Galactose-deficient IgA1-specific antibody or antigen-binding fragment thereof and related biomaterials and applications

Abstract

The present application discloses a galactose-deficient IgA1-specific antibody or its antigen-binding fragment and related biomaterials and applications, and belongs to the field of antibody technology. The main problem to be solved in this application is how to obtain a highly specific anti-Gd-IgA1 antibody. In order to solve the above technical problems, the present application provides an antibody or its antigen-binding fragment, which comprises a heavy chain variable region shown in positions 1-122 of SEQ ID NO:1 and a light chain variable region shown in positions 1 to 109 of SEQ ID NO:2. Compared with the existing antibody KM55, the obtained antibody has the advantage of high affinity with the antigen, and is expected to be more effectively used in the diagnosis of IgA nephropathy, monitoring of therapeutic efficacy, and judgment of prognosis.

Description

Technical Field

[0001] The present application belongs to the field of antibody technology, and specifically relates to galactose-deficient IgA1-specific antibodies or antigen-binding fragments thereof and related biomaterials and applications. Background Art

[0002] IgA nephropathy is the most common primary glomerular disease in the world and the most common cause of uremia in young and middle-aged people. In the past, immunosuppressive regimens based on supportive care and glucocorticoids have limited improvement in patient prognosis. In recent years, with the increasing understanding of the pathogenesis of IgA nephropathy, targeted treatments such as monoclonal antibodies, fusion proteins, and small molecule drugs that target B cells / plasma cells to reduce pathogenic IgA production have emerged one after another and have been approved for clinical use. At present, IgA nephropathy is undergoing a transition from traditional hormone immunosuppressive therapy to the era of targeted therapy. However, due to the lack of specific non-invasive diagnostic methods, compared with the rapid progress of targeted treatments for IgA nephropathy, its diagnosis still relies on renal puncture biopsy, and treatment and prognosis monitoring still rely on traditional clinical indicators such as proteinuria and renal function, which is in sharp contrast. The Kidney Disease: Improving Global Outcomes (KDIGO) guidelines point out that these traditional clinical pathological indicators are not developed based on pathogenesis or therapeutic targets, and therefore cannot be used to guide the selection of targeted therapeutic drugs or to judge early efficacy. Clinically, the wide variety of new therapeutic drugs for IgA nephropathy and their high costs have made it urgent to establish non-invasive evaluation methods for biomarkers based on pathogenesis and therapeutic targets to improve the diagnosis of IgA nephropathy, monitor treatment effects, and judge prognosis.

[0003] IgA nephropathy is characterized by the deposition of immune complexes mainly composed of IgA1 molecules in the kidneys as a typical pathological manifestation. In patients with IgA nephropathy, there is a lack of galactose modification in IgA1 molecules, and the formation of galactose-deficient IgA1 (Gd-IgA1) is a key link in the onset of IgA nephropathy. Based on this, the four-hit theory of the onset of IgA nephropathy was proposed: Gd-IgA1 increases (the first hit), leading to the production of anti-Gd-IgA1 antibodies (the second hit), the two form immune complexes that are deposited in the kidneys (the third hit), and the activation of complement and other inflammatory reactions damage the kidneys (the fourth hit). There is a hinge region composed of 19 amino acids between the heavy chain constant region 1 (CH1) and constant region 2 (CH2) of the human IgA1 molecule. It is a sequence composed of tandem repeats of proline (Pro), threonine (Thr), and serine (Ser). Each hinge region contains 6 Ser / Thr potential O-glycosylation modification sites (asterisk positions) VPST*PPT*PS*PS*T*PPT*PSPS. Under normal circumstances, the O-sugar chain core sugar N-acetylgalactosamine (GalNAc) of the IgA1 molecule is linked to the Ser / Thr site under the catalysis of N-acetylgalactosaminyltransferase (GalNAcTs). On this basis, galactose (Gal) can be linked to GalNAc through a β1,3 bond, and sialic acid (SA) can be linked to GalNAc or Gal through an α2,6 bond or an α2,3 bond, thus forming six different glycoform structures. In the peripheral blood of patients with IgA nephropathy, there is a lack of galactose modification in the hinge region of the IgA1 molecule, which in turn exposes O-GalNAc (Tn antigen) to form Gd-IgA1. Gd-IgA1 is significantly increased in the peripheral blood of patients with IgA nephropathy and is an independent risk factor affecting the prognosis of patients. The reduction of Gd-IgA1 level is closely related to the treatment response and is an important non-invasive diagnostic and therapeutic marker for IgA nephropathy.

[0004] However, the detection of Gd-IgA1 in the past has many difficulties, such as limited promotion of mass spectrometry detection of large samples, poor antigen-antibody binding ability of snail lectin HAA (which can recognize and bind to Tn antigen) method, and large batch effect of natural lectin. The development of highly specific anti-Gd-IgA1 antibodies has always been a core issue that has attracted much attention in the field of IgA nephropathy research. In the past, Japanese scholars Yasutake J and others immunized rats and used hybridoma cell technology to screen and obtain an IgG2b type mouse monoclonal anti-Gd-IgA1 antibody KM55. However, KM55 is only a reagent product for scientific research, and there are problems such as high overlap rate of detection values ​​between patients and healthy controls and poor binding ability with antigens. At present, there is still a lack of detection methods for high specificity and high sensitivity. With the development of antibody research and development technology, single B cell sequencing has shown great advantages and potential in antibody discovery and clone screening, providing new ideas for the development of anti-Gd-IgA1 monoclonal antibodies. Summary of the invention

[0005] The main problem to be solved by this application is how to obtain highly specific anti-Gd-IgA1 antibodies. To solve the above technical problems, this application provides the following technical solutions:

[0006] The present application provides an antibody or an antigen-binding fragment thereof, which comprises three heavy chain variable region complementarity determining regions (CDRs) named HCDR1, HCDR2 and HCDR3, and three light chain variable region complementarity determining regions named LCDR1, LCDR2 and LCDR3; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region comprise positions 26 to 33, positions 51 to 58 and positions 97 to 111 of SEQ ID NO:1, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody comprise positions 26 to 34, positions 52 to 54 and positions 91 to 99 of SEQ ID NO:2, respectively.

[0007] In this application, the complementarity determining regions (CDRs) are defined based on the IMGT numbering scheme.

[0008] The antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof that binds to the Tn5 antigen.

[0009] The Tn5 antigen is a compound of formula 1:

[0010] VPST*PPT*PS*PS*TPPT*PSPS-NH2 formula 1,

[0011] In formula 1, VPST represents the amino acid residues of valine, proline, serine and threonine respectively, * is the group of formula 2, the group of formula 2 is connected to the amino acid residue before *, the amino acid residue before * is a serine residue or a threonine residue, the H in the -OH in the serine residue or threonine residue before * is replaced by the group of formula 2, and the wavy line in formula 2 indicates the position connected to the amino acid residue.

[0012]

[0013] The antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof that binds to Gd-IgA1.

[0014] In the antibody or antigen-binding fragment thereof, the binding may be specific binding.

[0015] The specific binding refers to the affinity of one molecule binding to another molecule that is significantly higher than the affinity of binding to any cross-reactive antigen or off-target antigen (collectively referred to as non-target antigen). The affinity is determined using experimental techniques such as surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, kinetic exclusion assay (KinExA), isothermal titration calorimetry (ITC), radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, the specific or selective reaction will be at least 1.5 times the non-target signal or non-target binding noise.

[0016] In the above antibodies or antigen-binding fragments thereof, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are positions 26 to 33, positions 51 to 58, and positions 97 to 111 of SEQ ID NO: 1, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are positions 26 to 34, positions 52 to 54, and positions 91 to 99 of SEQ ID NO: 2, respectively.

[0017] Furthermore, the antibody or antigen-binding fragment thereof further comprises four heavy chain variable region framework regions (FR) named HFR1, HFR2, HFR3 and HFR4, and four light chain variable region framework regions named LFR1, LFR2, LFR3 and LFR4, respectively. In the antibody, HFR1 comprises an amino acid sequence selected from the following:

[0018] a111) positions 1 to 25 of SEQ ID NO: 1,

[0019] a112) has a sequence with more than 75% identity with a111);

[0020] HFR2 comprises an amino acid sequence selected from the group consisting of:

[0021] a121) 34 to 50 of SEQ ID NO: 1,

[0022] a122) has a sequence with more than 75% identity with a121);

[0023] HFR3 comprises an amino acid sequence selected from the group consisting of:

[0024] a131) positions 59 to 96 of SEQ ID NO: 1,

[0025] a132) has a sequence with more than 75% identity with a131);

[0026] HFR4 comprises an amino acid sequence selected from the group consisting of:

[0027] a141) positions 112 to 122 of SEQ ID NO: 1,

[0028] a142) has a sequence with more than 75% identity with a141);

[0029] The amino acid sequence of LFR1 includes an amino acid sequence selected from the group consisting of:

[0030] a151) positions 1 to 25 of SEQ ID NO: 2,

[0031] a152) has a sequence with more than 75% identity with a151);

[0032] The amino acid sequence of LFR2 includes an amino acid sequence selected from the group consisting of:

[0033] a161) positions 35 to 51 of SEQ ID NO: 2,

[0034] a162) has a sequence with more than 75% identity with a151);

[0035] The amino acid sequence of LFR3 includes an amino acid sequence selected from the group consisting of:

[0036] a171) positions 55 to 90 of SEQ ID NO: 2,

[0037] a172) has a sequence with more than 75% identity with a171);

[0038] The amino acid sequence of LFR4 includes an amino acid sequence selected from the group consisting of:

[0039] a181) positions 100 to 109 of SEQ ID NO: 2,

[0040] a182) has a sequence identity of more than 75% with a181).

[0041] In the present application, the light chain variable region (VL or VL domain) or the heavy chain variable region (VH or VH domain) of the antibody consists of three CDRs and a "framework region" separated by three CDRs. The framework region is used to align the CDRs that specifically bind to antigen epitopes. CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The VL domain and the VH domain both contain the following framework regions (FRs) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0042] Herein, the identity may specifically be 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% or 100% identity.

[0043] The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequence of the heavy chain variable region comprises positions 1-122 of SEQ ID NO:1, or has at least 75% identity with positions 1-122 of SEQ ID NO:1; and / or the amino acid sequence of the light chain variable region comprises positions 1-109 of SEQ ID NO:2, or has at least 75% identity with positions 1-109 of SEQ ID NO:2.

[0044] In the antibody of the above antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, positions 1-122; and / or the amino acid sequence of the light chain variable region is SEQ ID NO: 2, positions 1-109.

[0045] In the above-mentioned antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof may further include a constant region.

[0046] In the antibody or antigen-binding fragment thereof, the constant region may be a heavy chain constant region and a light chain constant region.

[0047] In the antibody or antigen-binding fragment thereof, the heavy chain constant region may be IgG, IgM, or IgA.

[0048] In the antibody or antigen-binding fragment thereof, the IgG may be IgG1, IgG2, IgG3 or IgG4.

[0049] In the antibody or antigen-binding fragment thereof, the light chain constant region may be a lamda (λ) constant region or a kappa (κ) constant region.

[0050] The antibody or antigen-binding fragment thereof may be murine, human, chimeric or humanized.

[0051] The term "antibody" in this application is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL) and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0052] The term "antigen binding fragment" refers to an antigen binding fragment of an antibody, which generally includes at least a portion of the antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antigen binding fragment retains at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parental antibody to the target.

[0053] The antigen binding fragments include Fab, Fab' fragments, F(ab')2 fragments, nanobodies, minimal recognition units (MRU), Fv antibodies, single-chain antibodies and heavy-chain antibodies.

[0054] The term "Fab" is an antigen binding fragment (Fab), which is composed of a complete antibody light chain and the VH (heavy chain variable region) and CH1 (heavy chain constant region 1) domains of the heavy chain. That is, it is a heterodimer formed by the heavy chain Fd and the complete light chain of the antibody bound by disulfide bonds.

[0055] The term "Fab' fragment" contains a complete antibody light chain and a portion of an antibody heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains. Thus, an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0056] The term "F(ab')2 fragment" consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0057] The term "nanoantibody" refers to a heavy chain single-domain antibody VHH (variable domain of heavy chain ofheavy-chain antibody), which is a protein composed of the variable region of the antibody heavy chain.

[0058] The term "minimum recognition unit (MRU)" refers to a single CDR structure in the variable region, with a molecular mass of only about 1% of the complete antibody, which can bind to the corresponding antigen.

[0059] The term "Fv antibody" is a protein consisting only of the heavy chain variable region and the light chain variable region of an antibody. The heavy chain variable region and the light chain variable region are linked by a non-covalent bond.

[0060] The term "single-chain antibody" (ScFv) refers to a protein composed of a short peptide linking the heavy chain variable region and the light chain variable region of an antibody.

[0061] The term "heavy chain antibody" (HCAb) refers to a protein consisting of only two heavy chains, without the presence of light chains, and without a CH1 region between the heavy chain variable region and the hinge region.

[0062] The present application also provides a biological material: the biological material is any of the following:

[0063] (A1) a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof,

[0064] (A2) an expression cassette containing the nucleic acid molecule described in (A1),

[0065] (A3) a recombinant vector containing the nucleic acid molecule described in (A1),

[0066] (A4) a recombinant cell containing the nucleic acid molecule described in (A1),

[0067] (A5) A recombinant microorganism containing the nucleic acid molecule described in (A1).

[0068] In the above biological material, the nucleic acid molecule described in (A1) can be any of the following:

[0069] A nucleic acid molecule encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody, wherein the nucleic acid molecule comprises positions 891 to 914, 966 to 989 and 1104 to 1148 from the 5' end of SEQ ID NO:3, respectively;

[0070] And / or, nucleic acid molecules encoding LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody, the nucleic acid molecules respectively comprise positions 133 to 159, 211 to 219, and 328 to 354 from the 5' end of SEQ ID NO:4.

[0071] In the above biological material, the nucleic acid molecule described in (A1) can be any of the following:

[0072] Nucleic acid molecules encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody, wherein the nucleic acid molecules are respectively positions 891 to 914, 966 to 989 and 1104 to 1148 from the 5' end of SEQ ID NO:3;

[0073] And / or, nucleic acid molecules encoding LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody, wherein the nucleic acid molecules are positions 133 to 159, 211 to 219 and 328 to 354 from the 5' end of SEQ ID NO: 4, respectively.

[0074] In the above biological material, the nucleic acid molecule described in (A1) can also be any of the following:

[0075] A nucleic acid molecule encoding the heavy chain variable region of the antibody, the nucleic acid molecule comprising positions 816 to 1181 of SEQ ID NO: 3;

[0076] And / or, a nucleic acid molecule encoding the light chain variable region of the antibody, wherein the nucleic acid molecule comprises positions 97 to 423 of SEQ ID NO:4.

[0077] In the above biological material, the nucleic acid molecule described in (A1) can also be any of the following:

[0078] A nucleic acid molecule encoding the heavy chain variable region of the antibody, the nucleic acid molecule being SEQ ID NO: 3, positions 816 to 1181;

[0079] And / or, a nucleic acid molecule encoding the light chain variable region of the antibody, wherein the nucleic acid molecule is positions 97 to 423 of SEQ ID NO:4.

[0080] In the above biological material, the nucleic acid molecule described in (A1) can also be any of the following:

[0081] A nucleic acid molecule encoding the heavy chain of the antibody, the nucleic acid molecule comprising positions 816 to 2156 of SEQ ID NO: 3;

[0082] And / or, a nucleic acid molecule encoding the light chain of the antibody, wherein the nucleic acid molecule comprises SEQ ID NO:4.

[0083] In the above biological material, the nucleic acid molecule described in (A1) can also be any of the following:

[0084] A nucleic acid molecule encoding the heavy chain of the antibody, the nucleic acid molecule being SEQ ID NO: 3, positions 816 to 2156;

[0085] And / or, a nucleic acid molecule encoding the light chain of the antibody, wherein the nucleic acid molecule is SEQ ID NO:4.

[0086] In the above biological materials, the nucleic acid molecule (A1) may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0087] In the above biological materials, the expression cassette (A2) refers to a DNA capable of expressing the antibody in a host cell, which may include not only a promoter for initiating transcription of the antibody encoding gene, but also a terminator for terminating transcription of the antibody encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0088] Existing expression vectors can be used to construct a recombinant vector containing the expression cassette.

[0089] In the above biological materials, the vector may be a plasmid, a cosmid, a phage or a virus vector.

[0090] In the above-mentioned biomaterial, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule described in (A1) into the antibody protein expression vector pcDNA3.4.

[0091] In a specific embodiment, taking the antibody as an example, a heavy chain expression vector pcDNA3.4-H and a light chain expression vector pcDNA3.4-L were constructed.

[0092] The full sequence of the pcDNA3.4-H is as shown in SEQ ID NO: 3. SEQ ID NO: 3 has CMVenhancer at positions 50 to 429, CMV promoter at positions 430 to 633, signal peptide coding genes at positions 759 to 815, heavy chain variable region (VH) coding genes at positions 816 to 1181, and heavy chain constant region coding genes at positions 1182 to 2156. pcDNA3.4-H can express the heavy chain of the antibody, and its amino acid sequence is as shown in SEQ ID NO: 1. SEQ ID NO: 1 has amino acid sequences of variable regions at positions 1 to 122, and constant regions at positions 123 to 446.

[0093] The structure of the pcDNA3.4-L is as follows: the sequence between positions 759 to 2156 of pcDNA3.4-H (i.e., SEQ ID NO:3) is replaced with the DNA fragment shown in SEQ ID NO:4, and the other sequences of pcDNA3.4-H are kept unchanged to obtain a recombinant vector. Positions 1 to 57 of SEQ ID NO:4 are the coding genes for the signal peptide, positions 58 to 384 are the coding genes for the light chain variable region (VL), and positions 385 to 705 are the coding genes for the light chain constant region. pcDNA3.4-L can express the light chain of the antibody, and its amino acid sequence is SEQ ID NO:2. Positions 1 to 109 of SEQ ID NO:2 are the amino acid sequences of the variable region, and positions 110 to 215 are the amino acid sequences of the constant region.

[0094] In the above biological materials, the microorganism can be bacteria (such as Escherichia coli), yeast, algae or fungi.

[0095] The full-length nucleotide sequence of the antibody described in the present application or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For example, the relevant sequence is synthesized by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them.

[0096] The present application also provides a method for preparing an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is the antibody or the antigen-binding fragment thereof, and the method comprises the step of expressing the coding gene of the antibody or the antigen-binding fragment thereof in a mammalian cell to obtain the antibody or the antigen-binding fragment thereof.

[0097] In the above method, the mammalian cell can be an isolated animal cell, such as a cell in a culture medium. The mammalian cell can be selected from ExpiCHO-S™ cells, Chinese hamster ovary cells (CHO), 293F cells, 293E cells, 293-6E cells, etc.

[0098] In the above method, expressing the coding gene of the antibody or antigen-binding fragment thereof in mammalian cells comprises infecting, transfecting or transforming the coding gene into a mammalian cell to obtain a recombinant mammalian cell containing the coding gene, culturing the recombinant mammalian cell to obtain a cell culture, and isolating and purifying the antibody or antigen-binding fragment thereof from the cell culture. The antibody or antigen-binding fragment thereof can be produced from a large-scale cell culture by methods known in the art. For example, Li et al., Cell culture processes for monoclonal antibody production. Mabs. 2010 September-October; 2 (5): 466-477. In certain embodiments, the mammalian cell is a Chinese hamster ovary cell (CHO) cell, a NS0 mouse myeloma cell, a HEK293 (human embryonic kidney 293) cell or a PER.C6 ® cell.

[0099] The present application also provides a reagent or a kit, wherein the reagent or the kit contains the antibody or the antigen-binding fragment thereof, and the reagent or the kit has at least one of the following uses:

[0100] (B1) Recognize or assist in the recognition of Gd-IgA1;

[0101] (B2) binding or assisting in binding to Gd-IgA1;

[0102] (B3) Recognize or assist in recognizing Tn antigen;

[0103] (B4) bind or assist in binding to Tn antigen;

[0104] (B5) detecting or assisting in detecting whether the sample to be tested contains Gd-IgA1;

[0105] (B6) detecting or assisting in detecting the content of Gd-IgA1 in the sample to be tested;

[0106] (B7) Detecting or assisting in detecting whether the sample to be tested contains Tn antigen;

[0107] (B8) detecting or assisting in detecting the content of Tn antigen in the sample to be tested;

[0108] (B9) Identify or assist in identifying IgA nephropathy;

[0109] (B10) targeting Gd-IgA1;

[0110] (B11) targeting Tn antigen;

[0111] (B12) Diagnosis of diseases related to Gd-IgA1;

[0112] (B13) Diagnosis of diseases related to Tn antigen.

[0113] The present application also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof.

[0114] The above-mentioned pharmaceutical composition can be a drug for treating diseases related to Gd-IgA1, and can also be a drug for treating diseases related to Tn antigen.

[0115] The present application also provides an application of an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is the antibody or the antigen-binding fragment thereof, and the application is an application in any of the following:

[0116] (C1) Recognize or assist in recognizing Gd-IgA1;

[0117] (C2) binds or assists in binding to Gd-IgA1;

[0118] (C3) Recognize or assist in recognizing Tn antigen;

[0119] (C4) binds or assists in binding to Tn antigen;

[0120] (C5) detecting or assisting in detecting whether the sample to be tested contains Gd-IgA1;

[0121] (C6) preparing products for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1;

[0122] (C7) detecting or assisting in detecting the content of Gd-IgA1 in the sample to be tested;

[0123] (C8) preparing a product for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be tested;

[0124] (C9) Detecting or assisting in detecting whether the sample to be tested contains Tn antigen;

[0125] (C10) Preparing products for detecting or assisting in detecting whether a sample to be tested contains Tn antigen;

[0126] (C11) detecting or assisting in detecting the content of Tn antigen in a sample to be tested;

[0127] (C12) preparing products for detecting or assisting in detecting the content of Tn antigen in a sample to be tested;

[0128] (C13) Identify or assist in identifying IgA nephropathy;

[0129] (C14) Preparation of products for identifying or assisting in the identification of IgA nephropathy;

[0130] (C15) preparing a product targeting Gd-IgA1;

[0131] (C16) preparing products targeting Tn antigen;

[0132] (C17) Preparation of products for diagnosis or treatment of diseases related to Gd-IgA1;

[0133] (C18) Preparation of products for diagnosis or treatment of diseases associated with Tn antigen.

[0134] The product may be a reagent, a kit, a drug or a medicament.

[0135] The present application also provides the application of the above-mentioned biomaterial, which is an application in any of the following:

[0136] (P1) preparing products for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1;

[0137] (P2) preparing products for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be tested;

[0138] (P3) Preparation of products for detecting or assisting in detecting whether a sample contains Tn antigen;

[0139] (P4) preparing products for detecting or assisting in detecting the content of Tn antigen in a sample to be tested;

[0140] (P5) Preparation of products for identifying or assisting in the identification of IgA nephropathy;

[0141] (P6) Preparation of products targeting Gd-IgA1;

[0142] (P7) Preparation of products for diagnosis or treatment of diseases related to Gd-IgA1;

[0143] (P8) Preparation of products targeting Tn antigen;

[0144] (P9) Preparation of products for diagnosis or treatment of diseases associated with Tn antigen.

[0145] In the above applications, IgA nephropathy is usually manifested as an increase in Gd-IgA1. Gd-IgA1 refers to the absence of galactose modification in the hinge region of the IgA1 molecule, which exposes O-GalNAc (Tn antigen).

[0146] Compared with the existing antibody KM55, the antibody provided in the present application is cloned using single B cell technology after immunizing mice. It has the advantages of high affinity with the antigen and improved patient diagnostic ability. The obtained antibody is expected to be more effectively used for the diagnosis of IgA nephropathy, monitoring of treatment efficacy and judgment of prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] Figure 1 The EC50 curves of the screened monoclonal antibodies and KM55 binding to Tn0 or Tn5, wherein A is the result of the screened monoclonal antibody B0033, B is the result of KM55, and C is the EC50 value of binding to Tn5. Compared with the existing antibody KM55, the monoclonal antibody obtained in this application can effectively distinguish between the two antigens Tn0 and Tn5, and has a higher binding ability to Tn5 than KM55.

[0148] Figure 2 The ELISA results of the screened monoclonal antibodies and KM55 binding to Gd-IgA1 / IgA1 are shown in Figure 1, where A is the result of the screened monoclonal antibody B0033 and B is the result of KM55. Compared with the existing antibody KM55, the monoclonal antibody obtained in this application has a stronger binding force with Gd-IgA1 and IgA1 molecules and can more effectively distinguish them.

[0149] Figure 3 The ELISA results of the screened monoclonal antibodies for detecting IgA1 and Gd-IgA1 are shown in Figure 1, where A is the result of the screened monoclonal antibody B0033 and B is the result of KM55. The results show that compared with the existing antibody KM55, the monoclonal antibody obtained in this application can more effectively distinguish IgA1 and Gd-IgA1 molecules.

[0150] Figure 4 The results are compared between the screened monoclonal antibody and KM55 in detecting Gd-IgA1 in IgA nephropathy patients and healthy controls, where A is the result of the screened monoclonal antibody B0033 and B is the result of KM55. Compared with the existing antibody KM55, the plasma Gd-IgA1 level detected by the monoclonal antibody obtained in this application is significantly higher than that of KM55.

[0151] Figure 5 This is the diagnostic curve of the screened monoclonal antibodies for IgA nephropathy patients.

[0152] Figure 6 This is the diagnostic curve of KM55 for patients with IgA nephropathy. DETAILED DESCRIPTION

[0153] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0154] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0155] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0156] The information of some experimental materials in the following examples is as follows: FITC anti-mouse CD19 (115506, Biolegend), PE / Cyanine7 anti-mouse IgM Antibody (406514, Biolegend), PE / Cyanine7anti-mouse IgD Antibody (405720, Biolegend), Bio-Tn5 (Shanghai Aminlian Biotechnology Co., Ltd.), and PE-streptavidin (405204, BioLegend).

[0157] Example 1. Obtaining a monoclonal antibody that specifically recognizes Gd-IgA1

[0158] 1. Design of Tn antigen in Gd-IgA1 molecule

[0159] GalNAc was added to the 5 serine and threonine sites where galactose was most frequently missing from the 19-amino acid peptide in the hinge region of IgA1 to form a high-density Tn antigen (hereinafter referred to as Tn5 antigen) as an immunization antigen, sorting antigen, and detection antigen.

[0160] The Tn5 antigen is a compound of formula 1:

[0161] VPST*PPT*PS*PS*TPPT*PSPS-NH2 formula 1,

[0162] In Formula 1, VPST represents amino acid residues of valine, proline, serine, and threonine, respectively, and * represents a group of Formula 2. The group of Formula 2 is connected to the amino acid residue before *, the amino acid residue before * is a serine residue or a threonine residue, the H in -OH in the serine residue or threonine residue before * is replaced by the group of Formula 2, and the wavy line in Formula 2 indicates the position connected to the amino acid residue.

[0163]

[0164] 2. Immunization of mice with Tn antigen

[0165] Tn5 was coupled to the carrier protein KLH (Shanghai Aminlian Biotechnology Co., Ltd.) and combined with adjuvant (CFA, sigma, F5881) / incomplete Freund's adjuvant (IFA, sigma, F5506) to 6-7 week old BALB / c female mice for 4 immunizations at intervals of two weeks. Specifically: Tn5-KLH (100μg / 100μL) + CFA (100μL), Tn5-KLH (100μg / 100μL) + IFA (100μL), Tn5-KLH (100μg / 100μL) + IFA (100μL), Tn5-KLH (100μg / 100μL) + IFA (100μL). The mice were killed and the spleen was taken out 3 days after the immunization.

[0166] 3. Isolation of single B cells bound to Tn5 from mouse spleen cells

[0167] Mouse spleen was ground and filtered through a 70 mm filter to obtain a single cell suspension. B cells were selected with FITC anti-mouse CD19 (115506, Biolegend), PE / Cyanine7 anti-mouse IgM Antibody (406514, Biolegend), and PE / Cyanine7 anti-mouse IgD Antibody (405720, Biolegend); single B cells bound to Tn5 were sorted with Bio-Tn5 (Shanghai Aminlian Biotechnology Co., Ltd.) and PE-streptavidin (405204, BioLegend).

[0168] 4. Antibody Sequencing

[0169] Single B cells obtained by lysis are used to obtain RNA; RNA is used as a template for reverse transcription to obtain cDNA; cDNA is used as a template and heavy chain / light chain variable region gene primers are used for PCR, and the V region gene sequence is obtained by sequencing. The amino acid residue sequence of the heavy chain / light chain variable region is obtained after the gene sequence is translated. The complementarity determining regions (CDRs) of the heavy chain variable region and the light chain variable region are defined according to the IMGT numbering scheme. The heavy chain variable region (VH) or light chain variable region (VL) of an antibody consists of a framework region separated by three complementarity determining regions. The framework region is used to align the CDRs that specifically bind to the antigen epitope. The CDR includes the amino acid residues in the antibody that are primarily responsible for antigen binding. The VH domain and the VL domain both contain the following framework regions (FRs) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2 and CDR3 of the VH domain are also referred to as HCDR1, HCDR2 and HCDR3, respectively, herein; the FR1, FR2, FR3 and FR4 of the VH domain are also referred to as HFR1, HFR2, HFR3 and HFR4, respectively, herein; the CDR1, CDR2 and CDR3 of the VL domain are also referred to as LCDR1, LCDR2 and LCDR3, respectively, herein; the FR1, FR2, FR3 and FR4 of the VL domain are also referred to as LFR1, LFR2, LFR3 and LFR4, respectively, herein.

[0170]

[0171] 5. Construction of recombinant expression vector

[0172] The heavy chain variable region gene of the monoclonal antibody obtained in step 4 is constructed between the signal peptide and the mouse IgG1 constant region of the modified pcDNA3.4 vector to obtain a recombinant expression vector pcDNA3.4-H. The full sequence of the recombinant expression vector pcDNA3.4-H is as shown in SEQ ID NO: 3. SEQ ID NO: 3 is CMV enhancer at positions 50 to 429, CMV promoter at positions 430 to 633, signal peptide coding genes at positions 759 to 815, heavy chain variable region (VH) coding genes at positions 816 to 1181, and heavy chain constant region (i.e., mouse IgG1 constant region) coding genes at positions 1182 to 2156. The pcDNA3.4-H vector can express the heavy chain of the antibody, and its amino acid sequence is SEQ ID NO: 1. SEQ ID NO: 1 is amino acid sequence of the variable region at positions 1 to 122, and amino acid sequence of the constant region (i.e., mouse heavy chain IgG1 constant region) at positions 123 to 446.

[0173] The monoclonal antibody light chain variable region gene obtained in step 4 is constructed between the signal peptide and the mouse light chain lambda constant region of the modified pcDNA3.4 vector to obtain a recombinant expression vector pcDNA3.4-L. The structure of the light chain expression vector pcDNA3.4-L is as follows: the sequence between positions 759 to 2156 of pcDNA3.4-H (i.e., SEQ ID NO:3) is replaced with the DNA fragment shown in SEQ ID NO:4. Positions 1 to 57 of SEQ ID NO:4 are the coding genes for the signal peptide, positions 58 to 384 are the coding genes for the light chain variable region, and positions 385 to 705 are the coding genes for the light chain constant region (i.e., the mouse light chain lambda constant region). The pcDNA3.4-L vector can express the light chain of the antibody, and its amino acid sequence is SEQ ID NO:2. Positions 1 to 109 of SEQ ID NO:2 are the amino acid sequences of the variable region, and positions 110 to 215 are the amino acid sequences of the constant region (i.e., the mouse light chain lambda constant region).

[0174]

[0175] 6. Antibody expression in vitro

[0176] ExpiCHO-S™ cells (ThermoFisher, Cat. No. A29127) were diluted to 6 × 10 6 Cell / mL. Aliquot 2.5 mL of cells per well of a 24-well deep-well plate for transfection.

[0177] Add 200 μL of ExpiFectamine reagent-DNA complex to each well containing culture in a 24-well deep-well plate, and cover the reaction plate with a gas-permeable sealing film. The ExpiFectamine reagent-DNA complex is prepared by mixing the heavy chain and light chain plasmids constructed in step 5 in a 1:1 ratio, 9.0 μL of ExpiFectamine CHO reagent (Gibco, A29129), and 189 μL of OptiPRO SFM (Gibco, 12309-050).

[0178] Incubate the 24-well deep-well plate on an orbital shaker at 37°C with 8% CO2 in a humidified atmosphere. Add ExpiFectamine CHO Enhancer (Gibco, A29129) and ExpiCHO Supplement (Gibco, A29129) 18-22 hours after transfection. For the entire 24-well deep-well plate, mix 400 μL of ExpiFectamine CHO Enhancer and 16 mL of ExpiCHO Supplement in a conical tube. Add 600 μL of this mixture to each well of the 24-well deep-well plate. Return the 24-well deep-well plate to a 37°C incubator with 8% CO2 and shake. On day 8 after transfection, centrifuge the plate (500 g, 10 min) and collect the culture supernatant for antibody purification.

[0179] 7. Antibody purification and detection

[0180] Protein A beads (GenScript, L00695) for purification were added to the expression supernatant (400 μL magnetic beads / 5 mg protein) in proportion and incubated for 2 h. After incubation, the beads were transferred to a 24-well deep-well plate (BIOLAND, SBS24-10-V) and placed in a Kingfisher flex fully automatic nucleic acid extractor to perform the purification procedure (2 PBS washes, 1 distilled water wash, 1 Elution elution of the antibody). Tris reagent was added to the Elution reagent containing the antibody for pH neutralization (62.6 μL Tris reagent / 1 mL Elution reagent). After centrifugation (3000 g, 5 min), the supernatant was taken to obtain the purified antibody solution, i.e., monoclonal antibody B0033, which was used for subsequent experiments.

[0181] Example 2: Antibody Binding Ability Test

[0182] 1. Binding specificity of ELISA antibody to Tn5

[0183] Antigen Tn5 and control antigen Tn0 (Tn0 is the same amino acid sequence as Tn5 without GalNAc modification) were coated at a concentration of 1 μg / mL (4°C, overnight), and blocked at 37°C for 2 hours with 5% casein sodium salt (Biyuntian, ST1132-100g). The antibody solution obtained in Example 1 (containing monoclonal antibody B0033) was added as the primary antibody, and a total of 10 concentrations of primary antibody were set (starting concentration 3 μg / mL, 3-fold dilution). The commercial KM55 antibody (IBL, #10777) was used as the positive control antibody. Goat anti-mouse IgG-HRP was used as the secondary antibody for detection (Invitrogen, 1:1000, G21040, 1:1000 dilution), rabbit anti-rat IgG (h+l)-HRP (Immunoway, RS030226, 1:10000 dilution) was used as the secondary antibody for detection of KM55, TMB color development (Solebold, PR1200), termination reaction (Solebold, C1058), and absorbance value (OD value) at 450nm was read. GraphPad Prism statistical software was used to process the data, and the experimental results of antibody binding to Tn5 and Tn0 were fitted with curves and EC50 was calculated.

[0184] The results are as follows Figure 1 As shown, the results show that compared with the existing antibody KM55, the monoclonal antibody obtained in the present application binds to Tn5 but not to Tn0. Compared with KM55, B0033 has a higher binding affinity to Tn5, wherein the EC50 of B0033 binding to Tn5 is 0.0225 μg / mL, and the EC50 of KM55 binding to Tn5 is 19.5400 μg / mL.

[0185] 2. Binding to Gd-IgA1

[0186] Gd-IgA1 was prepared as follows: commercial human IgA1 (Abcam, ab91020) was purchased and enzymatically digested in vitro with 4000U α2-3,6,8 neuraminidase (P0720L, New England Biolabs) and 400U β1-3 galactosidase (P0726S, New England Biolabs) at pH 7.4 and 37°C for 2 hours to obtain Gd-IgA1; IgA1 from the same batch without enzymatic digestion was used as a control.

[0187] The prepared Gd-IgA1 and commercial IgA1 were coated at a concentration of 3 μg / mL (4°C, overnight), blocked with 5% BSA (Solebol, A8020) at 37°C for 2 hours, and then the antibody solution obtained in Example 1 (containing monoclonal antibody B0033) was added as the primary antibody. The primary antibody was treated with 6 concentrations (initial concentration 9 μg / mL, 3-fold dilution). The commercial KM55 antibody (IBL, #10777) was used as the positive control antibody. Goat anti-mouse IgG-HRP was used as the secondary antibody for detection (Invitrogen, 1:1000, G21040, 1:1000 dilution), and rabbit anti-rat IgG (h+l)-HRP (Immunoway, RS030226, 1:10000 dilution) was used as the secondary antibody for detection of KM55. TMB color development (Solabo, PR1200) was used, and the reaction was terminated (Solabo, C1058). The absorbance value (OD value) at 450 nm was read.

[0188] The results are as follows Figure 2 As shown, the results show that the monoclonal antibody obtained in the present application can specifically bind to Gd-IgA1. Compared with the existing antibody KM55, the monoclonal antibody obtained in the present application has a higher degree of discrimination between IgA1 and Gd-IgA1.

[0189] 3. ELISA detection of Gd-IgA1

[0190] The monoclonal antibodies B0033 and KM55 were coated at a concentration of 2 μg / mL, respectively. After blocking with 5% BSA (Solebol, A8020) at 37 degrees for 2 hours, the primary antibodies were added with gradient dilutions (5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, and 0.3125 μg / mL) of IgA1 or Gd-IgA1, and goat anti-human IgA1-HRP was used as the secondary antibody for detection (abcam, AB97215, diluted 1:1000 when used). TMB was used for color development (Solebol, PR1200), and the reaction was terminated with ELISA stop solution (Solebol, C1058), and the absorbance at 450 nm (OD450) was read.

[0191] The results are as follows Figure 3 As shown, the results show that compared with the existing antibody KM55, the monoclonal antibody obtained in the present application can more effectively distinguish IgA1 and Gd-IgA1 molecules.

[0192] Example 3: Antibody diagnostic capability test

[0193] The plasma samples of IgA nephropathy patients or healthy controls were collected prospectively at Peking University First Hospital, and the project was approved by the Ethics Committee of Peking University First Hospital. All subjects signed informed consent forms;

[0194] The criteria for IgA nephropathy patients were IgA nephropathy confirmed by renal puncture biopsy at Peking University First Hospital. The inclusion criteria were: ① Immunofluorescence of renal puncture biopsy showed mainly IgA deposition; ② Immunoelectron microscopy showed immune complex deposition in the mesangial area; ③ No glucocorticoids or immunosuppressants were taken for at least half a year.

[0195] The standard for healthy controls was healthy controls matched in age, gender, and region. The inclusion criteria were: ① no history of kidney disease; ② no abnormality in renal function in biochemical examinations; ③ no nephritis symptoms such as hematuria and proteinuria in urine examinations.

[0196] There were 115 plasma samples from patients with IgA nephropathy.

[0197] Plasma samples from 124 healthy controls.

[0198] 1. Detection of Gd-IgA1 in plasma

[0199] Monoclonal antibody B0033 was coated at a concentration of 2 μg / mL (4°C, overnight) and blocked with 5% BSA (Solebol, A8020) at 37°C for 2 hours. Then, plasma samples of IgA nephropathy patients or healthy controls were added at a 400-fold dilution (diluted with PBS). Goat anti-human IgA1-HRP was used as the secondary antibody for detection (abcam, AB97215, diluted 1:1000 when used), TMB was used for color development (Solebol, PR1200), and the ELISA stop solution was used to stop the reaction (Solebol, C1058), and the absorbance at 450nm (OD450) was read. KM55 was used as the positive control antibody, and the method was the same as B0033. GraphPad Prism statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation, and the t-test was used; (*) represents P < 0.05 level, indicating that the difference is significant, (**) represents P < 0.01 level, (***) represents P < 0.001 level, and (****) represents P < 0.0001 level, indicating that the difference is extremely significant.

[0200] The results are as follows Figure 4 As shown, the results showed that the detection value of Gd-IgA1 in the plasma of IgA nephropathy patients by monoclonal antibody B0033 was significantly higher than that of healthy controls, and compared with the existing antibody KM55, the detection OD450 value of Gd-IgA1 was significantly improved.

[0201] 2. Diagnostic value for IgA nephropathy

[0202] 2.1 Diagnosis of IgA nephropathy using monoclonal antibody B0033

[0203] The OD values ​​of plasma samples from healthy controls were used as controls (negative), and the OD values ​​of plasma samples from patients with IgA nephropathy were analyzed by ROC curve analysis using SPSS 16.0 software. Figure 5 The area under the curve of B0033 is AUC = 0.7902. The optimal cut-off value is 0.2219 when the Youden index (sensitivity + specificity - 1) is the largest (sensitivity is 80%, specificity is 67.74%, Youden index is 0.4774). Based on this, patients with OD values ​​greater than or equal to 0.2219 are considered to be IgA nephropathy patients (positive), and patients with OD values ​​less than 0.2219 are considered to be non-IgA nephropathy patients (negative).

[0204] 2.2 KM55 antibody diagnosis of IgA nephropathy

[0205] The OD values ​​of plasma samples from healthy controls were used as controls (negative), and the OD values ​​of plasma samples from patients with IgA nephropathy were analyzed by ROC curve analysis using SPSS 16.0 software. Figure 6 The area under the curve of antibody KM55 shown is AUC = 0.6745. The optimal cut-off value is selected as 0.1077 when the Youden index (sensitivity + specificity - 1) is the largest (sensitivity is 77.39%, specificity is 50%, Youden index is 0.2739), and the OD value greater than or equal to 0.1077 is determined as IgA nephropathy patients (positive), and the OD value less than 0.1077 is non-IgA nephropathy patients (negative).

[0206] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. An antibody or antigen-binding fragment thereof that binds to Gd-IgA1, characterized in that: The antibody or antigen-binding fragment thereof comprises three heavy chain variable region complementary determining regions named HCDR1, HCDR2 and HCDR3, and three light chain variable region complementary determining regions named LCDR1, LCDR2 and LCDR3; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown at positions 26 to 33, positions 51 to 58 and positions 97 to 111 of SEQ ID NO:1, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are shown at positions 26 to 34, positions 52 to 54 and positions 91 to 99, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises four heavy chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4, and four light chain variable region framework regions named LFR1, LFR2, LFR3 and LFR4, wherein: The amino acid sequence of HFR1 is shown in positions 1 to 25 of SEQ ID NO: 1, The amino acid sequence of HFR2 is shown in positions 34 to 50 of SEQ ID NO: 1, The amino acid sequence of HFR3 is shown in positions 59 to 96 of SEQ ID NO: 1, The amino acid sequence of HFR4 is shown in positions 112 to 122 of SEQ ID NO: 1, The amino acid sequence of LFR1 is shown in positions 1 to 25 of SEQ ID NO: 2, The amino acid sequence of LFR2 is shown in positions 35 to 51 of SEQ ID NO: 2, The amino acid sequence of LFR3 is shown in positions 55 to 90 of SEQ ID NO: 2, The amino acid sequence of LFR4 is shown in positions 100 to 109 of SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in positions 1 to 122 of SEQ ID NO:1; and / or the amino acid sequence of the light chain variable region is as shown in positions 1 to 109 of SEQ ID NO:

2.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment thereof also includes a constant region.

5. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. An expression cassette, characterized in that: The expression cassette contains the nucleic acid molecule of claim 5.

7. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 5.

8. A recombinant cell, characterized in that: The recombinant cell contains the nucleic acid molecule of claim 5.

9. A recombinant microorganism, characterized in that: The recombinant microorganism contains the nucleic acid molecule according to claim 5.

10. A method for preparing an antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and the method comprises the step of expressing the coding gene of the antibody or antigen-binding fragment thereof in a mammalian cell to obtain the antibody or antigen-binding fragment thereof.

11. A reagent or a kit, characterized in that: The reagent or kit contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and the reagent or kit has at least one of the following uses: (B1) Recognize or assist in the recognition of Gd-IgA1; (B2) binding or assisting in binding to Gd-IgA1; (B3) detecting or assisting in detecting whether the sample to be tested contains Gd-IgA1; (B4) detecting or assisting in detecting the content of Gd-IgA1 in the sample to be tested; (B5) Diagnosis or auxiliary diagnosis of IgA nephropathy; (B6) Targeting Gd-IgA1.

12. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in binding or assisting binding to Gd-IgA1 for purposes other than disease diagnosis or treatment.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1 for purposes other than disease diagnosis or treatment.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a product for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in detecting or assisting in detecting the content of Gd-IgA1 in a sample for purposes other than disease diagnosis or treatment.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a product for detecting or assisting in the detection of the content of Gd-IgA1 in a sample to be tested.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a product targeting Gd-IgA1.

20. Use of the nucleic acid molecule of claim 5 in at least one of the following: (P1) preparing products for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1; (P2) preparing products for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be tested; (P3) Preparation of products for diagnosis or auxiliary diagnosis of IgA nephropathy; (P4) Preparation of products targeting Gd-IgA1.

21. Use of the expression cassette of claim 6 in at least one of the following: (P1) preparing products for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1; (P2) preparing products for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be tested; (P3) Preparation of products for diagnosis or auxiliary diagnosis of IgA nephropathy; (P4) Preparation of products targeting Gd-IgA1.

22. Use of the recombinant vector according to claim 7 in at least one of the following: (P1) preparing products for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1; (P2) preparing products for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be tested; (P3) Preparation of products for diagnosis or auxiliary diagnosis of IgA nephropathy; (P4) Preparation of products targeting Gd-IgA1.

23. Use of the recombinant cell of claim 8 in at least one of the following: (P1) preparing products for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1; (P2) preparing products for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be tested; (P3) Preparation of products for diagnosis or auxiliary diagnosis of IgA nephropathy; (P4) Preparation of products targeting Gd-IgA1.

24. Use of the recombinant microorganism according to claim 9 in at least one of the following: (P1) preparing products for detecting or assisting in detecting whether a sample to be tested contains Gd-IgA1; (P2) preparing products for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be tested; (P3) Preparation of products for diagnosis or auxiliary diagnosis of IgA nephropathy; (P4) Preparation of products targeting Gd-IgA1.

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