VH-CDR3 in the heavy chain variable region shown in SEQ ID NO.1; and VL-CDR1 has an amino acid sequence identical to that of VL-CDR1 in the light chain variable region shown in SEQ ID NO.2; VL-CDR2 has an amino acid sequence identical to that of VL-CDR2 in the light chain variable region shown in SEQ ID NO.2; VL-CDR3 has an amino acid sequence identical to that of VL-CDR3 in the light chain variable region shown in SEQ ID NO.2.

[0038] It is understood that the amino acid sequences of the variable regions shown in SEQ ID NO. 1 and 2, excluding the CDR region, are not used to define the GAD antibody or its antigen-binding fragment provided in this application. For example, if the GAD antibody or its antigen-binding fragment provided in this application contains a backbone region, it may differ from the backbone region in the variable regions shown in SEQ ID NO. 1 or 2. The CDR region in the variable regions shown in SEQ ID NO. 1 and 2 can be divided according to any optional manner known in the art. Optionally, the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the variable regions are defined by any one of the Kabat, Chothia, IMGT, ABM, or Contact systems. Taking the Kabat, Chothia, IMGT, ABM, or Contact definition methods as examples, the amino acid sequences of the CDRs are shown in Table 1:

[0039] Table 1 shows the heavy chain CDRs of VH as indicated by SEQ ID NO.1.

[0040]

[0041]

[0042] Table 2 shows the light chain CDRs of VL as indicated by SEQ ID NO.2.

[0043]

[0044] In an optional embodiment, the GAD antibody or its antigen-binding fragment has VH-CDR1, VH-CDR2 and VH-CDR3, heavy chain variable regions as defined in Table 1, and VL-CDR1, VL-CDR2 and VL-CDR3, light chain variable regions as defined in Table 2.

[0045] Taking the IMGT definition as an example: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.19; the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.22, the amino acid sequence of VL-CDR2 is YTS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.25.

[0046] In an optional implementation, the source species of the antibody sequence excluding the CDR region includes one or more of the following: rabbit, cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, human, and mutants thereof.

[0047] In an optional implementation, the heavy chain variable region further includes at least one skeleton region, such as one skeleton region, two skeleton regions, three skeleton regions, or four skeleton regions.

[0048] In an optional implementation, the skeleton region of the heavy chain variable region conforms to at least one of the following (i) to (iv):

[0049] (i) The backbone region VH-FR1 contains an amino acid sequence identical to that of the VH-FR1 of the heavy chain variable region shown in SEQ ID NO.1;

[0050] (ii) The backbone region VH-FR2 contains an amino acid sequence identical to that of the VH-FR2 of the heavy chain variable region shown in SEQ ID NO.1;

[0051] (iii) The backbone region VH-FR3 contains an amino acid sequence identical to that of VH-FR3 in the heavy chain variable region shown in SEQ ID NO.1; and,

[0052] (iv) The backbone region VH-FR4 contains an amino acid sequence consistent with the VH-FR4 of the heavy chain variable region shown in SEQ ID NO.1.

[0053] In an optional implementation, the light chain variable region further includes at least one skeleton region, such as one skeleton region, two skeleton regions, three skeleton regions, or four skeleton regions.

[0054] In an optional implementation, the skeletal region of the light chain variable region conforms to at least one of (i) to (iv) below:

[0055] (i) The backbone region VL-FR1 contains an amino acid sequence identical to that of the VL-FR1 of the light chain variable region shown in SEQ ID NO.2;

[0056] (ii) The backbone region VL-FR2 contains an amino acid sequence identical to that of the VL-FR2 of the light chain variable region shown in SEQ ID NO.2;

[0057] (iii) The backbone region VL-FR3 contains an amino acid sequence identical to that of VL-FR3 in the light chain variable region shown in SEQ ID NO.2; and,

[0058] (iv) The backbone region VL-FR4 contains an amino acid sequence consistent with the VL-FR4 of the light chain variable region shown in SEQ ID NO.2.

[0059] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the GAD antibody or its antigen-binding fragment is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0060] In an optional implementation, the GAD antibody or its antigen-binding fragment is an antibody or antigen-binding fragment containing a constant region.

[0061] In an optional implementation, the GAD antibody or its antigen-binding fragment is a monoclonal antibody.

[0062] In an optional implementation, at least a portion of the constant region sequence of the GAD antibody or its antigen-binding fragment is a human constant region sequence.

[0063] In an optional embodiment, the constant region sequence of the GAD antibody or its antigen-binding fragment is selected from the sequence of part or all of the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD include their subclasses and mutant forms.

[0064] In an optional implementation, the GAD antibody or its antigen-binding fragment contains a heavy chain constant region.

[0065] In an optional embodiment, the heavy chain constant region sequence of the GAD antibody or its antigen-binding fragment is derived from part or all of the constant region sequence of human IgG1, preferably including CH1, CH2 and CH3 of the constant region of human IgG1.

[0066] In an optional embodiment, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3.

[0067] In an optional implementation, the GAD antibody or its antigen-binding fragment contains a light chain constant region.

[0068] In an optional implementation, the light chain constant region sequence is derived from the light chain constant region of a mouse.

[0069] In an optional embodiment, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.4.

[0070] In an optional embodiment, the GAD antibody or its antigen-binding fragment is a human-mouse chimeric antibody, the heavy chain amino acid sequence of the GAD antibody or its antigen-binding fragment is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.6.

[0071] Secondly, a biomaterial is also provided, comprising polynucleotides, carriers, cells or conjugates;

[0072] The polynucleotide encodes the aforementioned GAD antibody or its antigen-binding fragment;

[0073] The vector carries polynucleotides;

[0074] Cells carry polynucleotides, or contain vectors, or are capable of expressing GAD antibodies or their antigen-binding fragments. By linking a polynucleotide encoding a GAD antibody or its antigen-binding fragment to a vector, the vector can be introduced into eukaryotic cells, especially mammalian cells, to construct cell lines capable of expressing GAD antibodies or their antigen-binding fragments, and the corresponding proteins can be obtained through cellular expression.

[0075] In an optional embodiment, the cells used to express the GAD antibody or its antigen-binding fragment are 293 cells (human kidney epithelial cell line), preferably 293F cells.

[0076] In an optional implementation, the cells used to express GAD antibodies or their antigen-binding fragments are CHO cells (Chinese hamster ovary cells).

[0077] The conjugate is an antibody-drug conjugate containing the first aspect of the GAD antibody or its antigen-binding fragment.

[0078] In an optional embodiment, the conjugate is a GAD antibody or its antigen-binding fragment conjugated with a solid-phase carrier or signal.

[0079] Thirdly, a method for preparing the GAD antibody or its antigen-binding fragment as described in the first aspect is also provided, including culturing cells capable of expressing the GAD antibody or its antigen-binding fragment.

[0080] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding a GAD antibody or its antigen-binding fragment into cells, and obtaining the GAD antibody or its antigen-binding fragment through purification.

[0081] In optional embodiments, the preparation method further includes synthesizing a polynucleotide containing a gene encoding a GAD antibody or its antigen-binding fragment as needed, and / or preparing a suitable expression vector as needed, transforming the expression vector into the desired cells and expressing it, and obtaining the GAD antibody or its antigen-binding fragment by purification.

[0082] In an optional embodiment, the cells are prepared by converting cells with a polynucleotide encoding a GAD antibody or an antigen-binding fragment thereof, as described in the first aspect. The polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0083] In an optional implementation, the preparation method includes fusing the C-terminus of the heavy chain variable region with a constant region fragment to construct a complete heavy chain expression plasmid.

[0084] In an optional implementation, the constant region segment comprises one or more of CH1, CH2 and CH3, preferably CH1, CH2 and CH3.

[0085] In an optional implementation, the cell is a eukaryotic cell, preferably a mammalian cell.

[0086] In an optional embodiment, the mammalian cells are 293 cells or CHO cells, preferably 293F cells.

[0087] Fourthly, a composition is also provided, comprising the GAD antibody of the first aspect or its antigen-binding fragment, or the biological material of the second aspect.

[0088] In an optional embodiment, the composition is a reagent or kit for detecting anti-GAD antibodies, or for diagnosing and / or assisting in the diagnosis of anti-GAD antibody-positive diseases; the kit contains standards and / or quality control products containing GAD antibodies or their antigen-binding fragments.

[0089] In an optional implementation, the kit is used for the diagnosis and / or auxiliary diagnosis of anti-GAD antibody-positive diseases.

[0090] In an optional implementation, the kit is used for the diagnosis and / or auxiliary diagnosis of at least one of type 1 diabetes, stiff-person syndrome, and autoimmune encephalitis.

[0091] In an optional implementation, the kit further includes a detection reagent, which includes an anti-GAD antibody detection reagent.

[0092] In an optional implementation, the kit may further include a detection reagent for detecting at least one of ICA (anti-islet cell autoantibody), IAA (anti-insulin autoantibody), and IA-2 (anti-islet tumor-associated antigen-2 autoantibody).

[0093] In an optional implementation, the kit may also include a solid support.

[0094] In optional embodiments, the GAD antibody or its antigen-binding fragment in the reagent or kit is coupled to a solid-phase carrier; or the GAD antibody or its antigen-binding fragment in the kit is packaged separately from the solid-phase carrier. By coupling the GAD antibody or its antigen-binding fragment to the solid-phase carrier, it can be used to capture GAD antigen in the sample to be tested. Alternatively, by coupling the GAD antibody or its antigen-binding fragment to the solid-phase carrier, it can be used to purify GAD antigen.

[0095] In an optional embodiment, the anti-GAD antibody detection reagent comprises a solid-phase carrier conjugated with GAD antigen.

[0096] In an optional implementation, the kit may also contain a signaling agent.

[0097] In optional embodiments, the GAD antibody or its antigen-binding fragment in the reagent or kit is conjugated with the signal molecule; the GAD antibody or its antigen-binding fragment in the reagent or kit and the signal molecule are packaged separately. By conjugating the GAD antibody or its antigen-binding fragment with the signal molecule, it can be used for localized detection of GAD antigen or for detection of GAD antigen in a sample via Western blotting.

[0098] The reagents or kits described above may optionally include reagents and / or consumables well known to those skilled in the art for use in detecting reactions or purifying proteins, including, but not limited to, one or more of buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, coupling agents, negative controls, positive controls, standards, quality control products, and markers.

[0099] The above-mentioned reagents or kits can be used in general immunoassay methods acceptable in the art, including but not limited to immunofluorescence staining, flow cytometry, immunoblotting, immunohistochemistry, ELISA, immunochromatography, or immunomagnetic beads. Those skilled in the art can formulate other reagents in the reagents or kits according to the corresponding detection methods, and this application does not limit this.

[0100] Fifthly, the application of the GAD antibody of the first aspect or its antigen-binding fragment, or the biological material of the second aspect, or the composition of the fourth aspect, in any of the following (I) to (VII):

[0101] (I) Non-diagnostic and treatment-oriented detection of anti-GAD antibodies;

[0102] (II) Preparation of products for detecting anti-GAD antibodies;

[0103] (III) Prepare products for the diagnosis and / or auxiliary diagnosis of diseases with positive anti-GAD antibodies;

[0104] (IV) Non-diagnostic and therapeutic target testing for GAD antigen;

[0105] (V) Prepare products for detecting GAD antigen;

[0106] (VI) Used for the isolation, enrichment and / or purification of GAD antigen;

[0107] (VII) Prepare products for the isolation, enrichment and / or purification of GAD antigens;

[0108] In the applications described in (I) to (III) above, GAD antibodies or their antigen-binding fragments can be used as standards and / or quality control materials during detection to provide a reliable reference for the test results, and can also be used to construct standard curves.

[0109] In optional implementations, anti-GAD antibody-positive diseases include, but are not limited to, type 1 diabetes, stiff-person syndrome, and autoimmune encephalitis.

[0110] In an optional implementation, diagnosing and / or assisting in the diagnosis of anti-GAD antibody-positive diseases includes distinguishing between type 1 diabetes and type 2 diabetes, identifying anti-GAD antibody-positive subjects as type 1 diabetes patients, or identifying them as type 1 diabetes patients in combination with other diagnostic results.

[0111] The applications described in (IV) to (VII) above can utilize the ability of GAD antibodies or their antigen-binding fragments to specifically target and bind to GAD antigens, thereby achieving at least one of the following: detection, separation, enrichment, and purification of GAD antigens.

[0112] In an optional implementation, when the GAD antibody or its antigen-binding fragment is an immunoconjugate, such as one linked to a signaling substance, the location or real-time detection of the GAD antigen can be achieved by detecting the signaling substance.

[0113] In an optional implementation, at least one of the following is achieved: detection, separation, enrichment, and purification of GAD antigen by isolating the anti-GAD antibody-GAD antigen immune complex.

[0114] In optional embodiments, in aspects (II), (III), (V) or (VII) above, those skilled in the art can prepare corresponding products (such as the immunoconjugates mentioned above) according to actual uses, and select other reagents to be included in the product, including but not limited to one or more of the following: signaling agents, solid-phase carriers, buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, conjugates, negative controls, positive controls, standards, quality control products, and markers.

[0115] The signaling agent in any of the above embodiments includes, but is not limited to, one or more of the following: enzymes, luminescent labels, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radioactive contrast agents, paramagnetic ions, metals, and photosensitizers.

[0116] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Luminescent labels include, but are not limited to, fluorescent proteins, synthetic small molecules, or polymer dyes. Specific examples include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, and Cascade. Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C, Phycocyanin R, Thiamine, Phycoerythrin, Phycoerythrin R, REG, Rhodamine Green, Rhodamine Isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red, or one or more of these. Fluorescent microspheres, colored microspheres, and latex microspheres are each independently selected from products acceptable in the art, such as those derived from commercially available products. Radionuclides include, but are not limited to, those expressed as radionuclides. 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68Ga、 86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and 83 One or more of Sr. Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0117] The solid support in any of the above embodiments includes, but is not limited to, microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices, or side-flow devices; more specifically, it can be, but is not limited to, enzyme-labeled wells, immunochromatographic test strips, or magnetic beads. The chromatography matrix can be any known chromatography matrix acceptable in the art, including, but not limited to, polystyrene, polysaccharide polymers, or silica gel. In optional embodiments, the chromatography matrix comprises gel particles.

[0118] This application discloses a GAD antibody or its antigen-binding fragment that specifically recognizes and binds to the GAD antigen, which is further used to prepare a human-mouse chimeric recombinant monoclonal antibody. This application has the following beneficial effects:

[0119] 1) The preparation method of the GAD antibody or its antigen-binding fragment is simple and time-saving. The CDR sequence of the antibody is known, and it can be expressed in vitro at any time by cells with high expression level. The production process is controllable and the batch-to-batch variation of the product is small.

[0120] 2) The GAD antibody or its antigen-binding fragment has good affinity and a KD of 1.28E-11M. It can be used as a standard and quality control in the test kit, which can alleviate the problems of complicated operation of polyclonal antibodies and low subsequent conjugation efficiency, reduce production costs, stabilize product quality, and significantly improve reaction values. On the other hand, compared with using human serum directly, it can also avoid the problems of difficult sample sources and high costs.

[0121] 3) The GAD antibody or its antigen-binding fragment can be coupled with a signaling agent for the detection of GAD antigen.

[0122] 4) The GAD antibody or its antigen-binding fragment can be coupled to a solid-phase carrier for immunoaffinity chromatography purification of GAD antigen, resulting in higher protein purity. Attached Figure Description

[0123] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0124] Figure 1 This is an electrophoresis diagram of the heavy chain (Fd) and light chain (L) used to construct the library in Example 1;

[0125] Figure 2 This is an electrophoresis image of the Fab gene fragment used to construct the library in Example 1;

[0126] Figure 3 The images show SDS-PAGE electrophoresis results of the four anti-GAD recombinant monoclonal antibodies in Example 2. GAD-Ab-non-reduced: the result of non-reduced SDS-PAGE electrophoresis, with a size of approximately 150 kDa; GAD-Ab-reduced: the result of reduced SDS-PAGE electrophoresis, with heavy and light chains of 50 kDa and 25 kDa, respectively; GAD-Ab-1 represents recombinant monoclonal antibody 1, GAD-Ab-2 represents recombinant monoclonal antibody 2, GAD-Ab-3 represents recombinant monoclonal antibody 3, and GAD-Ab-4 represents recombinant monoclonal antibody 4.

[0127] Figure 4 This is a line graph of recombinant monoclonal antibody 1 in the example;

[0128] Figure 5 In this example, the SPR method was used to determine the affinity between recombinant monoclonal antibody 1 and GAD antigen. Detailed Implementation

[0129] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0130] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0131] In all the following examples, the GAD antigen (Glutamate Decarboxylase 65kDa (GAD65), catalog number 13800) was purchased from Diarect GmbH, Germany.

[0132] Example 1: GAD antibody screening

[0133] (I) Preparation of phage display library

[0134] 1) Spleens were harvested from mice immunized with GAD antigen, and lymphocytes were separated using mouse lymphocyte separation fluid.

[0135] 2) RNA extraction: Take 1×10 6 Total RNA was extracted from lymphocytes using the Trizol method.

[0136] 3) Reverse transcription: The extracted total RNA is reverse transcribed to synthesize cDNA.

[0137] 4) Antibody gene fragment amplification: Using cDNA as a template, specific amplification primers are used to amplify the κ and λ light chains and the VH-CH1(Fd) region of the heavy chain of the antibody.

[0138] 20 μL reaction system: 1 μL cDNA, 0.8 μL Prime F, 0.8 μL Prime R, 10 μL 2×phanta maxmaster mix, 7.4 μL Nuclease-Free Water; reaction program: denaturation 95℃ 30 s, annealing 55℃ 30 sec, extension 72℃ 45 s, 30 cycles.

[0139] After the reaction was complete, loading buffer was added to the system, and the mixture was identified by 1% agarose gel electrophoresis. The electrophoresis results are shown below. Figure 1 As shown.

[0140] The target band was excised, and the antibody heavy chain (Fd) gene fragment and light chain gene fragment (L) were recovered separately.

[0141] 5) Antibody light chain and heavy chain gene fragments are combined into complete Fab gene fragments by overlapping PCR.

[0142] 25 μL reaction system and procedure: 30 ng light chain, 4.3 ng linker, 30 ng heavy chain, 0.5 μL sfi IF (upstream primer), 0.5 μL sfi IR (downstream primer), 12.5 μL 2×phanta max master mix, and Nuclease-Free Water to a total volume of 25 μL. Reaction procedure: Depolymerization at 95℃ for 30 s, annealing at 55℃ for 30 sec, extension at 72℃ for 90 s, 30 cycles. After the reaction, loading buffer was added, and the system was identified by 1% agarose gel electrophoresis. The electrophoresis image is shown below. Figure 2 As shown, the target band is approximately 1500 bp. The target band was excised, and the antibody Fab fragment was recovered.

[0143] Upstream primer sfiⅠF: 5'>GAGCAGGAGCATAGGAGGATCGGGCGGCGGCC<3' (SEQ ID NO.27);

[0144] Downstream primer sfiⅠR: 5'>CCATGGCAATGGTGATTCTGCTGCGCGGCCTGGCC<3' (SEQ ID NO. 28).

[0145] 6) Plasmid construction: Digest the antibody Fab fragment and pComb3xSS plasmid with sfiI enzyme, mix them at a molar ratio of 3:1 between the digested fragment and the plasmid, add T4 DNA ligase, and ligate overnight at 16°C.

[0146] 7) Library Construction: Recover the ligation product from the previous step. Take 500 ng of the recovered product, add 40 μL of TG1 competent cells, mix well, and transfer to an electroporation cuvette for electroporation. Parameter selection: Bac-Ec3. After electroporation, activate and incubate at 37℃ for 1 h. Transfer the activated bacterial culture to 2×YT medium, add 1 / 1000 ampicillin antibiotic and a final concentration of 2% glucose solution, and incubate at 37℃ and 220 rpm until OD600 = 0.6. Add 20 times the number of helper phage M13K07. Mix well and place in a shaker at 37℃ for static infection for 45 min. Centrifuge for 15 min to collect the bacteria, resuspend the pellet in fresh 2×YT+Amp+Kana medium, and incubate at 30℃ and 220 rpm for 14 h for phage amplification. The next day, centrifuge to collect the supernatant, add 1 / 4 volume of 20% PEG6000 to precipitate the phage, and resuspend in PBS to obtain the phage display library.

[0147] (II) Screening anti-GAD antibodies using phage display libraries

[0148] 1) Using biotin-conjugated GAD protein as the target antigen, after incubating with SA magnetic beads for 1 hour, 1×10⁻⁶ g of the protein was added to the reaction system. 12 The phages obtained in Example 1 were incubated for 1 hour, and the specific phages were captured by the antigen. They were then washed 10 times with PBST (PBS + 0.05% Tween-20).

[0149] 2) Add the antigen-binding magnetic beads to the TG1 bacterial culture, incubate at 37°C for 45 min, then activate and culture at 37°C and 220 rpm for 1 h. Take an appropriate amount of the incubated bacterial culture, serially dilute it and spread it on ampicillin plates. Add glucose (final concentration 2%) and 1 / 1000 ampicillin antibiotic to the remaining bacterial culture, and shake at 37°C and 220 rpm for 3 h.

[0150] 3) Add 10 μL of M13K07 to the bacterial culture and incubate at 37°C for 45 min for infection.

[0151] 4) Centrifuge at 6000×g for 10 min, resuspend in 10 mL of 2×YT+Amp+Kana medium, and amplify the phage by shaking at 30℃ and 220 rpm for 14 h. On the second day, centrifuge to collect the supernatant, add 1 / 4 volume of 20% PEG6000 to precipitate the phage, and resuspend in PBS to obtain the phages selected in the first round of screening.

[0152] 5) Using the phages obtained in the first round of screening, repeat steps 1 to 4 to perform the second round of screening.

[0153] 6) Single clone identification: Single clones were selected from the second round of screening and plated onto 96-well deep-well plates, and the phages were amplified by overnight shaking.

[0154] Plate coating: GAD antigen was used to coat ELISA plates, and BSA was used as a control protein for plate coating. The plates were incubated overnight at 4°C.

[0155] Blocking: On the second day, discard the coating solution, wash the plate 3 times with PBST, pat dry, add 3% milk, and block at 37°C for 2 hours.

[0156] Primary antibody preparation: Centrifuge 96-well deep-well plates, take the supernatant and dilute it in a final concentration of 1% milk, mix well and use it as the primary antibody for later use.

[0157] Primary antibody incubation: Discard the blocking solution, wash the plate 3 times with PBST, pat dry, add the primary antibody, and incubate at 37°C for 2 hours.

[0158] Secondary antibody incubation: Discard the primary antibody, wash the plate 5 times with PBST, pat dry, add 1:5000 diluted Anti-M13 Antibody (HRP) secondary antibody, and incubate at 37°C for 1 hour. Discard the secondary antibody, wash the plate 5 times with PBST, pat dry, add the chromogenic substrate for color development, and add stop solution after 15 minutes to stop the reaction. Read the value using a microplate reader.

[0159] 7) Select clones with high read values ​​(GAD antigen well OD value > 1) and low non-specific binding (BSA control protein well OD value < 0.5) for sequencing.

[0160] Light chain sequencing primers: Bomp: 5'>GTGTGGAATTGTGAGCGG<3' (SEQ ID NO.29);

[0161] Heavy chain sequencing primers: PELB: 5'>ACCTATTGCCTACGGCAGCCG<3' (SEQ ID NO.30).

[0162] Ten monoclonal antibodies were selected for sequencing, and the sequencing results showed that there were four different GAD antibody sequences, including antigen-binding domains.

[0163] Example 2: Expression and purification of anti-GAD recombinant monoclonal antibody

[0164] After obtaining the Fab region sequence of the candidate antibody through sequencing, gene synthesis is performed.

[0165] The PTT5 plasmid was selected as the vector, and the EcoRI+BamHI gene fragment was inserted using the EcoRI+BamHI cloning site. The light chain constant region sequence is shown in SEQ ID NO.4 and is a mouse-derived sequence.

[0166] The CH1 region of the heavy chain was replaced with the CH1 region of human IgG1, and the Fc region of human IgG1 was fused at the C-terminus. Using EcoRI+BamHI as the cloning site, the complete heavy chain fragment was inserted into the PTT5 plasmid. The heavy chain constant region sequence is shown in SEQ ID NO.3 and is a human-derived sequence.

[0167] Heavy chain constant region (SEQ ID NO.3):

[0168] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0169] Light chain constant region (SEQ ID NO.4):

[0170] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0171] Expression was performed using the mammalian cell 293F expression system.

[0172] 1) Plasmid extraction: The synthesized plasmids were transformed into TOP10 competent cells, activated for 1 hour, and then transferred to LB medium. The cells were cultured overnight at 37°C. The plasmids were extracted the next day using an endotoxin-free plasmid extraction kit to obtain the corresponding heavy chain plasmids and light chain plasmids.

[0173] 2) One day before transfection, 293F cells were seeded into suspension cell culture flasks, and the cell density was controlled at 1×10⁻⁶ cells / year. 6 per ml.

[0174] 3) On the second day, dilute 40 μg of heavy chain plasmid and 80 μg of light chain plasmid in 6 mL of transfection buffer and mix gently. Add 480 μL of PEI and mix gently. Incubate at room temperature for 20 minutes. Add the mixture dropwise to the cells and place the cells in an incubator for suspension culture at 98 rpm, 37°C, and 5% CO2.

[0175] 4) After 6 days, collect the culture medium supernatant, purify IgG using rProtein A packing material, elute with 0.1M Glycine (pH 3.0), and neutralize with 1M Tris (pH 8.0). After elution, replace the ultrafiltration centrifuge tubes with PBS buffer and concentrate the protein, determine the protein concentration, and verify its purity by SDS-PAGE. Figure 3 .

[0176] Example 3: Chemiluminescence assay for the binding activity of recombinant monoclonal antibody to GAD antigen

[0177] The detection performance of four monoclonal antibodies was tested using a chemiluminescence method, as follows:

[0178] 1) The four recombinant antibodies were diluted 250, 500 and 1000 times respectively and tested on the instrument (repeated twice). The concentration values ​​of each point were calculated and evaluated.

[0179] 2) Further linearity studies were conducted on antibodies with qualified titers: the average detected concentration was fitted to the theoretical concentration using the least squares method to obtain a linear regression equation.

[0180] 3) Verify the thermal stability of the antibody with the best titer and linearity after 7 days: Dilute the antibody to 40 IU / mL and 500 IU / mL respectively, and place it at 2-8℃ and 37℃ for 7 days respectively before testing (repeat 3 times) and calculate the deviation.

[0181] The test results are shown in Tables 3-5 and Figure 4 As shown, antibody No. 1 has a qualified titer, meets the linearity requirements, and exhibits good stability. The concentration value is calculated from the chemiluminescence value; antibody concentration = concentration value × dilution factor.

[0182] Table 3. Binding activities of antibodies numbered 1-4 with GAD antigen.

[0183]

[0184]

[0185] Based on the results in Table 3, antibody 1 was selected for further evaluation. The titers of the other three antibodies were too low to meet the requirements. A linearity study was conducted on antibody 1, and the results are shown in Table 4. Figure 4 As shown.

[0186] Table 4. Linearity of Antibody No. 1

[0187]

[0188] According to the results in Table 4, the reaction performance of antibody No. 1 meets the requirements. The next step is to verify its thermal stability. The results of the thermal stability verification are shown in Table 5.

[0189] Table 5. Thermal stability of antibody No. 1

[0190]

[0191]

[0192] The results in Table 5 show that the thermal stability is good and there is no significant difference between 2-8℃ and 37℃.

[0193] Sequencing of antibody 1 revealed that the heavy chain variable region sequence is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.2. Further analysis showed that the antigen-binding domain contains heavy and light chain CDRs as shown in Tables 6 and 7. The complete light chain sequence of the corresponding recombinant monoclonal antibody is shown in SEQ ID NO.6, and the complete heavy chain sequence is shown in SEQ ID NO.5.

[0194] Table 6 Heavy chain CDRs of recombinant monoclonal antibody No. 1

[0195]

[0196] Table 7 Light chain CDRs of recombinant monoclonal antibody No. 1

[0197]

[0198] Complete heavy chain sequence (SEQ ID NO.5):

[0199] EVKIEESSGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYSESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTRHWDDAMDCWGRG TSVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0200] Complete light chain sequence (SEQ ID NO.6):

[0201] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0202] Example 4: Determination of the affinity between recombinant monoclonal antibody 1 and GAD antigen using the SPR method

[0203] Further determination of the affinity between the screened recombinant antibody No. 1 and the GAD protein:

[0204] 1) Ligand preparation: Dilute the antibody to 10 μg / ml with sample diluent.

[0205] 2) Analyte preparation: Dilute GAD antigen to 32 μg / ml with sample diluent.

[0206] 3) Chip cleaning: Clean the chip twice with 150 μl / well of regeneration solution, sample diluent, and ultrapure water, and then pat dry.

[0207] 4) Ligand fixation: Set the ligand fixation parameters. According to the prompts, add 50 μl of sample diluent per well to run the baseline. After the baseline is run, remove the chip plate from the instrument and shake it dry. Add 50 μl of the prepared ligand solution per well to the chip plate to perform ligand fixation.

[0208] 5) Affinity determination: Immediately after ligand immobilization, remove the chip, discard the ligand solution, and pat dry. Set the affinity detection parameters, and according to the prompts, add 50 μl / well of the prepared analyte to perform binding and dissociation operations.

[0209] 6) Data Processing: Set calculation parameters and calculate antigen-antibody affinity. For example... Figure 5 The KD of recombinant antibody No. 1 with GAD antigen is 1.28E-11M.

[0210] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0211] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to GAD, characterized in that, It includes variable regions for heavy chains and variable regions for light chains; The heavy chain variable region includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3. The VH-CDR1, VH-CDR2, and VH-CDR3 have amino acid sequences that are identical to those of the VH-CDR1, VH-CDR2, and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.1, respectively. The VL-CDR1, VL-CDR2, and VL-CDR3 have amino acid sequences that are identical to those of the VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable region shown in SEQ ID NO.

2.

2. The antibody or antigen-binding fragment thereof that specifically binds to GAD according to claim 1, characterized in that, The antibody or its antigen-binding fragment VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 are defined by any one of the systems Kabat, Chothia, IMGT, ABM or Contact. Optionally, according to the IMGT definition: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.19; Optionally, the amino acid sequence of VL-CDR1 is as shown in SEQ ID NO.22, the amino acid sequence of VL-CDR2 is YTS, and the amino acid sequence of VL-CDR3 is as shown in SEQ ID NO.

25.

3. The antibody or antigen-binding fragment thereof that specifically binds to GAD according to claim 1, characterized in that, The antibody or its antigen-binding fragment, excluding the CDR region, is derived from one or more of the following species: rabbit, cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, human, and mutants thereof. Optionally, the heavy chain variable region further includes a skeleton region; Optionally, at least one backbone region of the heavy chain variable region contains an amino acid sequence consistent with the backbone region of the heavy chain variable region shown in SEQ ID NO.1; Optionally, the light chain variable region further includes a backbone region; Optionally, at least one backbone region of the light chain variable region contains an amino acid sequence consistent with the backbone region of the light chain variable region shown in SEQ ID NO.2; Optionally, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

2.

4. The antibody or antigen-binding fragment thereof that specifically binds to GAD according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment is a complete antibody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibody, or the smallest antibody recognition unit; Optionally, the antibody or its antigen-binding fragment is a monoclonal antibody; Optionally, the antibody or its antigen-binding fragment further includes a constant region; Optionally, at least a portion of the constant region sequence is a human constant region sequence; Optionally, the constant region sequence is selected from a portion or all of the constant region sequences of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Optionally, the antibody or its antigen-binding fragment contains a heavy chain constant region, the sequence of which is derived from the human heavy chain constant region; Optionally, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3; Optionally, the antibody or its antigen-binding fragment contains a light chain constant region; Optionally, the light chain constant region sequence is derived from the light chain constant region of a mouse; Optionally, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

4.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody or its antigen-binding fragment is a complete antibody, with the heavy chain amino acid sequence shown in SEQ ID NO.5 and the light chain amino acid sequence shown in SEQ ID NO.

6.

6. A biomaterial, characterized in that, It contains polynucleotides, carriers, cells, or conjugates; The polynucleotide encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is able to express the antibody or its antigen-binding fragment as described in any one of claims 1 to 5; The antibody conjugate is an antibody conjugate containing the antibody or its antigen-binding fragment as described in any one of claims 1 to 5.

7. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 1 to 5, characterized in that, Includes culturing cells as defined in claim 6; Optionally, the cell is prepared by converting a polynucleotide encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5 into the cell, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cell; Optionally, the cell is a eukaryotic cell, preferably a mammalian cell; Optionally, the mammalian cells are 293 cells or CHO cells, preferably 293F cells.

8. A composition, characterized in that, The composition comprises an antibody that specifically binds to GAD as described in any one of claims 1 to 5, or an antigen-binding fragment thereof, or the biological material as described in claim 6.

9. The composition according to claim 8, characterized in that, The composition is a reagent or kit; the reagent or kit is used to detect anti-GAD antibodies, or to diagnose and / or assist in the diagnosis of anti-GAD antibody-positive diseases; the kit contains standards and / or quality control products, the standards and / or the quality control products containing the antibody or its antigen-binding fragment; Optionally, the kit is used for the diagnosis and / or auxiliary diagnosis of at least one of type 1 diabetes, stiff-person syndrome, and autoimmune encephalitis; Optionally, the kit may further include a detection reagent for detecting at least one of anti-islet cell autoantibodies, anti-insulin autoantibodies, and anti-islet tumor-associated antigen-2 autoantibodies.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the biological material according to claim 6, or the composition according to claim 8, in any one of the following (I) to (VII): (I) Non-diagnostic and treatment-oriented detection of anti-GAD antibodies; (II) Preparation of products for detecting anti-GAD antibodies; (III) Prepare products for the diagnosis and / or auxiliary diagnosis of diseases with positive anti-GAD antibodies; (IV) Non-diagnostic and therapeutic target testing for GAD antigen; (V) Prepare products for detecting GAD antigen; (VI) Used for the isolation, enrichment and / or purification of GAD antigen; (VII) Prepare products for the isolation, enrichment and / or purification of GAD antigens; Optionally, the anti-GAD antibody-positive diseases include type 1 diabetes, stiff-person syndrome, and autoimmune encephalitis; Optionally, in any of (I) to (III), the antibody or its antigen-binding fragment is used as a standard or quality control.

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