Antibodies against galactose-deficient igal and complexes and uses thereof for targeted degradation

Abstract

The present disclosure relates to an anti-galactose-deficient IgA1 protein antibody or antigen-binding fragment thereof. The present disclosure also relates to a complex comprising an antibody binding portion to anti-galactose-deficient IgA1, a cell receptor binding portion to a hepatocyte via an asialoglycoprotein receptor (ASGPR) on the surface of the hepatocyte of a patient or subject, and a linking portion.

Description

Technical Field

[0001] This disclosure generally relates to a novel anti-galactose-deficient immunoglobulin antibody and a bifunctional molecule comprising a novel anti-galactose-deficient immunoglobulin antibody and a cell receptor binding moiety. Background Technology

[0002] IgA nephropathy is one of the most common types of glomerulonephritis worldwide and a leading cause of kidney failure in people under 40. Its clinical manifestations are diverse, ranging from occasional gross hematuria to microscopic hematuria and proteinuria; in rare cases, it can progress to nephrotic syndrome or acute kidney injury. IgA nephropathy has an early onset and a chronic, progressive course; with current treatments, almost all patients develop uremia within their lifetime, highlighting the urgent need for effective treatments to slow disease progression.

[0003] The pathogenesis of IgA nephropathy is not fully understood. Currently, the academic community believes that the pathogenesis originates from abnormal glycosylation of IgA1 O-glycans, leading to the production of galactose-deficient IgA1 (Gd-IgA1). Subsequently, the Gd-IgA1 hinge region is recognized as an autoantigen, triggering the production of anti-Gd-IgA1 autoantibodies, which in turn leads to the formation of circulating immune complexes. These immune complexes deposit in the renal mesangial area and activate inflammatory responses, causing kidney damage. (See Suzuki H et al., *Journal of Clinical Research*, 2009, 119(6), 1668-1677 (Appendix A); Stamellou E et al., *Nature Reviews Disease Primers*, 2023, 9, 67. These references are incorporated herein by reference in their entirety.)

[0004] In recent years, the emergence of lysosome-targeting chimera (LYTAC) technology has provided a new approach to the degradation of extracellular proteins. Currently, there are reports of applying LYTAC to the degradation of Gd-IgA1, utilizing anti-Gd-IgA1 antibodies to achieve targeted degradation of Gd-IgA1. Caianiello DF et al., *Nature Chemical Biology*, 2021, 17(9), 947953 (Appendix A), are cited and incorporated herein by reference in their entirety. CN121038815A discloses the use of an antibody that recognizes the hinge region of human galactose-deficient IgA1 to achieve targeted degradation of Gd-IgA1. Developing highly specific antibodies that recognize Gd-IgA1 will enable better targeted degradation of Gd-IgA1 in IgA nephropathy, promoting the development of precision treatment for IgA nephropathy. Summary of the Invention

[0005] This disclosure provides a novel monoclonal antibody against galactose-deficient IgA1 (GdIgA1) immunoglobulin, its amino acid and nucleotide sequences, and its uses. This disclosure relates to a bifunctional molecule capable of binding to and degrading GdIgA1 immunoglobulin.

[0006] In some embodiments, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to galactose-deficient IgA1. The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH and VL respectively comprise heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3). The amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 5, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 8. The CDRs are determined using the IMGT numbering scheme.

[0007] In some embodiments, this disclosure provides a polynucleotide that encodes an antibody or an antigen-binding fragment thereof provided in this disclosure.

[0008] In some embodiments, this disclosure provides a carrier comprising the polynucleotide provided in this disclosure.

[0009] In some embodiments, this disclosure provides a host cell that includes the vector provided in this disclosure.

[0010] In some embodiments, this disclosure provides a complex comprising an antibody or antigen-binding fragment thereof provided by this disclosure, the antibody or antigen-binding fragment thereof being specifically capable of binding to galactose-deficient IgA1; a cell receptor binding portion connected to the antibody or antigen-binding fragment thereof, the cell receptor binding portion being capable of binding to hepatocytes via the desialylate glycoprotein receptor ASGPR on hepatocytes; and a linker portion connecting the antibody or antigen-binding fragment thereof and the cell receptor binding portion.

[0011] In some embodiments, this disclosure provides a pharmaceutical composition comprising an antibody, a variant thereof, or an antigen-binding fragment thereof provided by this disclosure, or a complex thereof, and a pharmaceutically acceptable excipient.

[0012] In some embodiments, this disclosure provides the use of the pharmaceutical composition provided herein in the preparation of a medicament for removing galactose-deficient IgA1 in a patient or subject in need.

[0013] In some embodiments, this disclosure provides the use of the pharmaceutical composition provided herein in the preparation of a medicament for treating IgA nephropathy in a patient or subject in need. Attached Figure Description

[0014] The following figures form part of this specification and are intended to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by taking into account the detailed description of specific embodiments described herein and by referring to one or more of the accompanying drawings.

[0015] Figure 1 The affinity curves of the monoclonal antibody BD108-114 for the naked peptide (Tn0) of the IgA1 hinge region, the addition of GalNAc modification at 5 common modification sites (Tn5), the addition of GalNAc-Gal modification at 5 common modification sites (T5), the addition of GalNAc-Gal-sialic acid modification at 5 common modification sites (ST5), and the addition of one GalNAc modification at each of the 5 O glycosylation modification sites (Tn1) are shown.

[0016] Figure 2 The affinity assays of the monoclonal antibody BD108-114 for its target antigen glycopeptide and control naked peptide are shown.

[0017] Figure 3 VVL lectin blot analysis of monoclonal antibodies BD108-114 and BD108-114-triGalNAc is shown.

[0018] Figure 4 The flow cytometry fluorescence statistics show the expression levels of ASGPR1 in the constructed stable transgenic human desialylate glycoprotein receptor 1 (hASGPR1) 293T cells and hepG2 cell line.

[0019] Figure 5 Tn0 naked peptide, T 225 -GalNAc glycopeptide, T 228 - Flow cytometry fluorescence statistics of spontaneous endocytosis experiments of GalNAc glycopeptide and Tn5 glycopeptide.

[0020] Figure 6 The results show that BD108-480-triGalNAc and BD108-114-triGalNAc specifically mediate the target glycopeptide T. 228 -GalNAc's internalization.

[0021] Figure 7 A schematic diagram of drug administration in mice is shown.

[0022] Figure 8 The study demonstrated the selective degradation of Gd-IgA1 after administration of BD108-114-triGalNAc.

[0023] Figure 9A These are the results of IgA and DAPI staining in kidney sections after administration of BD108-114-triGalNAc.

[0024] Figure 9B This is the result of quantitative analysis of IgA fluorescence intensity after administration of BD108-114-triGalNAc.

[0025] Figure 10 HE staining of liver, spleen, lungs, and kidneys after administration of different concentrations of BD108-114-triGalNAc is shown. Detailed Implementation

[0026] Those skilled in the art will readily understand the features and advantages of the disclosed compositions and methods by reading the following detailed description. It should be understood that certain features of the disclosed compositions and methods, for clarity, have been described above and below in different embodiment contexts, but may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed compositions and methods described in a single embodiment may also be provided individually or in any sub-combination. Furthermore, unless the context clearly indicates otherwise, singular expressions may also include plural forms (e.g., one may refer to one or more).

[0027] Unless otherwise expressly stated, the values ​​within the various ranges specified in this disclosure are approximate, as are the minimum and maximum values ​​within the ranges, which are preceded by the word "approximately". In this way, slight deviations above and below the ranges can be used to achieve substantially the same results as the values ​​within the ranges. Furthermore, the disclosure of these ranges is intended to represent a continuous range, encompassing every value between the minimum and maximum values.

[0028] In this specification, the word “approximately” and its grammatical equivalents in relation to reference values ​​and their equivalent expressions may include a range of values ​​plus or minus 10%, such as a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. For example, the value “approximately 10” includes values ​​between 9 and 11.

[0029] definition As used in this disclosure, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, bispecific antibody, and antigen-binding fragment capable of binding to a specific antigen. A naturally occurring intact antibody consists of two heavy chains (H chains) and two light chains (L chains). Mammalian heavy chains are classified into α, δ, ε, γ, and μ types, each containing a variable region (VH) and first, second, and third constant regions (CH1, CH2, CH3); mammalian light chains are classified into λ and κ types, each containing a variable region (VL) and a constant region. Antibodies have a "Y"-shaped structure, with the stem formed by the second and third constant regions of the two heavy chains linked by disulfide bonds. Each arm of the "Y" consists of a variable region and a first constant region of one heavy chain, and a variable region and a constant region of the light chain bound to it. Antigen binding is handled by the variable regions of the light and heavy chains. The variable regions of two chains typically contain three high-variable rings, called complementarity-determining regions (CDRs). The CDRs for the light chain include LCDR1, LCDR2, and LCDR3, while the CDRs for the heavy chain include HCDR1, HCDR2, and HCDR3. The CDR boundaries of the antibodies described in this disclosure may be defined or marked according to conventions such as those of Kabat, IMGT, Chothia, or Al Lazikani (Al Lazikani, B., Chothia, C., Lesk, AM, *Journal of Molecular Biology*, 273(4), 927 (1997); Chothia, C. et al., *Journal of Molecular Biology*, Dec. 5; 186(3):65163 (1985); Chothia, C. and Lesk, AM, *Journal of Molecular Biology*, 196, 901 (1987); Chothia, C. et al., *Nature*, Dec. 2128; 342(6252):87783 (1989); Kabat EA et al., *National Institutes of Health*, Bethesda, Md.). (1991); Marie Paule Lefranc et al., Developmental and Comparative Immunology, 27:5577 (2003); Marie Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie Paule Lefranc, Molecular Biology of B cells (2nd Edition), Chapter 26, 481514, (2015).Interspersed among the three CDRs are flanking sequences called framework regions (FRs). FRs are more conserved than CDRs and form a scaffold structure supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but mediate various effector functions. Antibodies can be classified based on the amino acid sequence of the heavy chain constant region. The five major antibody classes or isotypes are IgA, IgD, IgE, IgG, and IgM, characterized by α, δ, ε, γ, and μ heavy chains, respectively. Some major antibody classes can be further subdivided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), and IgA2 (α2 heavy chain).

[0030] As used in this disclosure, the term "variable domain" refers to an antibody variable region or fragment thereof that contains one or more complementarity-determining regions (CDRs). While a variable domain may encompass an entire variable region (e.g., a heavy chain variable region HCVR or a light chain variable region LCVR), it may also contain a sequence less than the entire variable region, as long as the sequence retains the ability to bind to an antigen or form an antigen-binding site.

[0031] As used in this disclosure, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody (comprising one or more CDRs), or any other antibody fragment capable of binding to an antigen but not containing the complete structure of a native antibody. Examples of antigen-binding fragments include, but are not limited to: variable domains, variable regions, bifunctional antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), multispecific antibodies, camelified single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds. In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a specific human antibody, wherein said CDRs are grafted into a frame region from one or more different human antibodies. Further and more detailed forms of antigen-binding fragments (also known as antigen-binding moieties) are described in Spiess et al., Molecular Immunology, 67(2), 95106 (2015) and Brinkman et al., mAbs, 9(2), 182212 (2017), which are incorporated herein by reference in their entirety.

[0032] The "Fab" in antibody refers to the part of the antibody that consists of a single light chain (containing a variable region and a constant region) and a single heavy chain whose variable region and first constant region are linked by disulfide bonds.

[0033] “Fab” refers to a Fab segment that includes a portion of the hinge area.

[0034] “F(ab')2” refers to the dimer of Fab'.

[0035] For antibodies (such as IgG, IgA, or IgD isotypes), the term "Fc" refers to the portion of the antibody that consists of a second constant domain (CH2) and a third constant domain (CH3) of the first heavy chain bound to the CH2 and CH3 domains of the second heavy chain via disulfide bonds. For IgM and IgE isotype antibodies, the Fc portion further includes a fourth constant domain (CH4). The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not participate in antigen binding.

[0036] In the context of antibodies, the term "hinge region" refers to the heavy chain molecule portion that connects the CH1 and CH2 domains. This hinge region contains approximately 25 amino acid residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0037] As used in this disclosure, the term "CH2 domain" refers to the portion of a heavy chain molecule that is defined using a conventional numbering system, such as in IgG antibodies, extending from approximately amino acid 244 to amino acid 360 (amino acids 244–360, Kabat numbering system; or amino acids 231–340, EU numbering system); see portions of heavy chain molecules in Kabat, E. et al., U.S. Department of Health and Human Services (1983).

[0038] The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 amino acid residues. Some immunoglobulin classes (such as IgM) also further include a CH4 region.

[0039] In the context of antibodies, the term "Fv" refers to the smallest fragment of an antibody carrying an intact antigen-binding site. An Fv fragment consists of a variable region of a single light chain combined with a variable region of a single heavy chain. Various Fv designs have been provided in the prior art, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond; additionally, two domains can be linked together as a single polypeptide chain using a peptide linker, thus forming a single-chain Fv (scFv). Prior art has also produced Fv constructs comprising variable domains of heavy or light chain immunoglobulin chains associated with the variable and constant domains of the corresponding immunoglobulin heavy or light chains. Fvs can also be polymerized to form bifunctional and trifunctional antibodies (see Maynard et al., *Annual Review of Biomedical Engineering*, 2, 339-376 (2000)).

[0040] The terms “camelized single-domain antibody,” “heavy chain antibody,” or “HCAb” refer to antibodies containing two VH domains but not a light chain (see Riechmann L. and Muyldermans S., *Journal of Immunological Methods*, December 10; 231(1-2):25-38 (1999); Muyldermans S., *Journal of Biotechnology*, June; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies originally came from the Camelidae family, which includes camels, dromedary camels, and llamas. Despite lacking light chains, camel-type antibodies still possess a true antigen-binding spectrum (see Hamers Casterman C. et al., Nature, June 3; 363(6428):446-448 (1993); Nguyen VK. et al., Immunogenetics, April; 54(1):39-47 (2002); Nguyen VK. et al., Immunology, May; 109(1):93-101 (2003)). The variable domain (VHH domain) of heavy chain antibodies is the smallest known antigen-binding unit generated by adaptive immune responses (see Koch-Nolte F. et al., FISB Journal of Experimental Biology, November; 21(13):3490-3498, e.g., June 15, 2007 (2007)).

[0041] The term "bifunctional antibody" or "dAb" refers to a small antibody fragment having two antigen-binding sites, wherein the fragment contains a VH domain linked to a VL domain on the same polypeptide chain (i.e., VH-VL or VL-VH) (see, for example, Holliger P. et al., Proceedings of the National Academy of Sciences, July 15; 90(14):6444-6448 (1993); EP404097; WO93 / 11161). When a linker is used, if the linker is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, thereby creating two antigen-binding sites. These antigen-binding sites may target the same or different antigens (or epitopes). In some embodiments, the term "bispecific ds bifunctional antibody" refers to a bifunctional antibody that targets two different antigens (or epitopes).

[0042] As used in this disclosure, the term "valence" refers to the presence of a specified number of antigen-binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment having only one antigen-binding site; the term "polyvalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. Accordingly, the terms "bivalent," "trivalent," "tetravalent," and "hexavalent" indicate the presence of two, three, four, and six binding sites in an antigen-binding molecule, respectively. In some embodiments, the antibody or its antigen-binding fragment is bivalent.

[0043] As used in this disclosure, the term "bispecific" antibody refers to an artificial antibody having fragments derived from two different monoclonal antibodies and capable of binding to two different epitopes. The two epitopes may be present on the same antigen or on two different antigens.

[0044] As used in this disclosure, the term "multispecific" antibody refers to an artificial antibody having fragments derived from more than two different monoclonal antibodies and capable of binding to more than two different epitopes. The more than two epitopes may be present on the same antigen or on more than two different antigens.

[0045] As used in this disclosure, the term "chimerism" refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species, and the remainder of a heavy chain and / or light chain derived from a different species. In one exemplary embodiment, a chimeric antibody may comprise a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0046] As used in this disclosure, the term "humanized" refers to an antibody or antigen-binding fragment comprising a CDR derived from a non-human animal, an FR region derived from a human, and (where applicable) a constant region derived from a human.

[0047] As used in this disclosure, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0048] As used in this disclosure, the term "specific binding" refers to a non-random binding reaction between two molecules (e.g., antibody and antigen). Specific binding can be characterized by binding affinity, for example, expressed as a KD value, where KD is the ratio (koff / kon) of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium. The KD value can be determined by any conventional method known in the art, including but not limited to surface plasmon resonance, microthermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). A KD value ≤ 10... -6 M (e.g., ≤5×10) -7 M, ≤2×10 -7 M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M or ≤10 -9 M) can indicate a specific binding between an antibody or its antigen-binding fragment and the corresponding antigen.

[0049] As used in this disclosure, the term "epitope" refers to a specific set of atoms or amino acid residues on an antigen to which an antibody binds. If two antibodies exhibit competitive binding against an antigen, they bind to the same or closely related epitopes within the antigen. Epitopes can be linear or conformational (i.e., conformational epitopes consist of amino acid residues spaced apart on the primary sequence of the antigen).

[0050] The term "sequence identity percentage (%)" for amino acid sequences (or nucleic acid sequences) refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment, and, where necessary, the introduction of vacancies to achieve the maximum number of identical amino acid (or nucleic acid) residues. In other words, the sequence identity percentage (%) of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) identical to those in the reference sequence by the total number of amino acid residues (or bases) in either the candidate or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues can (but must) be considered identical residues. Alignments used to determine the percentage of sequence identity can be performed using tools well-known in the art, such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI); see also Altschul SF et al., *Molecular Biology Journal*, 215:403-410 (1990); Stephen F. et al., *Nucleic Acid Research*, 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute; see also Higgins DG et al., *Enzymological Methods*, 266:383-402 (1996); Larkin MA et al., *Bioinformatics*, 23(21):2947-2948 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the aforementioned tools, or can appropriately customize the parameters according to the needs of the alignment, for example, by selecting a suitable algorithm.

[0051] As used in this disclosure, the term “mutated” or “mutated” with respect to amino acid residues refers to the substitution, insertion, or deletion of one or more amino acid residues.

[0052] As used in this disclosure, the term “substitute” or “substituted” in relation to amino acid sequences means replacing a natural amino acid residue with a different amino acid residue.

[0053] The term "conservative substitution" in relation to amino acid sequences refers to the replacement of an existing amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitution can occur between amino acid residues of the following categories: amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile); amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln); amino acid residues with acidic side chains (e.g., Asp, Glu); amino acid residues with basic side chains (e.g., His, Lys, and Arg); and amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitution generally does not cause significant changes in protein conformation and structure, and therefore preserves the protein's biological activity. As used in this disclosure, the term "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof. Unless explicitly limited, this term encompasses polynucleotides containing known analogs of natural nucleotides, which have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Research, 19:5081 (1991); Ohtsuka et al., Journal of Biochemistry, 260:2605-2608 (1985); and Rossolini et al., Molecular and Cellular Probes, 8:91-98 (1994)).

[0054] Antibody IgA1 protein, or immunoglobulin A1, is one of the two subtypes of human IgA protein. It primarily participates in mucosal immune defense (respiratory and digestive tract defense), binding to viral and bacterial toxins and preventing their entry into cells. IgA1 protein is mainly distributed in serum and is the most abundant form of IgA in blood, accounting for approximately 80–90% of serum IgA. IgA1 has a typical antibody structure, namely a Y-shaped structure composed of two heavy chains and two light chains.

[0055] Unlike other antibodies (such as IgA2), IgA1 has a relatively long hinge region (i.e., a segment of amino acid sequence connecting the antigen-binding fragment (Fab) and the crystallizable fragment (Fc) of the IgA1 molecule). Generally, the hinge region of IgA1 is the proline residue (P221) at position 221.221 Histidine (H) up to position 243 243 The region between the two halves of the hinge is rich in proline, serine, and threonine, and contains a large number of O-glycosylation sites. It is a core structural feature that distinguishes IgA1 from other immunoglobulin subtypes.

[0056] Abnormal glycosylation of IgA1, especially galactose-deficient IgA1 (Gd-IgA1), is one of the core mechanisms in the development and progression of IgA nephropathy. Normally, the IgA1 molecule contains multiple O-glycosylation sites in its hinge region. These sites are initially modified by N-acetylgalactosamine (GalNAc), and subsequently further linked with galactose (Gal) by β1,3-galactosyltransferase (C1GALT1) and its molecular chaperone Cosmc, and may be modified by sialic acid to form a complete O-glycosylation structure. However, in some individuals, due to reduced C1GALT1 activity, abnormal Cosmc function, or premature action of sialyltransferases (such as ST6GalNAc-II), galactose cannot be added normally, resulting in Gd-IgA1 exposed to GalNAc. This abnormal structure is recognized as "non-self" by the immune system, inducing the production of autoantibodies (mostly IgG or IgA) against Gd-IgA1. Subsequently, Gd-IgA1 binds to these antibodies, forming large circulating immune complexes that are difficult to clear. These complexes eventually deposit in the glomerular mesangial area, activating the complement system and inducing an inflammatory response. This leads to mesangial cell proliferation and matrix expansion, ultimately causing kidney damage. This process represents a continuous pathological chain from abnormal molecular modification to immune dysregulation and then to organ damage, and is an important basis for the pathogenesis of IgA nephropathy.

[0057] Therefore, this disclosure provides, in one aspect, an antibody or antigen-binding fragment thereof that specifically binds to galactose-deficient IgA1, said antibody or antigen-binding fragment comprising a heavy chain variable region VH and a light chain variable region VL, said VH and VL comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, characterized in that the amino acid sequence of said HCDR1 is as shown in SEQ ID NO: 3, the amino acid sequence of said HCDR2 is as shown in SEQ ID NO: 4, the amino acid sequence of said HCDR3 is as shown in SEQ ID NO: 5, the amino acid sequence of said LCDR1 is as shown in SEQ ID NO: 6, the amino acid sequence of said LCDR2 is as shown in SEQ ID NO: 7, and the amino acid sequence of said LCDR3 is as shown in SEQ ID NO: 8, wherein said CDRs are determined by the IMGT numbering scheme. In some embodiments, said antibody or antigen-binding fragment thereof is capable of specifically binding to galactose-deficient IgA1 protein (Gd-IgA1) having said Tn antigen. In some embodiments, the antibodies or antigen-binding fragments provided in this disclosure can be used to remove Gd-IgA1 from patients or subjects in need, thereby treating or alleviating Gd-IgA1-induced nephropathy.

[0058] In some implementations, "O-glycosylation" refers to the addition of N-acetylgalactosamine residues to serine or threonine residues of a correctly folded protein substrate, catalyzed by tissue-specific N-acetylgalactosamine transferase.

[0059] As used in this disclosure, the term "Tn antigen" refers to a structure covalently linked to the monosaccharide N-acetylgalactosamine on a serine or threonine residue, namely GalNAcα1-O-Ser / Thr. The Tn antigen structure is the starting point for O-glycosylation and is formed through the catalysis of polypeptide α-galactosamine transferase (ppGalNAcT). Adding a galactose (Gal) residue to the Tn antigen structure further forms the T antigen; adding a sialic acid (Sia) residue to the T antigen structure forms the ST antigen.

[0060] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment have at least 70% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the paired VH and VL shown in Table 1, respectively.

[0061] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the antibody or its antigen-binding fragment are identified by any of the following numbering schemes: Kabat, IMGT, AbM, Chothia, Aho, and Contact.

[0062] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in the antibody or its antigen-binding fragment have the same amino acid sequence as the paired HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 shown in Table 2, or are variants thereof with no more than two or one amino acid substitutions.

[0063] In some embodiments, the VH and VL in the antibody or its antigen-binding fragment have the same amino acid sequence as the paired VH and VL shown in Table 1, or are homologous sequences having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with them.

[0064] Table 1. Amino acid sequences of the heavy and light chain variable regions of antibodies or their antigen-binding fragments. Table 2. CDR amino acid sequences of antibodies or their antigen-binding fragments In some embodiments, the antibody further comprises an immunoglobulin constant region, optionally a human immunoglobulin constant region, or optionally a human IgG constant region.

[0065] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0066] In some embodiments, the antibody is a bispecific antibody or a multispecific antibody.

[0067] In some embodiments, the antibody or its antigen-binding fragment is attached to one or more conjugate moieties.

[0068] In some embodiments, the conjugate portion comprises a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetic modulating portion, or a purified portion.

[0069] Polynucleotides In one aspect, this disclosure provides a polynucleotide encoding an antibody or antigen-binding fragment thereof provided in this disclosure.

[0070] In some embodiments, the antibody or its antigen-binding fragment may contain one or more modifications that introduce or remove glycosylation sites.

[0071] In some embodiments, the polynucleotide comprises one or more nucleotide sequences and / or homologous sequences encoding antibodies or antigen-binding fragments thereof provided herein, and / or variants having only degenerate substitutions, said homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity. The DNA encoding the monoclonal antibody is easily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[0072] In some embodiments, the polynucleotide sequences comprise SEQ ID NO: 9-10, and their homologous sequences, wherein the homologous sequences have at least 80% sequence identity with SEQ ID NO: 9-10.

[0073] In some embodiments, the homologous sequence encodes the same protein as encoded by SEQ ID NO: 9-10.

[0074] Table 3. Nucleic acid sequences encoding the heavy and light chain variable regions of antibodies or their antigen-binding fragments. In one aspect, this disclosure provides a carrier comprising the polynucleotide provided in this disclosure.

[0075] In some embodiments, polynucleotides may be inserted into a vector using recombination techniques known in the art for further cloning (DNA amplification) or expression. A variety of vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence. Vectors may also contain materials that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein envelopes.

[0076] In one aspect, this disclosure provides a host cell that includes the vector provided in this disclosure.

[0077] In some embodiments, a vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment may be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA of the vectors provided in this disclosure include prokaryotes, yeast, or higher eukaryotic cells. The host cell is transformed with a suitable expression or cloning vector to produce the antibody provided in this disclosure, and cultured in a modified conventional nutrient medium suitable for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequence. In another embodiment, the antibody may be prepared using homologous recombination methods known in the art.

[0078] complex In some embodiments, this disclosure provides a complex comprising (1) an antibody, a variant thereof, or an antigen-binding fragment thereof provided in this disclosure, the antibody or antigen-binding fragment thereof being specifically capable of binding to Gd-IgA1, (2) a cell receptor binding portion connected to the antibody, a variant thereof, or an antigen-binding fragment thereof, the cell receptor binding portion being capable of binding to hepatocytes via the desialylate glycoprotein receptor (ASGPR) of hepatocytes, and (3) a linker portion connecting the antibody, a variant thereof, or an antigen-binding fragment thereof and the cell receptor binding portion.

[0079] As used in this disclosure, the term "desialidated glycoprotein receptor" or "ASGPR" refers to a high-affinity C-type lectin receptor that is primarily located on the surface of hepatocytes, consists of ASGPR-1 and ASGPR-2 subunits, and binds to the galactose (Gal) and N-acetylgalactosamine (GalNAc) at the desialylated glycoprotein terminus, subsequently triggering internalization and degradation (see Ramírez-Cortés F et al. (2024), RSC Med Chem, 2024, 16, 525-544).

[0080] As used in this disclosure, the term "cell receptor" is a protein located on the surface of a cell that binds to a compound (e.g., a ligand, such as a protein) in solution or on another cell. Typically, ligand-receptor binding induces one or more biological responses. In this disclosure, the desialylate glycoprotein receptor (ASGPR) is a cell receptor located on the surface of hepatocytes or other cells that binds to desialylate glycoprotein or a derivative thereof. The term "cell receptor-binding moiety" has a generally accepted meaning in the biomedical field, and in this disclosure, the cell receptor-binding moiety is the desialylate glycoprotein receptor (ASGPR) binding group.

[0081] In some embodiments, the complex has the structure of formula [I]: [A] a -[L-[B] b ]c [I] Or a pharmaceutically acceptable salt thereof, wherein: a is 1, b is 3, c is 1; each A is an antibody, a variant thereof, or an antigen-binding fragment thereof provided in this disclosure; L is a linker portion comprising -[(CH2)] n -O] m - where n is 2 and m is 4; and each B is independently a cell receptor binding portion that is capable of binding to hepatocytes via ASGPR.

[0082] In some implementations, each B includes an N-acetyl-D-galactosamine (GalNAc) moiety.

[0083] In some embodiments, the Gd-IgA1 binding portion is a polypeptide having the complementarity-determining region (CDR) of the antibody provided in this disclosure.

[0084] In some embodiments, the cell receptor binding portion includes . residues.

[0085] In some implementations, L is reactively linked to the lysine residues exposed on the upper surface of A.

[0086] In some embodiments, L is covalently linked to the primary amine N atom on the lysine residue.

[0087] In some embodiments, L is connected to B via an azide-dibenzocyclooctyryl cycloaddition reaction.

[0088] Pharmaceutical Compositions and Applications In some embodiments, this disclosure provides a pharmaceutical composition comprising an antibody, a variant thereof, or an antigen-binding fragment thereof provided by this disclosure, or a composition of substances provided by this disclosure, and a pharmaceutically acceptable excipient.

[0089] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may comprise, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, clamping or chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[0090] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers (such as sugars and cyclodextrins). Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in this disclosure, the inclusion of one or more antioxidants (such as methionine) in a composition comprising an antibody or antigen-binding fragment and a substance as provided in this disclosure will reduce the oxidation of the antibody or antigen-binding fragment. This reduction in oxidation will prevent or reduce the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Therefore, in some embodiments, compositions comprising one or more antibodies disclosed in this disclosure and one or more antioxidants (such as methionine) are provided. Methods are also provided for preventing the oxidation of said antibody or antigen-binding fragment, extending its shelf life, and / or improving its efficacy by mixing said antibody or antigen-binding fragment as provided in this disclosure with one or more antioxidants (such as methionine).

[0091] Pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc. In some embodiments, unit doses of parenteral formulations are packaged in ampoules, vials, or syringes with needles. All formulations intended for parenteral administration shall be sterile and pyrogen-free, as is known and practiced in the art.

[0092] In some embodiments, the sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed in this disclosure in a suitable solvent. The solvent may contain excipients that will improve the stability of the powder or other pharmacological component or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable reagents. The solvent may contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art; in one embodiment, the buffer is substantially neutral pH. The solution is then sterilely filtered and subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of the antibody or a combination thereof provided in this disclosure. Overfilling the vials by a small amount (e.g., about 10%) beyond the amount required for a single dose or a set of doses is acceptable to facilitate accurate sample extraction and precise administration. The lyophilized powder can be stored under suitable conditions, such as at approximately 4°C to room temperature. The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. In one embodiment, for reconstitution, sterile and / or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder. The precise amount depends on the selected therapy and can be determined empirically.

[0093] In some embodiments, this disclosure provides the use of the pharmaceutical composition provided herein in the preparation of a medicament for removing galactose-deficient IgA1 in a patient or subject in need.

[0094] In some embodiments, this disclosure provides the use of the pharmaceutical composition provided herein in the preparation of a medicament for treating IgA nephropathy in a patient or subject in need.

[0095] The term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably in this disclosure.

[0096] The term "IgA nephropathy," also known as Berger's disease, has a generally accepted meaning in the biomedical field as the most common form of primary glomerulonephritis worldwide. The disease is named based on the pathological characteristics of IgA deposition in the glomeruli. It is estimated that 15–20% of patients with IgA nephropathy will develop end-stage renal disease within 20 years of disease onset (see Samuels JA et al. (2004), *Nephrology (Carlton)*, 2004, 9, 177–185). The IgA in the glomerular mesangial deposits originates solely from the IgA1 subclass and is aberrantly glycosylated, lacking galactose (Gal) in its hinge region O-linked glycan. Circulating IgA1 in patients with IgA nephropathy also carries galactose-deficient O-glycans, a type of galactose deficiency rarely found in serum IgA1 of normal individuals. These variants arise from altered expression of specific glycosyltransferases in the cells that produce IgA1. IgA1 immune complexes containing aberrantly glycosylated IgA1 bind to glomerular mesangial cells, inducing renal manifestations of IgA nephropathy (see Suzuki et al., Journal of Clinical Research, 119, 1668-1677 (2009)).

[0097] As used in this disclosure, the terms “treatment,” “treatment,” or “method” for a condition may be used interchangeably and include therapeutic treatments, preventive or defensive measures, such as preventing or alleviating the condition, slowing the onset or progression of the condition, reducing the risk of disease, preventing or delaying the development of disease-related symptoms, alleviating or eliminating disease-related symptoms, causing complete or partial remission of the disease, curing the disease, or any combination thereof.

[0098] The following embodiments are provided to better illustrate the claimed invention and should not be construed as limiting the scope of this disclosure. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of this disclosure. These specific compositions, materials, and methods are not intended to limit this disclosure but are merely illustrative of specific embodiments falling within the scope of this disclosure. Those skilled in the art can develop equivalent compositions, materials, and methods without departing from the scope of this disclosure and without exceeding the ordinary capabilities of the art. It should be understood that many changes can be made to the processes described herein while still remaining within the scope of this disclosure. It is the inventors' intention that these changes are also included within the scope of this disclosure.

[0099] Example Example 1. Development and characterization of Gd-IgA1-specific monoclonal antibodies 1. Development of Gd-IgA1-specific monoclonal antibodies 1.1 Immunization of mice with glycopeptide antigens Five common sites (T) in the hinge region of the in vitro synthesized IgA1 molecule were analyzed. 225 T 228 S 230 S 232 T 236 References for the above sites: Hui GK et al., Solution structures of natural and patient-derived monomeric human IgA1 reveal asymmetric extended conformations: implications for function and IgAN disease. Biochemical Journal (2015, Vol. 471, No. 2.) Modified GalNAc Tn5 glycopeptide (Shanghai Aminolink Biotechnology Co., Ltd.) was used as an immunogenic, sorting, and detection antigen. First, the target glycopeptide Tn5 synthesized in vitro was conjugated with the carrier protein KLH (Shanghai Aminolink Biotechnology Co., Ltd.) and combined with adjuvants (CFA, Sigma, F5881) / incomplete Freund's adjuvants (IFA, Sigma, F5506) to 6-7 week old female BALB / c mice, and immunized four times at two-week intervals. Specifically, the following glycopeptides were administered: target glycopeptide-KLH (100 μg / 100 μL) + CFA (100 μL), target glycopeptide-KLH (100 μg / 100 μL) + IFA (100 μL), target glycopeptide-KLH (100 μg / 100 μL) + IFA (100 μL), and target glycopeptide-KLH (100 μg / 100 μL) + IFA (100 μL). The mice were sacrificed and their spleens harvested 3 days after immunization.

[0100] 1.2 Sorting mouse spleen cells containing target glycopeptides as mono-B cells Mouse spleens were ground and filtered to obtain single-cell suspensions. B cells were screened using 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 that bound to the target glycopeptide were sorted using Biotin (Shanghai Ammonia Biotechnology Co., Ltd.) and PE-streptavidin (405204, BioLegend).

[0101] 1.3 Antibody Sequencing RNA was obtained from lysed B cells; cDNA was obtained by reverse transcription using the RNA as a template; PCR was performed using heavy / light chain variable region gene primers using the cDNA as a template, and the V region gene sequence was obtained by sequencing. The amino acid residue sequence of the heavy / light chain variable region was obtained after gene sequence translation. The complementarity-determining regions (CDRs) of the heavy and light chain variable regions were defined according to the IMGT numbering scheme. The heavy chain variable region (VH) or light chain variable region (VL) of the antibody consists of a frame region separated by three complementarity-determining regions. The frame region is used to align the CDRs that specifically bind to the antigen epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively, in this paper; the CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively, in this paper. The sequence information of the complementarity-determining region (CDR) of the heavy and light chain variable regions of the obtained monoclonal antibody BD108-114 is shown in Table 2, and the sequence information of the heavy chain variable region (VH) or light chain variable region (VL) is shown in Table 1.

[0102] 1.4 Construction of recombinant expression vectors The heavy chain variable region gene of the monoclonal antibody BD108-114 obtained in step 1.3 was constructed into pCMV3-CH (heavy chain), and the corresponding light chain variable region gene was constructed into pCMV3-CK (light chain kappa). Homologous recombination was performed using the ClonExpress II OneStep Cloning Kit (Novizan catalog number: C112-02), and the vectors were transformed into DH5α (Competentcell, Qingke catalog number: TSC-C01) to obtain expression vectors for the heavy and light chains of the monoclonal antibody numbered BD108-114.

[0103] 1.5 In vitro expression of antibodies Dilute ExpiCHO-S™ cells (ThermoFisher, catalog number A29127) to 6 × 10⁶ using ExpiCHO™ expression medium (Gibco, A29100-02). 6 Cell / mL. 2.5 mL of cells were dispensed into each well of a 24-well deep-well plate for transfection.

[0104] Add 200 μL of ExpiFectamine reagent-DNA complex to each well containing culture in a 24-well deep plate, and cover the reaction plate with a breathable sealing membrane. The ExpiFectamine reagent-DNA complex is prepared as follows: mix the heavy and light chain plasmids constructed in step 1.4 at a 1:1 ratio, add 9.0 μL of ExpiFectamine CHO reagent (Gibco, A29129), and 189 μL of OptiPRO SFM (Gibco, 12309-050).

[0105] Incubate the 24-well deep-well plate on an orbital shaker at 37 °C in humidified air containing 8% CO2. 18–22 hours after transfection, add ExpiFectamine CHO enhancer (Gibco, A29129) and ExpiCHO excipient (Gibco, A29129). For the entire 24-well plate, mix 400 μL of ExpiFectamine CHO enhancer and 16 mL of ExpiCHO excipient in a conical tube. Add 600 μL of this mixture to each well of the 24-well plate. Return the 24-well plate to the incubator at 37 °C with 8% CO2 and shake. On day 8 post-transfection, centrifuge the plate (500 g, 10 min) and collect the culture supernatant for antibody purification.

[0106] 1.6 Antibody purification and detection Protein A beads (GenScript, L00695) for purification were added to the expression supernatant (400 μL magnetic beads / 5 mg protein) at the specified ratio 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 automated nucleic acid extractor for purification (two PBS washes, one distilled water wash, and one Elution elution of 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 collected, which is the purified antibody solution, i.e., the required monoclonal antibody, for subsequent experiments.

[0107] 2. Antibody specificity for recognizing IgA1 hinge region glycoforms To test the recognition specificity of candidate antibodies for GalNAc glycoform modifications, different types of peptides / glycopeptides were synthesized, specifically including: IgA1 hinge region naked peptide (Tn0), five common sites (T 225 T 228 S 230 S 232T 236 The study synthesized Tn5 glycopeptides modified with GalNAc and glycopeptides modified with one GalNAc at each of the five sites (synthesized by Shanghai Aminolink Biotechnology Co., Ltd.). Furthermore, T5 was generated by chemically modifying Tn5 with galactose (Gal), and sT5 was generated by chemically modifying T5 with sialic acid, thus producing different types of glycopeptides.

[0108] The above nine peptides / glycopeptides were coated onto plates at a concentration of 1 µg / mL (4°C, overnight). Antigen-free coating solution served as a blank control. After blocking with 5% sodium casein (Beyotime, ST1132-100g) at 37°C for 2 hours, the previously obtained antibody solution (containing monoclonal antibody BD108-114) was added as the primary antibody. Eight primary antibody concentrations were prepared (starting at 1 μg / mL, serially diluted 3 times). Goat anti-human IgG(H+L)-HRP was used as the secondary antibody (Jackson, 109-035-003, 1:2000 dilution). TMB colorimetry (Solepro, PR1200) and a stop reaction (Solepro, C1058) were used. The absorbance (OD value) at 450 nm was read. GraphPad Prism statistical software was used to process the data, and the experimental results of antibody-peptide / glycopeptide binding were fitted to curves and the EC50 was calculated.

[0109] Results of the detection of binding specificity of different glycopeptides are shown in Figure 1 The corresponding EC50 affinity results are shown in Table 4. BD108-114 does not recognize Tn0, T5, and sT5, but specifically recognizes Tn5, and also recognizes T... 228 and T 236 Site modification of GalNAc.

[0110] Table 4. EC50 results of binding specificity detection for different glycopeptides. 3. Antibody affinity Surface plasmon resonance (SPR) experiments were performed on a Biacore 8K+ (12914229, Cytiva). Biotin-labeled glycopeptides or naked peptides were captured using an SA-conjugated chip. After binding for 60 s, gradient concentrations of the target antibody (0.78125 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) were introduced, with binding for 120 s and dissociation for 600 s. The signal values ​​(RU) generated by the sample-sample interaction were acquired at room temperature using Biacore 8K Control Software (v4.0.8.19879, Cytiva). The collected raw data were analyzed using Biacore 8K Evaluation Software 3.0 (v4.0.8.20368, Cytiva), and the affinity constant (KD) was obtained by fitting the raw data curves to a 1:1 binding model.

[0111] The results are as follows Figure 2 As shown, for the IgA1 hinge region, BD108-114 does not significantly bind to Tn0, and T 228 -GalNAc has a dissociation constant of 2.3 pM, indicating strong binding.

[0112] Example 2. Construction of a lysosomal-targeting chimera to degrade Gd-IgA1 1. Constructing a lysosomal-targeting chimera based on an anti-Gd-IgA1 monoclonal antibody. The BD108-114 antibody was chemically conjugated to triGalNAc via click. The antibody concentration was adjusted to 1 mg / mL with 100 mM NaHCO3. NHS-PEG4-azide (Bioruler, K90540) at a molar ratio of 1:25 was added to the antibody solution, and the reaction was allowed to proceed overnight with shaking at room temperature. Tris-HCl at pH 8.0 (50 mM) was added, and the reaction was allowed to proceed for 30 minutes with shaking at room temperature to quench the reaction. PBS was used as the replacement buffer. Then, Tri-GalNAc-DBCO (MCE, HY-148476) at a molar ratio of 1:100 was added to the antibody solution (1 mg / mL), and the reaction was allowed to proceed overnight with shaking at room temperature. PBS was used as the replacement buffer. The successful conjugation of triGalNAc was verified using VVL-biotin (vector laboratories, B-1235-2) and SA-HRP (vector laboratories, SA-5014-1) as the primary and secondary antibodies, respectively, for Western blotting. Figure 3 The VVL lectin (which specifically recognizes GalNAc) blot shows that triGalNAc was successfully conjugated to the antibody.

[0113] 2. Lysosomal targeting chimera-mediated in vitro degradation of target antigens 2.1 Construction of 293T cells stably expressing human desialyl glycoprotein receptor 1 (hASGPR1) First, a lentiviral expression plasmid carrying the full-length coding sequence of human hASGPR1 and the puromycin resistance gene was constructed. Using the polyethyleneimine (PEI) method, a plasmid mixture (10 µg human hASGPR1 expression plasmid, 7.5 µg pSPAX2, and 2.5 µg pMD2.G) was co-transfected into 293T cells for lentiviral packaging. The viral supernatant was collected and used to infect 293T cells. After puromycin-induced selection, a stable 293T cell line highly expressing hASGPR1 was obtained. Figure 4 The flow cytometry results showed that the ASGPR positivity rate was over 95%, significantly higher than that of hepG2 cells.

[0114] 2.2 Spontaneous endocytosis experiment of peptides / glycopeptides 293T cells expressing human ASGPR in good condition (viability ≥95%) were collected by digestion and cultured. The cell culture medium was adjusted to a concentration of 2 × 10⁻⁶ cells using DMEM (CORNING, 10-013-CVRC) + 1% FBS (NEWZERUM, FBS-E500). 6 50 μL was added to each well of a 96-well plate (1×10⁻⁶ cells / mL). 5 Cells / well), cultured at 37°C, 5% CO2 for 6 hours until adherent. Peptide labeling: Tn0, Tn1-T 228 The initial concentration of Tn5 was 1 mg / ml. 0.33 μL of each was mixed with 20.67 μL of water and 9 μL of PE Streptavidin (Biolegend, 405204) dye for labeling, and incubated at 4°C in the dark for 30 min. The labeled peptides were then serially diluted 5-fold with cell culture medium to a maximum final concentration of 250 ng / ml, and 50 μL was added to each well of the aforementioned cell culture plate. After overnight incubation, cells were collected by centrifugation, resuspended in 150 μL of PBS, and analyzed by flow cytometry (BD, LSR Tortessa X20). The mean fluorescence intensity of PE was recorded. The results showed that Tn5 exhibited spontaneous internalization, while Tn0 and Tn1-T... 228 The absence of spontaneous endocytosis suggests that this peptide can be used as a test peptide in endocytosis experiments. Figure 5 ).

[0115] 2.3 Internalization of test peptides by antibodies 293T cells expressing ASGPR were cultured and labeled with the test peptide as described above. The labeled peptide was then diluted with cell culture medium to a final concentration of 10 ng / mL, and 25 μL was added to each well of a new 96-well plate. 25 μL of serially diluted BD108-114 and BD108-114-triGalNAc were added, with a maximum final antibody concentration of 10 μg / mL. The antibody and peptide were mixed and incubated at room temperature for 30 minutes. Finally, the antibody-peptide complex was added to the wells of adherent cells, with a final volume of 100 μL. After overnight incubation, cells were collected by centrifugation, resuspended in 150 μL of PBS, and analyzed by flow cytometry (BD, LSR Tortessa X20). The mean fluorescence intensity of PE was recorded. The results showed that BD108-114-triGalNAc mediates Tn1-T... 228 Dose-dependent endocytosis; while control peptides Tn0 and Tn1-T 225 The control antibody BD108-114, which was not conjugated with triGalNAc, showed no significant endocytosis at any concentration. Figure 6 ).

[0116] Example 3. In vivo testing 1. Testing the degradation of Gd-IgA1 in a passive mouse model of IgA nephropathy IgA molecules were purified from the plasma of patients with IgA nephropathy using the method described above. Male BALB / c mice aged 6-8 weeks were injected intravenously with 500 μg (100 μL) of IgA derived from the plasma of patients with IgA nephropathy. Three minutes later, orbital venous blood was collected, and each group was then injected intravenously with 25 μL of BD108-114-triGalNAc (0 μg [PBS], 0.3125 μg, 0.625 μg, 1.25 μg, 2.5 μg, and 5 μg, respectively). Orbital venous blood was collected before administration, 30 minutes after administration, and 60 minutes after administration. Two hours later, the mice were sacrificed, and blood and tissue samples from the kidneys, liver, spleen, and lungs were collected for paraffin and frozen section preparation. Figure 7 Five mice were in each group.

[0117] Table 5. Drug concentrations in mice 2. Changes in circulating total IgA and Gd-IgA1 after drug administration ELISA was used to detect IgA and Gd-IgA1 levels in mouse plasma. Anti-human IgA antibody (Jackson, 109-006-011) and BD108-114 were coated onto ELISA plates and incubated overnight at 4 °C. After washing and blocking, diluted mouse plasma samples (10,000-fold dilution for total IgA detection and 50-fold dilution for Gd-IgA1 detection) were added and incubated at room temperature. After washing, enzyme-labeled secondary antibody (Abcam, Ab97215) was added and incubated at room temperature. Finally, after chromogenic development, the absorbance was measured, and the remaining amounts of total IgA and Gd-IgA1 recognized by the two different antibodies were calculated.

[0118] The experimental results show that BD108-114-triGalNAc does not significantly degrade total IgA, but selectively degrades Gd-IgA1 specifically by BD108-114. Figure 8 BD108-114-triGalNAc exhibits a hook effect in the selective degradation of the corresponding Gd-IgA1, with the optimal degradation concentration being 1.25 μg after 0.5 h–2 h of drug administration. Figure 8 ).

[0119] 3. Changes in renal IgA deposition after drug administration Fresh mouse kidney tissue was collected after culling and immediately fixed in 4% paraformaldehyde at room temperature for 48 hours. After fixation, the tissue was dehydrated with a gradient of sucrose solutions (10%, 30%), embedded in OCT embedding medium, flash-frozen in liquid nitrogen, and stored at -80°C. Sections with a thickness of 10 μm were prepared using a cryostat (Leica CM-1950) and attached to anti-detachment slides. Tissue sections were circled on slides using a Biosharp (BC004) histochemical pen. After washing with PBS, 3% BSA was added for blocking at room temperature for 1 hour. Mouse kidney sections were repaired with pepsin antigen retrieval solution (Solarbio, X1035) at 37°C for 15 minutes. After washing with PBS, IgA primary antibody (Abcam, ab124716, 1:100) was added and incubated overnight at 4°C. After washing with PBS, IgA fluorescent secondary antibody (Abcam, ab150079, 1:500) was added and incubated at room temperature for 1 hour. After washing with PBS, anti-quenching mounting medium (containing DAPI) (SouthernBiotech, 0100-20) was added and the sections were mounted with coverslips. Images were acquired using a confocal microscope (ZEISS, LSM 900), and quantitative analysis was performed using ImageJ software (v1.54p). The average fluorescence intensity of the target signal was statistically analyzed.

[0120] The results showed that when BD108-114-triGalNAc was administered at a dose of 1.25 μg, the level of IgA deposited in the kidneys was the lowest. Figure 9A -B).

[0121] 4. Safety evaluation of tissues and organs To assess safety, fresh mouse kidney, liver, spleen, and lung tissues were immediately fixed in 4% paraformaldehyde at room temperature for 48 hours. After fixation, the tissues were dehydrated with a gradient of ethanol (70%, 80%, 90%, 95%, 100%), cleared with xylene, and embedded in paraffin. Sections of 4 μm thickness were prepared using a microtome, mounted on glass slides, and dried. After dewaxing with xylene and hydration with a gradient of ethanol, the sections were stained with hematoxylin for 3-10 minutes, rinsed with tap water to regain blue color, differentiated with hydrochloric acid alcohol for a few seconds, and counterstained with eosin for 1-3 minutes. Finally, the sections were dehydrated with a gradient of ethanol, cleared with xylene, and mounted with neutral resin. Images were acquired using a digital slide scanner. Results showed that BD108-114-triGalNAc did not cause tissue damage, inflammatory infiltration, or morphological abnormalities. Figure 10 Specifically, under magnification, hepatocytes show a central vein, neatly arranged hepatocytes, hepatic cords, and sinusoids, with no inflammatory cell infiltration, cell necrosis, or significant pathological structural changes; the liver structure is intact. Under magnification, lung cells show alveoli, alveolar walls, and alveolar sacs, with no alveolar dilation, alveolar wall thickening, inflammatory cell infiltration, or significant pathological changes; the lung structure is intact. Under magnification, spleen cells show distinct trabeculae, red pulp (splenic sinusoids and cords), and white pulp (lymphoid follicles and marginal zone), with no inflammatory cell infiltration or other significant pathological structural changes; the spleen structure is intact. Under magnification, kidney cells show glomeruli, renal tubules, and renal interstitium, with clearly defined glomerular and tubular structures, no inflammatory cell infiltration, no lesions in the glomeruli or tubules, and no other significant pathological changes; the kidney structure is intact.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to galactose-deficient IgA1, said antibody or antigen-binding fragment comprising a heavy chain variable region VH and a light chain variable region VL, said VH and VL respectively comprising heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3, characterized in that, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 5, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

8. The CDRs are determined using the IMGT numbering scheme.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of VH is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO:

2.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The immunoglobulin constant region of the antibody is the constant region of human immunoglobulin.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a humanized antibody.

5. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or its antigen-binding fragment according to claim 1.

6. The polynucleotide according to claim 5, wherein the polynucleotide sequence comprises a heavy chain coding sequence and a light chain coding sequence, characterized in that, The heavy chain coding sequence is shown in SEQ ID NO: 9, and the light chain coding sequence is shown in SEQ ID NO:

10.

7. A carrier, characterized in that, The carrier comprises the polynucleotide according to claim 5 or 6.

8. A host cell, characterized in that, The host cell includes the vector according to claim 7.

9. A complex, characterized in that, The complex has the structure of formula [I]: [A] a -[L-[B] b ] c [I] Or its pharmaceutically acceptable salt, wherein: a is 1, b is 3, c is 1; Each A is an antibody or its antigen-binding fragment as described in claim 1; L is the connecting portion, which includes -[(CH2)] n -O] m - where n is 2 and m is 4; and Each B is an independent cell receptor-binding moiety that can bind to hepatocytes via ASGPR.

10. The complex according to claim 9, characterized in that, Each B includes the N-acetyl-D-galactosamine GalNAc moiety.

11. The complex according to claim 9, characterized in that, The cell receptor binding portion includes . residues.

12. The complex according to claim 9, characterized in that, L reacts and connects with the lysine residues exposed on the upper surface of A.

13. The complex according to claim 12, characterized in that, L is covalently linked to the primary amine N atom on the lysine residue.

14. The complex according to claim 9, characterized in that, L is linked to B via an azide-dibenzocyclooctyne cycloaddition reaction.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to claim 1, or the complex according to claim 9, and a pharmaceutically acceptable excipient.

16. Use of the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating IgA nephropathy.

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