CN119431594B

CN119431594B - Galactose-deleted IgA1 specific antibody or antigen binding fragment thereof, and related biological material and application thereof

Info

Publication number: CN119431594B
Application number: CN202510024726.7A
Authority: CN
Inventors: 张月苗; 曹云龙; 吕继成
Original assignee: Peking University First Hospital; Beijing Changping Laboratory
Current assignee: Wuxi Shenrui Bio Pharmaceuticals Co ltd
Priority date: 2025-01-08
Filing date: 2025-01-08
Publication date: 2025-03-28
Anticipated expiration: 2045-01-08
Also published as: CN119431594A

Abstract

The application discloses a galactose-deleted IgA1 specific antibody or an antigen binding fragment thereof and a related biological material and application thereof, belonging to the technical field of antibodies. The main problem to be solved by the application is how to obtain the anti-Gd-IgA1 antibody with high specificity. In order to solve the above technical problems, the present application provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region shown at positions 1 to 122 of SEQ ID NO. 1 and a light chain variable region shown at 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 antigen, and is expected to be more effectively used for diagnosis, monitoring treatment effect, prognosis judgment and the like of IgA nephropathy.

Description

Galactose-deleted IgA1 specific antibody or antigen binding fragment thereof, and related biological material and application thereof

Technical Field

The application belongs to the technical field of antibodies, and particularly relates to a galactose-deleted IgA1 specific antibody or an antigen binding fragment thereof, and a related biological material and application thereof.

Background

IgA nephropathy is the most common primary glomerular disease worldwide, and is also the most common cause of uremia in young and young adults. Past immunosuppressive regimens based on supportive treatment and glucocorticoid-based therapy have limited improvement in patient prognosis. In recent years, along with the increasing understanding of pathogenesis of IgA nephropathy, targeted therapeutic means for targeting key links such as B cells/plasma cells to reduce pathogenic IgA production by monoclonal antibodies, fusion proteins, micromolecules and the like are developed successively, and have been used in clinical practice in batches. Currently, igA nephropathy is undergoing a shift from traditional hormone immunosuppressive therapy to targeted therapy age. However, due to the lack of specific noninvasive diagnostic methods, compared with the rapid progress of IgA nephropathy targeted therapies, the diagnosis still depends on kidney puncture biopsy, and the treatment and prognosis monitoring still depend on traditional clinical indexes such as proteinuria, kidney function and the like, which are in sharp contrast. The global prognosis of improvement of kidney disease (KIDNEY DISEASE: improving Global Outcomes, KDIGO) guidelines indicate that these traditional clinical pathology indicators are not based on pathogenesis or therapeutic target development, and therefore cannot be used to guide targeted therapeutic drug selection nor to judge early efficacy. Clinically, the problems of various novel therapeutic drugs for IgA nephropathy, high cost and the like make it necessary to establish a noninvasive evaluation method for biomarkers based on pathogenesis and therapeutic targets so as to improve diagnosis, monitoring therapeutic effects and judging prognosis of IgA nephropathy.

IgA nephropathy is a typical pathological manifestation of IgA1 molecule-based immune complexes in renal deposition. The galactose modification deletion of IgA1 molecules in IgA nephropathy patients, and the formation of galactose deletion IgA1 (galactose-DEFICIENT IGA1, gd-IgA 1) is a key link leading to the onset of IgA nephropathy. Therefore, the four-hit theory of IgA nephropathy suggests that the increase of Gd-IgA1 (first hit) results in the generation of anti-Gd-IgA1 antibody (second hit), and immune complexes formed by the Gd-IgA1 antibody and the Gd-IgA antibody deposit on the kidney (third hit) and activate inflammatory reactions such as complement to damage the kidney (fourth hit). Between the heavy chain constant region 1 (CH 1) and constant region 2 (CH 2) of the human IgA1 molecule there is a hinge region consisting of 19 amino acids, which is a sequence consisting of tandem repeats of proline (Pro), threonine (Thr), serine (Ser), each hinge region comprising 6 Ser/Thr potential O-glycosylation modification sites (asterisk positions) VPST pptsps tspptsps. Normally, igA1 molecules O-sugar chain core sugar N-acetylgalactosamine (N-acetylgalacosamine, galNAc) will be linked to Ser/Thr site under the catalysis of N-acetylgalactosamine transferase (N-acetylgalactosaminyltransferase, galNAcTs), on the basis of which galactose (galactose, gal) can be linked to GalNAc via a.beta.1, 3 bond, sialic acid (SIALIC ACID, SA) is linked to GalNAc or Gal via an. Alpha.2, 6 bond or. Alpha.2, 3 bond, thereby forming six different glycoforms. The hinge region of the peripheral blood IgA1 molecule of the IgA nephropathy patient is deleted in galactose modification, so that O-GalNAc (Tn antigen) is exposed to form Gd-IgA1.Gd-IgA1 is significantly elevated in peripheral blood of IgA nephropathy patients and is an independent risk factor affecting prognosis of patients. Gd-IgA1 level reduction is closely related to treatment response, and is an important noninvasive diagnosis and treatment marker for IgA nephropathy.

However, the existing detection of Gd-IgA1 has the problems that mass spectrum detection of a large sample is limited in popularization, the binding force of snail lectin HAA (the lectin can recognize and bind with Tn antigen) is poor compared with that of antigen-antibody detection, the natural lectin batch effect is large, and the like, so that the research and development of the anti-Gd-IgA1 antibody with high specificity is a core problem of great attention in the research field of IgA nephropathy. A monoclonal anti-Gd-IgA1 antibody KM55 of IgG2b type is obtained by screening a rat immunized with Yasutake J of Japanese scholars and the like by utilizing a hybridoma cell technology. However, KM55 is only a reagent product for scientific research, and has the problems of high overlapping rate of detection values of patients and healthy controls, poor binding force with antigen, and the like, and a detection method aiming at high specificity and high sensitivity is still lacking at present. Along with development of antibody research and development technology, single B cell sequencing presents great advantages and potential in antibody discovery and clone screening, and provides a new thought for developing anti-Gd-IgA1 monoclonal antibodies.

Disclosure of Invention

The main problem to be solved by the application is how to obtain the anti-Gd-IgA1 antibody with high specificity. In order to solve the technical problems, the application provides the following technical scheme:

The present application provides an antibody or antigen binding fragment thereof comprising 3 heavy chain variable region Complementarity Determining Regions (CDRs) designated HCDR1, HCDR2 and HCDR3, respectively, and 3 light chain variable region complementarity determining regions designated LCDR1, LCDR2 and LCDR3, respectively, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 comprise positions 26 to 33, 51 to 58 and 97 to 111 of SEQ ID NO. 1, respectively, and wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 comprise positions 26 to 34, 52 to 54 and 91 to 99 of SEQ ID NO. 2, respectively.

In the present application, the Complementarity Determining Regions (CDRs) are defined based on the IMGT numbering scheme.

The antibody or antigen binding fragment thereof may be an antibody or antigen binding fragment thereof that binds to a Tn5 antigen.

The Tn5 antigen is a compound with a structure shown in a formula 1:

VPST PPT PS TPPT PSPS-NH ₂ formula 1,

In formula 1, VPST represents amino acid residues of valine, proline, serine and threonine, respectively, the group of formula 2 is linked to the amino acid residue before x, the amino acid residue before x is serine residue or threonine residue, H in-OH in serine residue before x or threonine residue is replaced with the group of formula 2, and the wavy line in formula 2 indicates the position linked to the amino acid residue.

The antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment thereof that binds Gd-IgA 1.

In the antibody or antigen binding fragment thereof, the binding may be specific binding.

By specific binding is meant that one molecule binds to another with a significantly higher affinity than any cross-reactive or off-target antigen (collectively referred to as non-target antigens). The affinities are 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 response will be at least 1.5 times that of the non-target signal or non-target binding noise.

In the antibody or the antigen binding fragment thereof, the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are 26 th to 33 th, 51 th to 58 th and 97 th to 111 th of SEQ ID NO. 1 in sequence, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are 26 th to 34 th, 52 th to 54 th and 91 th to 99 th of SEQ ID NO. 2 in sequence.

Further, the antibody or antigen binding fragment thereof further comprises 4 heavy chain variable region Framework Regions (FR) named HFR1, HFR2, HFR3 and HFR4, respectively, and 4 light chain variable region framework regions named LFR1, LFR2 and LFR3 and LFR4, respectively, wherein HFR1 comprises an amino acid sequence selected from the group consisting of:

a111 1 to 25 positions of SEQ ID NO.1,

A112 A) a sequence having 75% or more identity to a 111);

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

a121 34 th to 50 th positions of SEQ ID NO. 1,

A122 A) a sequence having greater than 75% identity to a 121);

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

a131 59 to 96 positions of SEQ ID NO. 1,

A132 A sequence having 75% or more identity to a 131);

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

a141 112 to 122 of SEQ ID NO. 1,

A142 A) a sequence having greater than 75% identity to a 141);

the amino acid sequence of LFR1 comprises an amino acid sequence selected from the group consisting of:

a151 1 to 25 positions of SEQ ID NO. 2,

A152 A) a sequence having 75% or more identity to a 151);

the amino acid sequence of LFR2 comprises an amino acid sequence selected from the group consisting of:

a161 35 to 51 positions of SEQ ID NO. 2,

A162 A) a sequence having 75% or more identity to a 151);

the amino acid sequence of LFR3 comprises an amino acid sequence selected from the group consisting of:

a171 55 to 90 positions of SEQ ID NO. 2,

A172 A) a sequence having greater than 75% identity to a 171);

The amino acid sequence of LFR4 comprises an amino acid sequence selected from the group consisting of:

a181 100 th to 109 th positions of SEQ ID NO. 2,

A182 A) a sequence having a identity of 75% or more to a 181).

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 the three CDRs. The framework regions are used to align CDRs that specifically bind to an epitope. CDRs include amino acid residues in antibodies that are primarily responsible for antigen binding. The VL and VH domains comprise, from amino-to carboxy-terminus, the Framework Regions (FR) and CDR regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDR1, CDR2 and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2 and LCDR3, respectively, and CDR1, CDR2 and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2 and HCDR3, respectively.

Herein, the identity (identity) may be specifically 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.

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.

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

In the above antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof may further comprise a constant region.

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

In the antibody or antigen binding fragment thereof, the heavy chain constant region may be IgG, igM, igA.

In the antibody or antigen binding fragment thereof, the IgG may be IgG1, igG2, igG3, or IgG4.

In the antibody or antigen binding fragment thereof, the light chain constant region may be a lamda (lambda) constant region or a Kappa (Kappa) constant region.

The antibody or antigen binding fragment thereof may be murine, human, chimeric or humanized.

The term "antibody" in the present application is an iso-tetralin protein of about 150000 daltons having the same structural characteristics, consisting 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 varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the constant region of the light chain being opposite the first constant region of the heavy chain and the variable region of the light chain being opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, which generally comprises at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody (parental antibody). The antigen binding fragments described above retain at least some of the binding specificity of the parent antibody. Typically, the antigen binding fragments retain at least 10% of the parent binding activity when expressed on a molar basis. In particular, the antigen binding fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.

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.

The term "Fab" is an antigen binding fragment (fragment of antigen binding, fab) consisting of VH (heavy chain variable region) and CH1 (constant region) 1 domains of a complete antibody light and heavy chain. I.e., heterodimers formed by the heavy chain Fd and the intact light chain of the antibody bound by disulfide bonds.

The term "Fab' fragment" contains the portion of an intact antibody light chain and an antibody heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains. Thereby, interchain disulfide bonds can be formed between the two heavy chains of the two Fab 'fragments to form the F (ab') ₂ molecule.

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

The term "nanobody", i.e. a heavy chain single domain antibody VHH (variable domain of HEAVY CHAIN of heavy-chain antibody) is a protein consisting of antibody heavy chain variable regions.

The term "Minimal Recognition Unit (MRU)" refers to a single CDR structure comprising only the variable domain, and has a molecular mass of only about 1% of that of an intact antibody, and can bind to the corresponding antigen.

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

The term "single chain antibody" (ScFv) is a protein in which the heavy and light chain variable regions of an antibody are linked by a short peptide.

The term "heavy chain antibody" (HCAb) is a protein consisting of only two heavy chains, no light chain present, and no CH1 region between the heavy chain variable region and the hinge region.

The application also provides a biological material, which is any one of the following:

(A1) A nucleic acid molecule encoding any of the antibodies or antigen binding fragments thereof,

(A2) An expression cassette comprising the nucleic acid molecule as described in (A1),

(A3) A recombinant vector comprising the nucleic acid molecule of (A1),

(A4) Recombinant cells comprising the nucleic acid molecule of (A1),

(A5) A recombinant microorganism comprising the nucleic acid molecule of (A1).

In the above biological material, the nucleic acid molecule of (A1) may be any of the following:

Nucleic acid molecules encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody, said nucleic acid molecules comprising positions 891 to 914, 966 to 989 and 1104 to 1148, respectively, from the 5' end of SEQ ID No. 3;

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

Nucleic acid molecules encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody, said nucleic acid molecules being positions 891 to 914, 966 to 989 and 1104 to 1148, respectively, from the 5' end of SEQ ID No. 3;

and/or nucleic acid molecules encoding LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody, said nucleic acid molecules being positions 133 to 159, 211 to 219 and 328 to 354, respectively, from the 5' end of SEQ ID No. 4.

In the above biological material, the nucleic acid molecule of (A1) may specifically be any of the following:

a nucleic acid molecule encoding the heavy chain variable region of said antibody, said nucleic acid molecule comprising positions 816 to 1181 of SEQ ID No. 3;

and/or a nucleic acid molecule encoding the light chain variable region of said antibody, said nucleic acid molecule comprising positions 97 to 423 of SEQ ID NO. 4.

a nucleic acid molecule encoding the heavy chain variable region of said antibody, said nucleic acid molecule being at positions 816 to 1181 of SEQ ID NO. 3;

And/or a nucleic acid molecule encoding the light chain variable region of said antibody, said nucleic acid molecule being at positions 97 to 423 of SEQ ID NO. 4.

a nucleic acid molecule encoding the heavy chain of said antibody, said nucleic acid molecule comprising positions 816 to 2156 of SEQ ID No. 3;

and/or a nucleic acid molecule encoding the light chain of said antibody, said nucleic acid molecule comprising SEQ ID NO. 4.

a nucleic acid molecule encoding the heavy chain of said antibody, said nucleic acid molecule being at positions 816 to 2156 of SEQ ID NO. 3;

And/or a nucleic acid molecule encoding the light chain of the antibody, said nucleic acid molecule being SEQ ID NO. 4.

In the above biological material, the nucleic acid molecule (A1) may be DNA such as cDNA, genomic DNA or recombinant DNA, or RNA such as mRNA or hnRNA.

In the above biological material, the expression cassette (A2) means a DNA capable of expressing the antibody in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the antibody but also a terminator for terminating transcription of the gene encoding the antibody. Further, the expression cassette may also include an enhancer sequence.

Recombinant vectors containing the expression cassette can be constructed using existing expression vectors.

In the above biological material, the vector may be a plasmid, cosmid, phage or viral vector.

In the above biological material, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule of (A1) into an antibody protein expression vector pcDNA3.4.

In a specific example, the antibody is used to construct heavy chain expression vector pcDNA3.4-H and light chain expression vector pcDNA3.4-L.

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

The structure of the pcDNA3.4-L is as follows, namely, the sequence between 759 th and 2156 th positions of the pcDNA3.4-H (namely SEQ ID NO: 3) is replaced by a DNA fragment shown in SEQ ID NO:4, and other sequences of the pcDNA3.4-H are kept unchanged to obtain the recombinant vector. SEQ ID NO.4 shows the coding gene of the signal peptide at positions 1 to 57, the coding gene of the light chain variable region (VL) at positions 58 to 384 and the coding gene of the light chain constant region at positions 385 to 705. The pcDNA3.4-L can express the light chain of the antibody, and the amino acid sequence of the pcDNA3.4-L is SEQ ID NO. 2.SEQ ID NO.2 shows the amino acid sequences of the variable regions at positions 1 to 109 and the amino acid sequences of the constant regions at positions 110 to 215.

In the above biological material, the microorganism may be bacteria (e.g., E.coli), yeast, algae, or fungi.

The full-length nucleotide sequence of the antibody or a fragment thereof of the present application can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. The sequences concerned are synthesized, for example, by artificial synthesis, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

The present application also provides a method for producing an antibody or an antigen-binding fragment thereof, which is the antibody or the antigen-binding fragment thereof, comprising the step of expressing a gene encoding the antibody or the antigen-binding fragment thereof in a mammalian cell to obtain the antibody or the antigen-binding fragment thereof.

In the above method, the mammalian cell may be an ex vivo animal cell, such as a cell in a culture medium. The mammalian cells may be selected from ExpiCHO-STM cells, chinese hamster ovary Cells (CHO), 293F cells, 293E cells, 293-6E cells, and the like.

In the above method, expressing the gene encoding the antibody or antigen-binding fragment thereof in mammalian cells comprises infecting, transfecting or transforming mammalian cells with the gene encoding to obtain recombinant mammalian cells containing the gene encoding, culturing the recombinant mammalian cells to obtain a cell culture, and isolating and purifying the antibody or antigen-binding fragment thereof from the cell culture. The antibodies or antigen binding fragments thereof may be produced from large scale cell cultures using 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, NS0 murine myeloma cell, HEK293 (human embryonic kidney 293) cell, or per.c ^® cell.

The application also provides a reagent or kit comprising said antibody or antigen binding fragment thereof, said reagent or kit having at least one of the following uses:

(B1) Identifying or assisting in identifying Gd-IgA1;

(B2) Binding or assisting in binding Gd-IgA1;

(B3) Recognizing or assisting in recognizing Tn antigen;

(B4) Binding or assisting in binding Tn antigen;

(B5) Detecting or assisting in detecting whether the sample to be detected contains Gd-IgA1;

(B6) Detecting or assisting in detecting the content of Gd-IgA1 in a sample to be detected;

(B7) Detecting or assisting in detecting whether a sample to be detected contains Tn antigen;

(B8) Detecting or assisting in detecting the Tn antigen content in the sample to be detected;

(B9) Identifying or aiding in the identification of IgA nephropathy;

(B10) Targeting Gd-IgA1;

(B11) Targeting Tn antigen;

(B12) Diagnosing diseases associated with Gd-IgA 1;

(B13) Diagnosing a disease associated with the Tn antigen.

The application also provides pharmaceutical compositions comprising said antibodies or antigen binding fragments thereof.

The pharmaceutical composition can be used for treating diseases related to Gd-IgA1 and Tn antigen.

The application also provides the use of an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof, in any of the following:

(C1) Identifying or assisting in identifying Gd-IgA1;

(C2) Binding or assisting in binding Gd-IgA1;

(C3) Recognizing or assisting in recognizing Tn antigen;

(C4) Binding or assisting in binding Tn antigen;

(C5) Detecting or assisting in detecting whether the sample to be detected contains Gd-IgA1;

(C6) Preparing a product for detecting or assisting in detecting whether a sample to be detected contains Gd-IgA 1;

(C7) Detecting or assisting in detecting the content of Gd-IgA1 in a sample to be detected;

(C8) Preparing a product for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be detected;

(C9) Detecting or assisting in detecting whether a sample to be detected contains Tn antigen;

(C10) Preparing a product for detecting or assisting in detecting whether a sample to be detected contains Tn antigen;

(C11) Detecting or assisting in detecting the Tn antigen content in the sample to be detected;

(C12) Preparing a product for detecting or assisting in detecting the Tn antigen content in a sample to be detected;

(C13) Identifying or aiding in the identification of IgA nephropathy;

(C14) Preparing a product that recognizes or aids in recognizing IgA nephropathy;

(C15) Preparing a Gd-IgA1 targeting product;

(C16) Preparing a Tn antigen-targeted product;

(C17) Preparing a product for diagnosing or treating diseases related to Gd-IgA1;

(C18) And preparing a product for diagnosing or treating the diseases related to the Tn antigen.

The product may be a reagent, kit, medicament or medicament.

The application also provides application of the biological material, wherein the application is applied to any one of the following:

(P1) preparing a product for detecting or assisting in detecting whether Gd-IgA1 is contained in a sample to be detected;

(P2) preparing a product for detecting or assisting in detecting the content of Gd-IgA1 in a sample to be detected;

(P3) preparing a product for detecting or assisting in detecting whether the sample to be detected contains Tn antigen;

(P4) preparing a product for detecting or assisting in detecting the Tn antigen content in the sample to be detected;

(P5) preparing a product that recognizes or aids in recognizing IgA nephropathy;

(P6) preparing a Gd-IgA 1-targeted product;

(P7) preparing a product for diagnosing or treating a disease associated with Gd-IgA 1;

(P8) preparing a Tn antigen-targeted product;

(P9) preparing a product for diagnosing or treating a Tn antigen-related disease.

IgA nephropathy is usually manifested as an increase in Gd-IgA1 in the above applications. Gd-IgA1 refers to the deletion of galactose modification in the hinge region of IgA1 molecule, thereby exposing O-GalNAc (Tn antigen).

Compared with the existing antibody KM55, the antibody provided by the application is cloned by adopting a single B cell technology after mice are immunized, has the advantages of high affinity with antigen and improved patient diagnosis capability, and is expected to be more effectively used for diagnosis, monitoring treatment effect, prognosis judgment and the like of IgA nephropathy.

Drawings

FIG. 1 is a graph showing the EC50 of the monoclonal antibody obtained by the screening and the binding of KM55 to Tn0 or Tn5, wherein A is the result of the monoclonal antibody B0033 obtained by the screening, B is the result of KM55, and C is the EC50 value of the binding to Tn 5. Compared with the existing antibody KM55, the monoclonal antibody obtained in the application can effectively distinguish Tn0 and Tn5 antigens, and the binding capacity with Tn5 is higher than that of KM55.

FIG. 2 is a graph showing ELISA results of the monoclonal antibody obtained by screening and the binding of KM55 and Gd-IgA1/IgA1, wherein A is the result of the monoclonal antibody B0033 obtained by screening, and B is the result of KM 55. Compared with the existing antibody KM55, the monoclonal antibody obtained by the application has stronger binding force with Gd-IgA1 and IgA1 molecules and can be effectively distinguished.

FIG. 3 is a graph showing ELISA results of IgA1 and Gd-IgA1 detection by the monoclonal antibody obtained by the screening, wherein A is the result of B0033 of the monoclonal antibody obtained by the screening, and B is the result of KM 55. The results show that compared with the existing antibody KM55, the monoclonal antibody obtained in the application can more effectively distinguish IgA1 and Gd-IgA1 molecules.

FIG. 4 shows the comparison of the effect of the selected monoclonal antibody on Gd-IgA1 in IgA nephropathy patients and healthy controls with KM55, wherein A is the result of the selected 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 the application is obviously higher than that of the KM55.

FIG. 5 shows a diagnostic curve of the monoclonal antibodies obtained by the screening against IgA nephropathy patients.

FIG. 6 is a diagnostic curve of KM55 for IgA nephropathy patients.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

Some of the experimental material information in the examples described below are ：FITC anti-mouse CD19（115506, Biolegend）、PE/Cyanine7 anti-mouse IgM Antibody（406514, Biolegend）、PE/Cyanine7 anti-mouse IgD Antibody （405720, Biolegend）、Bio-Tn5（ Shanghai Ammonia Biotechnology Inc.), PE-strepitavidin (405204, bioLegend).

EXAMPLE 1 acquisition of monoclonal antibodies specifically recognizing Gd-IgA1

1. Designing Tn antigen in Gd-IgA1 molecule

The IgA1 hinge region 19 amino acid peptide segments are added with GalNAc at serine and threonine sites where galactose deletion occurs most frequently to form a high-density Tn antigen (hereinafter abbreviated as Tn5 antigen) which is used as an immune antigen, a sorting antigen and a detection antigen.

The Tn5 antigen is a compound with a structure shown in a formula 1:

VPST PPT PS TPPT PSPS-NH ₂ formula 1,

In formula 1, VPST represents an amino acid residue of valine, proline, serine, threonine, respectively, and is a group of formula 2. The group of formula 2 is linked to a pre-amino acid residue, which is a serine residue or a threonine residue, and H in-OH in the pre-serine residue or threonine residue is replaced with a group of formula 2, and the wavy line in formula 2 indicates the position linked to the amino acid residue.

2. Tn antigen immunized mice

Tn5 was conjugated to carrier protein KLH (Shanghai Ammonia Biotechnology Co., ltd.) and was combined with adjuvant (CFA, sigma, F5881)/incomplete Freund's adjuvant (IFA, sigma, F5506) to immunize 6-7 week old BALB/c female mice two weeks apart. Specifically, the spleen was taken 3 days after ：Tn5-KLH（100μg/100μL）+CFA（100μL）、Tn5-KLH（100μg/100μL）+IFA（100μL）、Tn5-KLH（100μg/100μL）+IFA（100μL）、Tn5-KLH（100μg/100μL）+IFA（100μL）. immunization was completed.

3. Sorting single B cells combined with Tn5 by mouse spleen cells

The spleens of the mice were ground and filtered through a 70mm screen to give a single cell suspension. B cells were selected by FITC anti-mouse CD19 （115506, Biolegend）、PE/Cyanine7 anti-mouse IgM Antibody（406514, Biolegend）、PE/Cyanine7 anti-mouse IgD Antibody（405720, Biolegend） rounds, and single B cells binding to Tn5 were selected by Bio-Tn5 (Shanghai Ammonia Biotechnology Co., ltd.) and PE-strepitavidin (405204, bioLegend).

4. Antibody sequencing

Obtaining RNA by splitting single B cell, obtaining cDNA by reverse transcription with RNA as template, PCR with cDNA as template, heavy chain/light chain variable region gene primer, obtaining V region gene sequence by sequencing. The amino acid residue sequence of the heavy chain/light chain variable region is obtained after translation of the gene sequence. And defining Complementarity Determining Regions (CDRs) of the heavy chain variable region and the light chain variable region according to the IMGT numbering scheme. The heavy chain variable region (VH) or the light chain variable region (VL) of an antibody is composed of framework regions separated by three complementarity determining regions. The framework regions are used to align CDRs that specifically bind to an epitope. CDRs include amino acid residues in antibodies that are primarily responsible for antigen binding. The VH domain and VL domain each comprise, from amino terminus to carboxy terminus, the Framework Regions (FR) and CDR regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. CDR1, CDR2 and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2 and HCDR3, respectively, FR1, FR2, FR3 and FR4 of the VH domain are also referred to herein as HFR1, HFR2, HFR3 and HFR4, respectively, CDR1, CDR2 and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2 and LCDR3, respectively, and FR1, FR2, FR3 and FR4 of the VL domain are also referred to herein as LFR1, LFR2, LFR3 and LFR4, respectively.

5. Construction of recombinant expression vectors

And (3) constructing the heavy chain variable region gene of the monoclonal antibody obtained in the step (4) between the signal peptide of the modified pcDNA3.4 vector and the mouse IgG1 constant region to obtain a recombinant expression vector pcDNA3.4-H. The complete sequence of the recombinant expression vector pcDNA3.4-H is shown as SEQ ID NO. 3.SEQ ID NO. 3 shows CMV ENHANCER at positions 50 to 429, CMV promoter at positions 430 to 633, signal peptide encoding genes at positions 759 to 815, heavy chain variable region (VH) encoding genes at positions 816 to 1181, and heavy chain constant region (i.e., murine IgG1 constant region) encoding genes at positions 1182 to 2156. The pcDNA3.4-H vector can express the heavy chain of the antibody, and the amino acid sequence of the pcDNA3.4-H vector is SEQ ID NO. 1.SEQ ID NO. 1 shows the amino acid sequences of the variable regions at positions 1 to 122 and the amino acid sequences of the constant regions (i.e.the murine heavy chain IgG1 constant region) at positions 123 to 446.

And (3) constructing the monoclonal antibody light chain variable region gene obtained in the step (4) between the signal peptide of the modified pcDNA3.4 vector and a murine light chain lambda constant region to obtain a recombinant expression vector pcDNA3.4-L. The light chain expression vector pcDNA3.4-L has the structure that the sequence between 759 th and 2156 th positions of pcDNA3.4-H (namely SEQ ID NO: 3) is replaced by a DNA fragment shown as SEQ ID NO: 4. SEQ ID NO. 4 shows the coding genes of the signal peptide at positions 1 to 57, the coding genes of the light chain variable region at positions 58 to 384 and the coding genes of the light chain constant region (i.e. the murine light chain lambda constant region) at positions 385 to 705. The pcDNA3.4-L vector can express the light chain of the antibody, and the amino acid sequence of the pcDNA3.4-L vector is SEQ ID NO. 2.SEQ ID NO. 2 shows the amino acid sequences of the variable regions at positions 1 to 109 and the amino acid sequences of the constant regions (i.e.the murine light chain lambda constant region) at positions 110 to 215.

6. Antibody in vitro expression

ExpiCHO-STM cells (Thermo Fisher, cat. No. A29127) were diluted to 6X 10 ⁶ Cell/mL using ExpiCHOTM expression medium (Gibco, A29100-02). 24. 2.5 mL cells per well were packed in well deep plates for transfection.

200 Mu L ExpiFectamine of the reagent-DNA complex was added to each well of a 24-well deep-well plate containing the culture, and the reaction plate was covered with a gas-permeable sealing membrane. Wherein ExpiFectamine reagent-DNA complex was prepared by mixing the heavy and light chain plasmids 1:1 constructed in step 5, 9.0. Mu. L ExpiFectamine CHO reagent (Gibco, A29129), 189. Mu. L OptiPRO SFM (Gibco, 12309-050).

The 24-well deep-well plate was incubated on an orbital shaker at 37℃in humidified air with 8% CO ₂. ExpiFectamine CHO enhancers (Gibco, A29129) and ExpiCHO adjuvant (Gibco, A29129) were added 18-22 hours after transfection. For a monolithic 24-well deep well plate, 400 μ L ExpiFectamine CHO enhancer and 16 mL ExpiCHO adjuvant were mixed in a conical tube. 600 μl of the mixture was added to each well of a 24-well deep well plate. The 24-well deep-well plate was returned to the 37 ℃ incubator containing 8% CO ₂ and shaken. On day 8 post transfection, the well plates were centrifuged (500 g,10 min) and culture supernatants were collected for antibody purification.

7. Antibody purification and detection

Protein A beads for purification (GenScript, L00695) were added to the expression supernatant (400. Mu.L magnetic beads/5 mg Protein) in proportion and incubated for 2h. After incubation, the beads were transferred to a 24-well deep-well plate (BIOLAND, SBS 24-10-V) and placed in KINGFISHER FLEX full-automatic nucleic acid extractor to perform purification procedures (2 PBS washes, 1 distilled water wash, 1 antibody Elution). To the antibody-containing Elutation reagent, tris reagent was added for pH neutralization (62.6. Mu.L Tris reagent/1 mL Elutation reagent). After centrifugation (3000 g,5 min), the supernatant was taken to obtain a purified antibody solution, monoclonal antibody B0033, which was used in subsequent experiments.

Example 2 antibody binding Capacity test

1. Binding specificity of enzyme-linked immunosorbent assay (ELISA) antibodies to Tn5

Antigen Tn5 and control antigen Tn0 (Tn 0 is the same amino acid sequence as Tn5 without GalNAc modification) were each coated at a concentration of 1. Mu.g/mL (4 ℃ C., overnight), 5% casein sodium salt (Biyun day, ST1132-100 g) was blocked at 37℃for 2 hours, and then the antibody solution (containing monoclonal antibody B0033) obtained in example 1 was added as primary antibodies, and 10 concentration treatments (initial concentration of 3. Mu.g/mL, 3-fold dilution) were added to the primary antibodies. The commercial KM55 antibody (IBL, # 10777) was used as a positive control antibody. The absorbance at 450nm (OD value) was read using goat anti-mouse IgG-HRP as detection secondary antibody (Invitrogen, 1:1000, G21040,1:1000 dilution), KM55 selected rabit anti-rate IgG (h+l) -HRP (Immunoway, RS030226,1:10000 dilution) as detection secondary antibody, TMB developed (Soxhoba, PR 1200), terminated reaction (Soxhoba, C1058). Data were processed using GRAPHPAD PRISM statistical software, and experimental results of antibody binding to Tn5 and Tn0 were fitted to curves and EC50 was calculated.

The results are shown in FIG. 1, and the results show that the monoclonal antibody obtained in the present application binds to Tn5 and does not bind to Tn0, compared with the existing antibody KM 55. The binding force of B0033 to Tn5 was higher than that of KM55, wherein the EC50 of B0033 to Tn5 was 0.0225. Mu.g/mL, and the EC50 of KM55 to Tn5 was 19.5400. Mu.g/mL.

2. Binding to Gd-IgA1

Gd-IgA1 was prepared by purchasing commercial human IgA1 (Abcam, ab 91020), in vitro using 4000 U.alpha.2-3, 6,8 neuraminidase (P0720L, NEW ENGLAND Biolabs), 400 U.beta.1-3 galactosidase (P0726S, NEW ENGLAND Biolabs), and digestion for 2 hours at pH 7.4 at 37℃to obtain Gd-IgA1, and the same batch of non-digested IgA1 was used as a control.

Gd-IgA1 and commercial IgA1 prepared above were each coated with 3. Mu.g/mL (4 ℃ C., overnight), blocked with 5% BSA (Soy pal, A8020) at 37 ℃ C. For 2 hours, and then the antibody solution (containing monoclonal antibody B0033) obtained in example 1 was added as primary antibody, and 6 concentration treatments (initial concentration 9. Mu.g/mL, 3-fold dilution) were applied to the primary antibody. The commercial KM55 antibody (IBL, # 10777) was used as a positive control antibody. The absorbance at 450nm (OD value) was read using goat anti-mouse IgG-HRP as detection secondary antibody (Invitrogen, 1:1000, G21040,1:1000 dilution), KM55 selected rabit anti-rate IgG (h+l) -HRP (Immunoway, RS030226,1:10000 dilution) as detection secondary antibody, TMB developed (Soxhoba, PR 1200), terminated reaction (Soxhoba, C1058).

The results are shown in FIG. 2, and the results show that the monoclonal antibody obtained in the application can specifically bind Gd-IgA1. Compared with the existing antibody KM55, the monoclonal antibody obtained by the application has higher differentiation degree on IgA1 and Gd-IgA1.

3. ELISA detection of Gd-IgA1

Monoclonal antibodies B0033 and KM55 were each coated at a concentration of 2. Mu.g/mL, and after blocking at 37℃for 2 hours with 5% BSA (Soxhaustao, A8020), primary antibodies were each added in gradient dilutions (5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, and 0.3125. Mu.g/mL) to IgA1 or Gd-IgA1, and the absorbance at 450nm (OD 450) was read using goat anti-human IgA1-HRP as the detection secondary antibody (abcam, AB97215, diluted 1:1000 in use), TMB color development (Soxhao, PR 1200), ELISA terminator termination reaction (Soxhao, C1058).

The results are shown in FIG. 3, and demonstrate that the monoclonal antibody obtained in the present application is capable of distinguishing IgA1 and Gd-IgA1 molecules more effectively than the existing antibody KM 55.

Example 3 antibody diagnostic Capacity test

The source of plasma samples for IgA nephropathy patients or healthy controls was samples collected prospectively at the first hospital of the university of beijing, and the project was approved by the first hospital ethics committee of the university of beijing. All subjects signed informed consent;

The standard of IgA nephropathy patients is IgA nephropathy confirmed by kidney puncture biopsy in a first hospital of Beijing university, and the selected standard is ① that IgA deposition is mainly displayed by kidney puncture biopsy immunofluorescence, ② is used for prompting membranous region immune complex deposition, and ③ is not taken with glucocorticoid or immunosuppressant for at least half a year.

The standard of the healthy control is the healthy control with age, sex and region matching, and the selected standard is ① that no history of kidney diseases exists in the past, no abnormality exists in the ② biochemical examination of kidney functions, and no nephritis manifestations such as haematuria, proteinuria and the like exist in the ③ urine examination.

Plasma samples from IgA nephropathy patients were 115.

Plasma samples of healthy controls 124 cases.

1. Detection of Gd-IgA1 in plasma

Monoclonal antibody B0033 was blocked at a concentration of 2. Mu.g/mL for 2 hours at 37℃with a plate (4℃overnight), 5% BSA (Soy pal, A8020). Plasma samples (diluted with PBS) of 400-fold diluted IgA nephropathy patients or healthy controls were then added. The absorbance at 450nm (OD 450) was read using goat anti-human IgA1-HRP as the secondary detection antibody (abcam, AB97215, diluted 1:1000 in use), TMB chromogenic (Soxhibao, PR 1200), ELISA terminator stop reaction (Soxhibao, C1058). KM55 was used as a positive control antibody in the same manner as B0033. The data were processed using GRAPHPAD PRISM statistical software and the experimental results were expressed as mean ± standard deviation using t-test; (-) for P <0.05 level and indicated that the differences were significant, (-) for P <0.01 level and ((-) for P <0.001 level and ((-) for P <0.0001 level and indicated that the differences were significant.

The results are shown in fig. 4, and the results show that the detection value of the monoclonal antibody B0033 on Gd-IgA1 in plasma of IgA nephropathy patients is obviously higher than that of a healthy control, and compared with the existing antibody KM55, the detection OD450 value of Gd-IgA1 is obviously improved.

2. Diagnostic value for IgA nephropathy

2.1 Diagnosis of IgA nephropathy Using monoclonal antibody B0033

The ROC curve analysis was performed on the OD of plasma samples of IgA nephropathy patients using SPSS 16.0 software with the OD of plasma samples of healthy controls as control (negative), as shown in fig. 5, area under the B0033 curve auc= 0.7902. According to the maximum value of about dengue index (sensitivity + specificity-1) (sensitivity 80%, specificity 67.74%, about dengue index 0.4774), the optimal cut off value is 0.2219, and according to this, it is judged that the OD value is equal to or higher than 0.2219 is IgA nephropathy patient (positive), and the OD value is lower than 0.2219 is non-IgA nephropathy patient (negative).

2.2 Diagnosis of IgA nephropathy by KM55 antibody

The ROC curve analysis was performed on the OD of plasma samples of IgA nephropathy patients using SPSS 16.0 software with the OD of plasma samples of healthy controls as controls (negative), as shown in fig. 6, the area under the curve auc=0.6755 of antibody KM 55. According to the maximum value of about dengue index (sensitivity + specificity-1) (sensitivity: 77.39%, specificity: 50%, about dengue index: 0.2739), the optimal cut off value is 0.1077, and it is determined that the OD value is 0.1077 or more is IgA nephropathy patients (positive) and the OD value is less than 0.1077 is non-IgA nephropathy patients (negative).

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. An antibody or antigen-binding fragment thereof that binds Gd-IgA1, characterized in that the antibody or antigen-binding fragment thereof comprises 3 heavy chain variable region complementarity determining regions designated HCDR1, HCDR2, and HCDR3, respectively, and 3 light chain variable region complementarity determining regions designated LCDR1, LCDR2, and LCDR3, respectively, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable regions are shown at positions 26 to 33, 51 to 58, and 97 to 111, respectively, of SEQ ID NO:1, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable regions of the antibody are shown at positions 26 to 34, 52 to 54, and 91 to 99, respectively, of SEQ ID NO: 2.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises 4 heavy chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4, respectively, and 4 light chain variable region framework regions named LFR1, LFR2 and LFR3 and LFR4, respectively,

The amino acid sequence of the HFR1 is shown in positions 1 to 25 of SEQ ID NO. 1,

The amino acid sequence of the HFR2 is shown in 34 th to 50 th positions of SEQ ID NO. 1,

The amino acid sequence of the HFR3 is shown in the 59 th to 96 th positions of SEQ ID NO. 1,

The amino acid sequence of the HFR4 is shown in 112 to 122 positions of SEQ ID NO. 1,

The amino acid sequence of the LFR1 is shown in positions 1 to 25 of SEQ ID NO. 2,

The amino acid sequence of the LFR2 is shown in the 35 th to 51 th positions of SEQ ID NO. 2,

The amino acid sequence of the LFR3 is shown in 55 th to 90 th positions of SEQ ID NO. 2,

The amino acid sequence of the 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, wherein 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 shown in positions 1 to 122 of SEQ ID NO. 1 and/or the amino acid sequence of the light chain variable region is shown in positions 1 to 109 of SEQ ID NO. 2.

4. The antibody or antigen-binding fragment thereof of claim 1 to 3, wherein the antibody or antigen-binding fragment thereof further comprises a constant region.

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

6. An expression cassette comprising the nucleic acid molecule of claim 5.

7. A recombinant vector comprising the nucleic acid molecule according to claim 5.

8. A recombinant cell comprising the nucleic acid molecule of claim 5.

9. A recombinant microorganism comprising the nucleic acid molecule according to claim 5.

10. A method for producing an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is as claimed in any one of claims 1 to 4, which comprises the step of expressing a gene encoding the antibody or the antigen-binding fragment thereof in a mammalian cell to obtain the antibody or the antigen-binding fragment thereof.

11. A reagent or kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the reagent or kit has at least one of the following uses:

(B1) Identifying or assisting in identifying Gd-IgA1;

(B2) Binding or assisting in binding Gd-IgA1;

(B3) Detecting or assisting in detecting whether the sample to be detected contains Gd-IgA1;

(B4) Detecting or assisting in detecting the content of Gd-IgA1 in a sample to be detected;

(B5) Diagnosis or auxiliary diagnosis of IgA nephropathy;

(B6) Gd-IgA1 is targeted.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

13. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for binding or aiding in the binding of Gd-IgA1 for non-diagnostic or therapeutic purposes.

14. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for the detection or assisted detection of the presence or absence of Gd-IgA1 in a sample to be tested for non-disease diagnosis or treatment purposes.

15. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for the preparation of a product for detecting or aiding in the detection of the presence or absence of Gd-IgA1 in a sample to be tested.

16. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for the detection or assisted detection of Gd-IgA1 content in a sample to be tested for non-disease diagnosis or treatment purposes.

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

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

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

20. Use of the nucleic acid molecule of claim 5 in at least one of:

(P3) preparing a product for diagnosis or aiding diagnosis of IgA nephropathy;

(P4) preparation of Gd-IgA 1-targeted products.

21. Use of the expression cassette of claim 6 in at least one of:

(P3) preparing a product for diagnosis or aiding diagnosis of IgA nephropathy;

(P4) preparation of Gd-IgA 1-targeted products.

22. Use of the recombinant vector of claim 7 in at least one of:

(P3) preparing a product for diagnosis or aiding diagnosis of IgA nephropathy;

(P4) preparation of Gd-IgA 1-targeted products.

23. Use of the recombinant cell of claim 8 in at least one of:

(P3) preparing a product for diagnosis or aiding diagnosis of IgA nephropathy;

(P4) preparation of Gd-IgA 1-targeted products.

24. Use of the recombinant microorganism of claim 9 in at least one of:

(P3) preparing a product for diagnosis or aiding diagnosis of IgA nephropathy;

(P4) preparation of Gd-IgA 1-targeted products.