Phospholipase a2 receptor polypeptides and uses thereof

Abstract

Phospholipase A2 receptor polypeptides and uses thereof are provided. Polypeptides are described herein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 1-37, SEQ ID NO: 39, SEQ ID NO: 41-47. The polypeptides described herein are used for the treatment, prevention, and / or diagnosis of primary membranous nephropathy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically to polypeptide drugs for the treatment, prevention and / or diagnosis of primary membranous nephropathy. Background Technology

[0002] Primary membranous nephropathy (pMN) is a kidney-specific autoimmune disease, with the M-type phospholipase A2 receptor (PLA2R) as its main target antigen. Multiple studies have shown that the proportion of PLA2R-related MN in pMN patients varies between 44% and 82% across different ethnicities and regions. PLA2R is not expressed in the glomeruli of rodents, thus hindering the establishment of animal models of PLA2R-related membranous nephropathy, and direct evidence of pathogenicity from anti-PLA2R antibodies has remained unproven in animal models. Until 2020, researchers established a transgenic mouse strain expressing the full-length murine PLA2R gene in glomerular podocytes.

[0003] For pathogenic target antigens, the region that truly induces an immune response is the antigenic epitope (antigenic determinant). To date, research on the antigenic epitope of PLA2R has mainly focused on B-cell conformational epitopes. Kao et al. (Kao L, Lam V, Waldman M, et al. Identification of the immunodominant epitope region in phospholipase A2 receptor-mediating autoantibody binding in idiopathic membranous nephropathy. J Am Soc Nephrol. 2015, 26(2):291-301) designed and recombined the complete PLA2R protein and cleaved it into different domains using proteases.

[0004] Fresquet et al. (Fresquet M, Jowitt TA, Gummadova J, et al. Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy. J Am Soc Nephrol. 2015, 26(2):302-313) found that Western blot experiments had limitations in revealing antigen-antibody interactions, failing to further reveal the antigen reactivity of smaller N-terminal regions. Subsequently, Fresquet used surface plasmon resonance (SPR) experiments to find that the affinity of purified anti-PLA2R antibody for CysR was consistent with that of the intact recombinant PLA2R protein.

[0005] In addition to the dominant epitope, PLA2R may also contain extended epitopes associated with disease progression and outcome. Seitz-Polski et al. (Seitz-Polski B, Dolla G, Payré C, et al. Epitope Spreadingof Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy. J Am Soc Nephrol. 2016, 27(5):1517-1533) proposed that patient serum can recognize CysR, CysR-FnII-CTLD1, and CysR-FnII-CTLD1-7. The proportion of patients recognizing CysR is significantly higher than that recognizing CTLD1 or CTLD7. Therefore, it is inferred that in addition to the main CysR epitope, CTLD1 and CTLD7 are the second and third epitopes that have spread out. They also found that patients who recognized the spreading epitopes CTLD1 and / or CTLD7 had higher anti-PLA2R antibody titers, more severe disease, and worse prognosis than those who only recognized CysR. Patients who only recognized CysR were most likely to achieve spontaneous remission, while those who recognized CTLD1 and / or CTLD7 were more likely to present with persistent proteinuria and develop end-stage renal disease. Multivariate regression analysis showed that the spread of antigenic epitopes was an independent risk factor for clinical non-remission in patients. Subsequently, Reinhard's team (Reinhard L, Zahner G, Menzel S, et al. Clinical Relevance of Domain-Specific Phospholipase A2 Receptor 1 Antibody Levels in Patients with Membranous Nephropathy. J Am Soc Nephrol. 2020, 31(1):197-207) found that, in addition to CysR, CTLD1, and CTLD7, CTLD8 was the fourth epitope recognized in patient serum. At a serum dilution of 1:100, patients recognizing the C-terminal epitope had higher antibody titers compared to those recognizing the N-terminal epitope, consistent with the findings of Seitz-Polski et al. However, by adjusting the experimental methods and changing the serum dilution ratio, researchers found that 100% of patients could recognize both the N-terminal and C-terminal epitopes, inferring that the body's immune system had already completed epitope expansion against PLA2R at the onset of the disease. Therefore, PLA2R possesses different B-cell epitope repertoires, resulting in a variety of different anti-PLA2R antibodies.As immune disorders begin and progress, humoral immunity gradually matures, the epitope repertoire of B cells expands from a single cell to multiple cells, antibody titers increase, and eventually clinical manifestations appear.

[0006] There is a need in the art for peptides used to treat, prevent and / or diagnose membranous nephropathy, such as primary membranous nephropathy. Summary of the Invention

[0007] This invention is partly based on the inventors' discovery that, during their research on T-cell epitopes related to phospholipase A2 receptor-related primary membranous nephropathy, they unexpectedly discovered that multiple polypeptides of the phospholipase A2 receptor could be used to treat, prevent, and / or diagnose membranous nephropathy. Membranous nephropathy can be primary membranous nephropathy.

[0008] In one aspect, the present invention provides a polypeptide comprising one or more of the amino acid sequences shown in Table 1.

[0009] Table 1: Synthetic PLA2R peptides

[0010]

[0011]

[0012]

[0013] In one aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 1-37, SEQ ID NO: 39, SEQ ID NO: 41-47.

[0014] In another aspect, the present invention provides a nucleic acid comprising a polynucleotide sequence encoding the polypeptide described herein.

[0015] In another aspect, the present invention provides a carrier comprising the nucleic acid described herein.

[0016] In another aspect, the present invention provides a host cell comprising the nucleic acids or vectors described herein. The host cell may be a eukaryotic cell or a prokaryotic cell. The eukaryotic cell may be a yeast cell, an insect cell, or an animal cell. The prokaryotic cell may be a bacterial cell, such as an Escherichia coli cell.

[0017] In another aspect, the present invention provides compositions comprising the polypeptides, nucleic acids, vectors and / or host cells described herein.

[0018] In another aspect, the present invention provides the use of the polypeptides, nucleic acids, carriers, host cells, and compositions described herein for the preparation of medicaments for the treatment, prevention, and / or diagnosis of primary membranous nephropathy.

[0019] In another aspect, the present invention provides the use of polypeptides, nucleic acids encoding polypeptides, vectors containing the nucleic acids, host cells containing the vectors, or compositions containing one or more of these in the preparation of a kit for (1) predicting clinical remission or renal function in patients with membranous nephropathy after treatment; (2) determining the positivity status of anti-PLA2R antibodies in patients with membranous nephropathy; or (3) determining the severity of membranous nephropathy.

[0020] In one implementation, the polypeptide is a polypeptide described herein, such as the polypeptides in Table 1.

[0021] In one embodiment, the polypeptide is selected from SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 1-28, SEQ ID NO: 30, SEQ ID NO: 32-37, SEQ ID NO: 39, and SEQ ID NO: 41-43.

[0022] In one implementation, severity includes one or more of the following: higher levels of urinary protein, higher levels of albumin, more severe chronic tubulointerstitial damage, or more severe acute tubulointerstitial damage compared to patients with anti-PLA2R antibody-negative membranous nephropathy; or the treatment includes treatment with angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, immunosuppressants, calcineurin inhibitors, cyclophosphamide, and / or rituximab.

[0023] In one implementation, the patient expresses HLA DRB1. 1501 molecules and / or HLA DRB1 0301 molecule.

[0024] In one implementation, membranous nephropathy is primary membranous nephropathy.

[0025] In another aspect, the present invention provides the use of polypeptides, nucleic acids encoding polypeptides, vectors containing the nucleic acids, host cells containing the vectors, or compositions containing one or more of these in the preparation of medicaments for the prevention or improvement of membranous nephropathy in patients expressing these polypeptides.

[0026] In one embodiment, the polypeptide is a polypeptide described herein, such as the polypeptides in Table 1. In one embodiment, the polypeptide is selected from: SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:29, or SEQ ID NO:31.

[0027] In one implementation, the patient expresses 1, 2, 3, or 4 of the following: HLA DRB1 1501 molecule, HLA DRB1 0301 molecule, HLA DRB1 0901, HLA DRB1 0701.

[0028] In one implementation, membranous nephropathy is primary membranous nephropathy.

[0029] In another aspect, the present invention provides the use of polypeptides, nucleic acids encoding polypeptides, vectors containing said nucleic acids, host cells containing said vectors, or compositions containing one or more of these in the preparation of a medicament for use in expressing DRB1. 1501 and / or DRB1 Among patients with membranous nephropathy in 0301, and DRB1 1501 and DRB1 0301 binds but does not cause T cell activation, or may be used for the prevention or treatment of membranous nephropathy.

[0030] In one embodiment, the polypeptide is a polypeptide described herein, such as the polypeptides in Table 1. In one embodiment, the polypeptide is selected from SEQ ID NO:28, SEQ ID NO:32, and SEQ ID NO:6.

[0031] In one implementation, membranous nephropathy is primary membranous nephropathy.

[0032] In another aspect, the use of polypeptides described herein, such as the polypeptide of SEQ ID NO: 38, nucleic acids encoding the polypeptides, vectors containing the nucleic acids, host cells containing the vectors, or compositions containing one or more of these in the preparation of a medicament for inducing the release of anti-inflammatory factors or inducing a Treg response in patients with membranous nephropathy, or for the prevention or treatment of membranous nephropathy. In one embodiment, membranous nephropathy is primary membranous nephropathy.

[0033] In another aspect, it provides the ability to block HLA DRB1 in vitro. 1501 molecules and / or HLA DRB1 The 0301 molecule or a method of binding to the T cell receptor on the surface of T cells without activating T cells includes causing the polypeptide described herein to bind to HLA DRB1. 1501 molecules and / or HLA DRB1 0301 molecule or T-cell receptor contact. The polypeptide may be selected from: SEQ ID NO:28, SEQ ID NO:32, or SEQ ID NO:6. In one embodiment, the HLA DRB1... 1501 molecules and / or HLA DRB1 The 0301 molecule is present on the surface of cells (such as peripheral blood mononuclear cells).

[0034] The advantages of this invention include:

[0035] 1. The antibody recognition of the peptides described in this article, such as CysR2 / 4 / 6, CTLD6-6-1, and CTLD7-11, is related to the level of antibodies against the intact PLA2R antigen in patients and the severity of the disease. Antibodies targeting linear epitopes of the PLA2R autoantigen can be detected in patient plasma. The recognized continuous amino acid sequence CTLD8-8, CTLD8-10, and CTLD8-12 may represent a conformational structure and is associated with pathogenesis. Recognition of linear peptides CysR2 / 4 / 6, CTLD6-6-1, and CTLD7-11 is associated with disease severity and may be epitopes related to disease progression.

[0036] 2. The plasma of pMN patients showed a high positive recognition rate for the polypeptide described in this article.

[0037] 3. Some of the peptides described in this article are related to DRB1. 1501 and DRB1 Both 0301 exhibit high bonding strength.

[0038] 4. Some of the peptides described in this article can be bound by protective HLA molecules but cannot be bound by susceptible HLA molecules.

[0039] 5. Some of the peptides described in this article are related to DRB1. 1501 and DRB1 0301 binds but does not induce T cell activation or has an anti-inflammatory effect.

[0040] 6. The indices of T-cell proliferation induced by some of the peptides described in this article are correlated with patients' baseline clinical characteristics and prognosis. Attached Figure Description

[0041] Figure 1The recognition rate of PLA2R linear peptides in the plasma of patients with PLA2R-related pMN. The recognition rate of linear peptides in the patient's plasma for a. CysR and FnII regions; b. CTLD1 and CTLD2 regions; c. CTLD3 and CTLD4 regions; d. CTLD5 and CTLD6 regions; e. CTLD7 and CTLD8 regions.

[0042] Figure 2 Clinical relevance analysis of patients identifying peptides: a. Patients with pMN identified CysR2 / 4 / 6 had higher serum albumin levels; b. Patients with pMN identified CTLD6-6-1 had higher anti-PLA2R antibody titers; c. Patients with pMN identified CTLD7-11 had higher serum albumin levels; d. Patients with higher anti-PLA2R antibody titers.

[0043] Figure 3 PLA2R peptide co-localizes with DRB molecules rather than DQ molecules. CTLD7-1 and CTLD7-2 can bind to DRB1. b. Co-localization of HLA-DRB1 molecules on the surface of 1501 homozygous cells. CTLD7-1 and CTLD7-2 do not co-localize with DRB1. HLA-DQ molecules co-localize on the surface of 1501 homozygous cells.

[0044] Figure 4 The binding of HLA molecules to PLA2R peptides. Based on the full-length amino acid sequence of the PLA2R whole antigen, a group of 123 overlapping PLA2R peptides were chemically synthesized. The binding affinity of each peptide to HLA molecules expressed on the following four homozygous human B cell lines was detected by flow cytometry: susceptible DRB1 1501, Susceptibility DRB1 0301, Protective DRB1 0901, Protective DRB1 0701 homozygous human B cell line. Binding capacity (%) is expressed as the percentage of all B cells that are double-positive for both DRB1 and PLA2R peptides (%) minus the percentage of cells that bind nonspecifically (%). Binding capacity is categorized as high (dark), moderate (medium), low, and no binding (light).

[0045] Figure 5 Proliferation index of CD4+ T cells stimulated by different PLA2R peptides: a. Proliferation index of T cells in pMN patients stimulated by peptides; b. Proliferation index of T cells in healthy controls stimulated by peptides; c. Comparison of the proliferation index of T cells in pMN patients (left column of paired columns) and healthy controls (right column of paired columns) stimulated by peptides. p<0.05, p<0.01, p<0.001). e peptide proliferation index of T cells in different pMN patients (left side of paired bars: DRB1) 1501 1501, DRB1 1501 0301 Right side: DRB1 1501 / Other, DRB1 0301 / Other). Patients with higher SI levels of fCTLD7-1-stimulated CD4+ T cells had a slower antibody seroconversion time than patients with lower SI levels.

[0046] Figure 6 : The cytokine profile induced by PLA2R peptide in Th cells (a, IL-6; b, IL-10; c, IL-17F; d, TNF-α; e, IL-9; f, cytokines produced in the culture supernatant of PBMCs from patients carrying risk genes after stimulation by different peptides; most peptides induced higher levels of IL-4 secretion in cells from patients carrying two risk genes, while CTLD7-1 induced higher levels of secretion of multiple cytokines in cells from patients carrying two risk genes; g, the positivity rate of each cytokine after peptide stimulation of patient PBMCs).

[0047] Figure 7 The cytokine profile induced by PLA2R peptide from Th cells (a, IL-17A; b, IL-22; c, IL-13; d, IFN-γ; e, IL-5; f, IL-2; g, IL-4). Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0049] definition

[0050] As used herein, a polypeptide refers to a group of amino acid residues linked by peptide bonds. The polypeptides described herein are polypeptides derived from the M-type phospholipase A2 receptor. Polypeptides can be synthetic or recombinant. Polypeptides in this article encompass variants obtained by appropriate mutations of the parent polypeptide, such as substitution, addition, deletion, or insertion. The number of mutated amino acid residues can be one or more.

[0051] As used herein, "nucleic acid" refers to a plurality of nucleotides linked together by nucleotides. This linkage can be, for example, a phosphodiester bond. Nucleic acid as used herein may comprise a polynucleotide encoding a polypeptide of the present invention.

[0052] As used herein, the term "vector" is a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may contain the nucleic acids of this invention for introduction into cells for expression. Vectors may contain expression control elements operatively linked to the nucleic acids, such as promoters, terminators, and / or enhancers.

[0053] As used herein, the term "host cell" refers to a cell into which nucleic acid molecules have been introduced using molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle gun techniques. Host cells can be eukaryotic or prokaryotic cells. For example, eukaryotic cells include yeast cells, animal cells, and / or insect cells. Prokaryotic cells can be E. coli cells.

[0054] As used in this article, the term "membranous nephropathy (MN)" is a glomerular disease clinically characterized by varying degrees of proteinuria, mostly nephrotic syndrome, and may be accompanied by hematuria. Globally, membranous nephropathy is a leading cause of nephrotic syndrome in adults. In 75%–80% of cases of membranous nephropathy, the cause is unknown and is termed primary membranous nephropathy (pMN); 20%–25% are caused by various other diseases: infections (hepatitis B, hepatitis C, etc.), medications (nonsteroidal anti-inflammatory drugs, metals, etc.), malignancies (lung, kidney, stomach, colon, etc.) or autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), urticarial vasculitis, sarcoidosis, thyroiditis, Sjögren's syndrome, systemic sclerosis, or ankylosing spondylitis. The clinical presentations are similar between primary and secondary membranous nephropathy, but there are significant differences in immunohistochemistry and disease progression. In primary membranous nephropathy, immune complexes are deposited only in the subepithelial and intramembranous regions, while secondary membranous nephropathy is characterized by mesangial proliferation and usually also has mesangial and subendothelial deposits.

[0055] As used in this article, PLA2R is a glycoprotein encoded by the PLA2R gene. PLA2R is a member of the C-type lectin superfamily and the mannose receptor family, which in mammals also includes the cation-dependent mannose-6-phosphate receptor, C-type mannose receptor 2, Endo180, and dendritic cell receptor DEC-205. This family is a group of transmembrane glycoproteins with similar domain structures, including an N-terminal cysteine-rich domain, a type II fibronectin domain, and 8–10 C-type lectin-like domains. They have a short cytoplasmic domain associated with endocytosis, enabling the internalization of extracellular ligands. Two types of PLA2R have been identified based on the ligand sPLA2: M-type (muscular type or PLA2R1) and N-type (neuronal type or PLA2R2). Of these two types, the M-type receptor, PLA2R1, has been the most studied, and it is also the M-type receptor, PLA2R1, that is associated with primary membranous nephropathy. However, for convenience, most studies on membranous nephropathy often refer to it as PLA2R. PLA2R is a 180 kDa protein. Its extracellular domain begins with a cysteine-rich domain (CysR) as its head, followed by a fibronectin type II domain (FnII), and then eight C-type lectin-like domains (CTLDs). These domains exhibit at least two conformations: an extended conformation, where the N-terminal domain extends outward from the cell surface; and a bent conformation, where the N-terminal domain folds backward and contacts the CTLDs. Conformational changes, and whether the structure is compact or loose, are mainly regulated by pH. Cryo-electron microscopy shows that PLA2R forms a dense bicyclic conformation under acidic pH conditions and an expanded conformation under alkaline pH conditions. PLA2R is expressed in various human tissues. In the kidney, PLA2R is expressed only in podocytes. The primary physiological function of PLA2R is as a receptor for soluble phospholipase A2 (sPLA2). pH-dependent structural changes in PLA2R allow it to bind to its ligand at physiological pH, releasing the ligand in acidic lysosomes. Upon binding to sPLA2, PLA2R downregulates the associated inflammatory cascade by inhibiting sPLA2 phospholipase activity. Furthermore, the binding of sPLA2 to PLA2R enhances the release of lipid mediators and / or activates specific signaling pathways, such as cell proliferation, migration, contraction, and secretion. Studies have found that the FnII and CTLD1-2 domains of PLA2R1 play a role in activating cell signaling, such as inducing cell death and mediating collagen-dependent migration and proliferation, while CTLD3-5 is the region where sPLA2 binds. Under pathological conditions, such as bacterial infection, asthma, and renal failure causing excessive inflammatory responses, the expression and signaling of PLA2R can change.PLA2R expression may be upregulated under various inflammatory stimuli (including cytokines and bacterial wall lipopolysaccharides), and elevated PLA2R levels can lead to autoimmune diseases. For example, there is a close link between elevated PLA2R levels or polymorphisms at this locus in human glomerular podocytes and the development of primary membranous nephropathy. However, to date, the regions and mechanisms by which PLA2R plays a role in the pathogenesis of primary membranous nephropathy remain unclear.

[0056] As used in this article, anti-PLA2R antibodies are antibodies against PLA2R produced by patients with primary membranous nephropathy. Circulating anti-PLA2R antibodies are specific to primary membranous nephropathy but can also be found in secondary membranous nephropathy. Several studies in China have shown that the detection rate of anti-PLA2R antibodies in pMN ranges from 59% to 82%. International studies, based on their research populations, have found that anti-PLA2R antibody titers are closely related to disease activity, can predict treatment response and renal prognosis, and can predict disease relapse, among other things.

[0057] As used in this article, the HLA region is located on chromosome 6 and is one of the most densely populated parts of DNA. Its gene products include antigen-binding molecules, receptors, and signaling factors. HLA molecules can be further divided into classes I, II, and III. GWAS studies on primary membranous nephropathy have found that variations in the HLA region are closely related to the disease. Currently, HLA-DRB1 has been identified. 1501 and HLA-DRB1 The 0301 genotype is an independent risk factor for membranous nephropathy in the Han Chinese population. A study (Cui Z, Xie LJ, Chen FJ, et al. MHC Class II Risk Alleles and Amino Acid Residues in Idiopathic Membranous Nephropathy. J Am Soc Nephrol. 2017, 28(5):1651-1664) also found that DRB1... 0901 was the strongest protective factor against membranous nephropathy in the Han Chinese population (OR=0.16, P<0.001), suggesting that DRB1 0901 is a protective genotype for membranous nephropathy in the Han Chinese population; GWAS (Xie J, Liu L, Mladkova N, et al. The genetic architecture of membranous nephropathy and its potential to improve non-invasive diagnosis. Nat Commun. 2020, 11(1):1600) studies have shown that DRB1 0701 is a protective genotype for membranous nephropathy in European populations. In this study, patients expressed HLA-DRB1. 1501, HLA-DRB1 0301, DRB1 0701 or DRB1 One or more of 0901, and / or the patient may be homozygous or heterozygous.

[0058] The peptides in this article

[0059] This invention provides polypeptides with multiple functions. These polypeptides can function in patients carrying risk genes.

[0060] 1. Prognostic peptides

[0061] This article presents prognostic peptides. Antibody recognition of peptides CysR2 / 4 / 6, CTLD6-6-1, and CTLD7-11 is associated with the level of antibodies against the intact anti-PLA2R antigen and the severity of the disease. CTLD6-6-1 showed the highest recognition rate, which was correlated with higher anti-PLA2R antibody titers at diagnosis; the latter is a prognostic indicator of clinical non-remission and renal insufficiency. In autoimmune responses, autoreactive B cells targeting CTLD6-6-1 may continuously stimulate autoreactive T cells, leading to persistent autoimmune reactions and the continuous production of autoantibodies, thus causing more severe kidney damage. Therefore, CTLD6-6-1 may be a linear epitope associated with disease progression. Anti-PLA2R antibodies are closely related to clinical presentation; patients with positive antibodies have more severe disease and are less likely to achieve clinical remission.

[0062] Antibodies against linear epitopes of PLA2R autoantigens can be detected in patient plasma. The recognized sequence of CTLD8-8, CTLD8-10, and CTLD8-12 is a continuous amino acid sequence that may be a conformational structure and is associated with the pathogenesis.

[0063] The identification of peptides such as CysR2 / 4 / 6, CTLD6-6-1, and CTLD7-11 is related to the severity of the disease. The amino acid sequences of various peptides are provided below.

[0064] CTLD6-6-1:FGQTTSVWIG (SEQ ID NO:1)

[0065] CysR2:SESLKKCIQAGKSVL (SEQ ID NO:2)

[0066] CysR4:LTLENCKQANKHMLW (SEQ ID NO:3)

[0067] CysR6:MLWKWVSNHGLFNIGGS (SEQ ID NO:4)

[0068] CysR2 / 4 / 6: SESLKKCIQAGKSVLTLENCKQANKHMLWKW VSNHGLFNIGGS (SEQ ID NO:5)

[0069] CTLD7-11:TACESFLQGAIC (SEQ ID NO:6)

[0070] CTLD8-8:KWFDGTPTDQSNWGIRKPDT (SEQ ID NO:7)

[0071] CTLD8-10:DTDYFKPHHCVALRIPEGLWQL (SEQ ID NO:8)

[0072] CTLD8-12:SPCQEKKGFICK (SEQ ID NO:9)

[0073] 2. Plasma from pMN patients showed a high positive recognition rate for the following peptides.

[0074] The peptides described in this article can be identified with high recognition rates from pMN patient plasma, such as the following peptides:

[0075] CysR10:LSLYECDSTLVSLRWRCNRK(SEQ ID NO:10)

[0076] CTLD3-12: KHCRHFKAMSLC (SEQ ID NO:11)

[0077] CTLD8-8 / 10 / 12:KWFDGTPTDQSNWGIRKPDTDYFKPHHCVALRIPEG LWQLSPCQEKKGFICK(SEQ ID NO:12)

[0078] FnII1:THGMPCMFPFQYNHQWHHEC(SEQ ID NO:13)

[0079] FnII3:TREGREDDLLWCATTSRYER(SEQ ID NO:14)

[0080] FnII1 / 3:THGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYER(SEQ ID NO:15)

[0081] CTLD2-10:SQLCVSAEQSEGHWKVKNCE(SEQ ID NO:16)

[0082] CTLD2-11:EGHWKVKNCEERLFYICK(SEQ ID NO:17)

[0083] CTLD2-10 / 11:SQLCVSAEQSEGHWKVKNCEERLFYICK(SEQ ID NO:18)

[0084] CTLD7-3: IKTCLMHKAQLVSITDQYHQSF (SEQ ID NO:19)

[0085] CTLD7-4:SITDQYHQSFLTVVLNRLGY(SEQ ID NO:20)

[0086] CTLD7-6:AHWIGLFTDTDNGLNFDWSDG(SEQ ID NO:21)

[0087] CTLD7-8:TKSSFTFWKDEESSLLGDCV(SEQ ID NO:22)

[0088] CTLD7-3 / 4 / 6 / 8:IKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHW IGLFTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCV(SEQ ID NO:23)

[0089] CTLD8-9: SNWGIRKPDTDYFKPHHCVA (SEQ ID NO:24)

[0090] CTLD8-11:LRIPEGLWQLSPCQEKKGFI(SEQ ID NO:25)

[0091] CTLD8-9 / 11:SNWGIRKPDTDYFKPHHCVALRIPEGLWQLSPCQEKKGFI(SEQ ID NO:26)

[0092] The peptide described in this article can interact with DRB1. 1501 and DRB1 0301 all exhibit high binding affinity. The polypeptide sequence may include:

[0093] CysR1:KGIFVIQSESLKKCI(SEQ ID NO:27)

[0094] CysR3:IQAGKSVLTLENCKQ(SEQ ID NO:28)

[0095] CysR10:LSLYECDSTLVSLRWRCNRK(SEQ ID NO:10)

[0096] CysR12:LRWRCNRKMITGPLQYS(SEQ ID NO:30)

[0097] FnII-3:TREGREDDLLWCATTSRYER(SEQ ID NO:14)

[0098] CTLD2-9:TLEPHIFPNRSQLCVSAEQS(SEQ ID NO:32)

[0099] CTLD3-4:VTITNRFEQAFITSLISSVVKM(SEQ ID NO:33)

[0100] CTLD3-9:QYTHWNTHQPRYSGGCVAMR(SEQ ID NO:34)

[0101] CTLD3-10:RYSGGCVAMRGRHPLGRWEV(SEQ ID NO:35)

[0102] CTLD3-11:GRHPLGRWEVKHCRHFKAMS(SEQ ID NO:36)

[0103] CTLD4-10:DNTYFGEDARNCAVYKANKT(SEQ ID NO:37)

[0104] CTLD5-2-1:FASEWLNFEF(SEQ ID NO:38)

[0105] CTLD6-5:VEQAFITMNLFGQTTSVWIG(SEQ ID NO:39)

[0106] CTLD7-1:YGNRTYKIINANMTWYAAIK(SEQ ID NO:40)

[0107] CTLD7-2:INANMTWYAAIKTCLMHKAQLV(SEQ ID NO:41)

[0108] CTLD8-1-1:IKFKSNCYSFST(SEQ ID NO:42)

[0109] CTLD8-5-2:GSSVQMVWLN(SEQ ID NO:43)

[0110] 3. Peptides that are bound by protective HLA molecules but cannot be bound by susceptible HLA molecules.

[0111] The peptides described in this article can be bound only to protective HLA molecules but not to susceptible HLA molecules. The reason they can play a protective role may be that the protective HLA molecules present antigenic peptides, which activate anti-inflammatory T cells and prevent disease.

[0112] CTLD3-5: TSLISSVVKMKDSYFWIALQ (SEQ ID NO:44)

[0113] CTLD3-12: KHCRHFKAMSLC (SEQ ID NO:45)

[0114] CTLD5-1:YQDAEYLFHTFASEWLNFEF(SEQ ID NO:46)

[0115] CTLD6-2:PKDPSSWKNWTHAQHFCAEE(SEQ ID NO:47)

[0116] CysR7: GLFNIGGSGCLGLNF (SEQ ID NO:29)

[0117] CTLD2-6:SETWIGLSSNKIPVSFEWSN (SEQ ID NO:31).

[0118] 4. With DRB1 1501 and DRB1 0301 binds to peptides that do not induce T cell activation

[0119] This article provides information related to DRB1. 1501 and DRB1 0301 binds to a polypeptide that does not induce T cell activation.

[0120] CysR3:IQAGKSVLTLENCKQ(SEQ ID NO:28)

[0121] CTLD2-9:TLEPHIFPNRSQLCVSAEQS(SEQ ID NO:32)

[0122] CTLD7-11:TACESFLQGAIC (SEQ ID NO:6)

[0123] 5. Peptides with anti-inflammatory effects

[0124] This article presents peptides with anti-inflammatory effects. For example, the peptide CTLD5-2-1 primarily induces the secretion of the cytokine IL-10, an anti-inflammatory cytokine. Generally, Treg cells maintain immune tolerance by producing IL-10, inhibiting B cell maturation and antibody production, suppressing the secretion of pro-inflammatory factors, and so on. In pMN, Tregs are known to play a crucial role; patients with pMN show a significantly reduced percentage of Treg cells at baseline, which increases after rituximab treatment.

[0125] CTLD5-2-1:FASEWLNFEF (SEQ ID NO: 38)

[0126] 6. Able to induce higher levels of CD4 in the patient group. + T cell proliferation peptides

[0127] The following peptides in this article can induce higher levels of CD4 in the patient group. + T cell proliferation: CysR1, CysR10, CysR12, FnII-3, CTLD3-9, CTLD3-10, CTLD3-11, CTLD5-2-1, CTLD7-1, and CTLD7-2. The index of T cell proliferation induced by peptides can be correlated with patient baseline clinical characteristics and prognosis.

[0128] CysR1:KGIFVIQSESLKKCI(SEQ ID NO:27)

[0129] CysR10:LSLYECDSTLVSLRWRCNRK(SEQ ID NO:10)

[0130] CysR12:LRWRCNRKMITGPLQYS(SEQ ID NO:30)

[0131] FnII-3:TREGREDDLLWCATTSRYER(SEQ ID NO:14)

[0132] CTLD3-9:QYTHWNTHQPRYSGGCVAMR(SEQ ID NO:34)

[0133] CTLD3-10:RYSGGCVAMRGRHPLGRWEV(SEQ ID NO:35)

[0134] CTLD3-11:GRHPLGRWEVKHCRHFKAMS(SEQ ID NO:36)

[0135] CTLD5-2-1:FASEWLNFEF(SEQ ID NO:38)

[0136] CTLD7-1:YGNRTYKIINANMTWYAAIK(SEQ ID NO:40)

[0137] CTLD7-2:INANMTWYAAIKTCLMHKAQLV(SEQ ID NO:41)

[0138] Example

[0139] The following embodiments are provided to illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative and not restrictive. The invention is limited only by the scope of the appended claims.

[0140] Example 1: Study of anti-PLA2R antibody in Chinese patients with primary membranous nephropathy

[0141] This study detected the level of anti-PLA2R antibodies in pMN patients at our center (Peking University First Clinical Medical College) and analyzed their impact on prognosis. Furthermore, it explored the recognition of PLA2R linear B-cell epitopes by anti-PLA2R antibodies in the patients' plasma.

[0142] 1. Materials and Methods

[0143] 1.1 Research Subjects and Specimens

[0144] The study included patients diagnosed with pMN via renal biopsy in the Nephrology Department of our hospital who underwent regular follow-up. Patients with membranous nephropathy caused by secondary factors such as hepatitis B virus, hepatitis C virus, systemic lupus erythematosus, rheumatoid arthritis, malignant tumors, drugs, and heavy metals were excluded. Clinical data were collected from the day of the renal biopsy until the end of the follow-up. Plasma samples collected during the renal biopsy and follow-up were stored at -80℃ for later use.

[0145] In this study, eGFR was calculated using the MDRD formula for the Chinese population: eGFR = 175 x serum creatinine - 1.234 x age - 0.179 x 0.79 (for females).

[0146] The use of glucocorticoids and immunosuppressants, as well as the definitions of remission and relapse, conformed to the 2012 KDIGO (Nephropathy: Improving Global Efficacy) guidelines for glomerulonephritis. Efficacy assessment of clinical outcomes included complete remission, partial remission, and no remission. Complete remission was defined as proteinuria less than 0.3 g / day and normal serum albumin and creatinine; partial remission was defined as proteinuria less than 3.5 g / day, a decrease of more than 50% from the highest level, and elevated or normalized albumin levels, with stable serum creatinine; failure to meet these criteria was defined as no remission. The primary endpoint for assessing renal outcomes was end-stage renal disease; the secondary endpoint was renal insufficiency, defined as a decrease in eGFR of more than 50% from baseline, with a final eGFR below 60 ml / min / 1.73 m².

[0147] This study was approved by our hospital's ethics committee and complies with the Helsinki Declaration. Informed consent was obtained from the patients.

[0148] 1.2 Renal pathological assessment

[0149] Kidney biopsies were performed on all patients upon admission, and the kidney specimens were independently evaluated by at least two nephrologists using optical and electron microscopes.

[0150] The fluorescence intensity of IgG, IgM, IgA, C3, C1q, and IgG subclasses was semi-quantitatively labeled using a scale of 0 to 4: 0 represents negative, 1 represents weak staining power, 2 represents moderate staining, 3 represents strong staining power, and 4 represents obvious staining. According to the Ehrenreich and Churg classification, glomerular lesions are divided into four stages. Chronic tubulointerstitial injury is defined as tubular atrophy and interstitial fibrosis, while acute tubulointerstitial injury includes loss of the tubular brush border and interstitial mononuclear cell infiltration. Semi-quantitative labeling using a scale of 0 to 4 was used: 0 represents 0-5% interstitial involvement, 1 represents 6%-25% interstitial involvement, 2 represents 26%-50% interstitial involvement, 3 represents more than 50% interstitial tissue involvement, and 4 represents more than 75% interstitial tissue involvement.

[0151] 1.3 Experimental Reagents and Materials

[0152] Anti-PLA2R Antibody Detection Kit (Indirect Immunofluorescence Assay): EUROIMMUN

[0153] Anti-PLA2R Antibody IgG Detection Kit (ELISA Method): EUROIMMUN

[0154] Anti-THSD7A Antibody Detection Kit (Indirect Immunofluorescence Assay): EUROIMMUN

[0155] Rabbit anti-human PLA2R antibody: Sigma-Aldrich

[0156] Horseradish peroxidase-labeled goat anti-rabbit IgG: Dako Biotechnology

[0157] Diaminobenzidine (DAB) substrate colorimetric solution: Beijing Zhongshan Jinqiao Company

[0158] Citrate buffer (pH 6.1): Beijing Zhongshan Jinqiao Company

[0159] PBS buffer (pH 7.2): Beijing Zhongshan Jinqiao Company

[0160] Polystyrene 96-hole plate: Nunc Corporation

[0161] Affinity: Sigma-Aldrich

[0162] ALP-labeled goat anti-human IgG: Sigma-Aldrich

[0163] p-Nitrobenzene: Sigma-Aldrich

[0164] Dimethyl sulfoxide (DMSO): Sigma-Aldrich

[0165] Tween-20: Beijing Chemical Reagent Company

[0166] 1.4 Experimental Apparatus

[0167] Benchtop Low-Temperature Centrifuge: Sorvall Biofuge Fresco model, DuPont

[0168] Optical microscope (CKX41): Olympus Corporation

[0169] Fluorescence microscope: Carl Zeiss AG

[0170] Chemiluminescence imaging system: GE Healthcare

[0171] Microplate reader: Model 680, Bio-RAD

[0172] Low-temperature refrigerator: MDF-U52V model, SANYO company

[0173] Microwave oven: LG Corporation

[0174] Pipettes: Thermo Fisher Scientific

[0175] Low-temperature centrifuge (Sorvall RT-6000D model): DuPont

[0176] Water bath (HH) W21 model): Beijing Changfeng Instrument Company

[0177] Shaking Insulator (TS-2000A): Qilin Medical Instrument Factory

[0178] Magnetic stirrer (S6 type): Jinbeide Industry and Trade Co., Ltd.

[0179] Electronic balance (PB303 model): Mettler Corporation

[0180] 1.5 Experimental Methods and Procedures

[0181] 1.5.1 Qualitative detection of cyclic anti-PLA2R antibodies

[0182] Qualitative detection of circulating antibodies was performed using an anti-PLA2R antibody detection kit (indirect immunofluorescence assay). The principle is as follows: human embryonic kidney cells are transfected with complementary DNA encoding PLA2R, and the resulting material is added to the reaction wells as a substrate. If anti-PLA2R antibodies are present in the plasma, the cell substrate will exhibit a fluorescent reaction. Main operational steps:

[0183] Patient plasma was diluted 1:10 and added to the reaction wells, then incubated at room temperature for 30 min. After washing the plate, FITC-labeled secondary antibody was added to the reaction wells and incubated at room temperature for 30 min. After mounting with mounting medium, the fluorescence intensity of cells in the reaction wells was observed using a fluorescence microscope.

[0184] 1.5.2 Quantitative detection of circulating anti-PLA2R antibody IgG

[0185] Quantitative detection of circulating antibodies was performed using an anti-PLA2R antibody IgG detection kit (enzyme-linked immunosorbent assay). The principle is as follows: a microplate is coated with recombinant PLA2R antigen. Calibrators and plasma samples are added separately. Different concentrations of anti-phospholipase A2 receptor antibody IgG in the samples specifically bind to the antigen coated on the microplate. Subsequently, peroxidase (HRP)-labeled rabbit anti-human IgG antibody and 3,3',5,5'-tetramethylbenzidine (TMB) are added. The color intensity is proportional to the analyte concentration in the sample. Detection is performed at a wavelength of 450 nm using an ELISA reader. A standard curve is plotted based on the OD values ​​corresponding to the calibrators, and the concentration of anti-phospholipase A2 receptor antibody IgG in each sample can be calculated from the standard curve. Main operating steps:

[0186] (1) Dilute the patient's plasma 1:100, add 100 μl of the diluted sample to the reaction well, and incubate at 37°C for 60 min;

[0187] (2) Discard the reaction solution, wash each well three times with 300 μl of washing solution, and pat dry;

[0188] (3) Add 100 μl of HRP-labeled rabbit anti-human IgG antibody to each well and incubate at 37°C for 30 min;

[0189] (4) Discard the reaction solution, wash each well three times with 300 μl of washing solution, and pat dry;

[0190] (5) Add 100 μl of TMB substrate to each microwell and incubate at room temperature for 30 min;

[0191] (6) Add 100 μl of stop solution to each microwell and mix well;

[0192] (7) Place the microplate on the microplate reader and use a wavelength of 450 nm to detect the OD value of each sample; make a standard curve according to the OD value corresponding to the calibrator, and use the OD value of the sample to calculate the concentration of anti-PLA2R antibody IgG in the sample.

[0193] 1.5.3 Qualitative detection of cyclic anti-THSD7A antibodies

[0194] Qualitative detection of circulating antibodies was performed using an anti-THSD7A antibody detection kit (indirect immunofluorescence assay). The principle is as follows: human embryonic kidney cells are transfected with complementary DNA encoding THSD7A, and the resulting material is added to the reaction wells as a substrate. If anti-THSD7A antibodies are present in the plasma, the cell substrate will exhibit a fluorescent reaction. Main operational steps:

[0195] (1) Dilute the plasma to be tested at a ratio of 1:10, add it to the reaction wells coated with antigen, and incubate at room temperature for 30 min;

[0196] (2) Wash the plate three times, add FITC-labeled secondary antibody to the reaction wells, and incubate at room temperature for 30 min;

[0197] (3) After sealing with the mounting medium, observe the fluorescence intensity of cells in the reaction wells using a fluorescence microscope.

[0198] 1.5.4 Peptide Preparation

[0199] The peptides listed in Table 1 were synthesized by Beijing Zhongke Yaguang Company. Peptide synthesis was performed using the F-moc method on an automated peptide synthesizer, with biotin labeled at the N-terminus of the sequence. Purification was achieved by HPLC, and mass spectrometry analysis confirmed the correct sequence. The purity of all peptides used in the experiments was above 98%.

[0200] 1.5.5 ELISA detection of autoantibodies recognizing the PLA2R linear antigenic epitope in the plasma of patients with PLA2R-related membranous nephropathy

[0201] Reagent preparation

[0202] (1) 0.01M PBS (pH 7.4)

[0203] (2) 0.1% PBST

[0204] (3) 1% PBS / BSA

[0205] (4) PBST / BSA

[0206] (5) 0.05M carbonate buffer (pH 9.6)

[0207] (6) Alkaline phosphatase substrate chromogenic buffer (pH 9.8)

[0208] (7) Alkaline phosphatase substrate colorimetric solution (prepare fresh before use)

[0209] Methods and Steps

[0210] All peptides were dissolved in 10% DMSO and 90% ultrapure water, then aliquoted (concentration 500 μg / ml) and stored at -40℃ until use.

[0211] (1) Dilute avidin to 5 μg / ml with 0.05 mol / L carbonate buffer (pH 9.6), coat the microplate with 100 μl per well, and incubate overnight at 4°C for 14-16 h.

[0212] (2) After incubation, rinse each well three times with 300 μl PBST for three minutes each time, and then pat dry.

[0213] (3) Block with 1% PBS / BSA, 100 μl per well, and incubate at 37°C for 60 min.

[0214] (4) After incubation, rinse each well three times with 300 μl PBST for three minutes each time, and then pat dry.

[0215] (5) Taking the 17 peptides in the CysR region as an example, the peptides were diluted to 10 μg / mL with 0.05 mol / L carbonate buffer (pH 9.6). 34 wells of the microplate were used as antigen-coated wells, and the other 2 wells were coated with only 0.05 mol / L carbonate buffer as non-antigen-coated wells.

[0216] (6) Dilute the serum to be tested with 1% PBST / BSA at a dilution ratio of 1:100, and add it to 17 antigen-coated wells and 1 non-antigen-coated well as the sample group. Add only 1% PBST / BSA to the other 17 antigen-coated wells and 1 non-antigen-coated well as the blank control group. 100 μl per well, incubate at 37°C for 60 min.

[0217] (7) After incubation, rinse each well three times with 300 μl PBST for three minutes each time, and then pat dry.

[0218] (8) Use alkaline phosphatase-labeled goat anti-human IgG diluted at a ratio of 1:5000, add 100 μl to each well, and incubate at 37°C for 60 min.

[0219] (9) After incubation, rinse each well three times with 300 μl PBST for three minutes each time, and then pat dry.

[0220] (10) Dissolve p-nitrobenzene in alkaline phosphatase substrate chromogenic buffer, add 100 μl to each well of the microplate, and develop for 30 min.

[0221] (11) The absorbance value is read by the microplate reader at a wavelength of 405 nm. ΔOD = [OD(sample group antigen-coated wells) − OD(sample group non-antigen-coated wells)] - [OD(blank group antigen-coated wells) − OD(blank group non-antigen-coated wells)]

[0222] Plasma from 17 healthy blood donors was used as a control group, and the above method was used for testing. The normal value was defined as the mean plus two standard deviations. A value exceeding this threshold was considered positive.

[0223] 1.5.6 Statistical Methods

[0224] Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Normally distributed data were expressed as mean ± standard deviation, and t-tests were used for comparisons between groups. Non-normally distributed data were expressed as interquartile ranges, and nonparametric rank-sum tests were used for comparisons between groups. Qualitative data were expressed as absolute values ​​and percentages, and chi-square tests were used for statistical analysis. Logistic regression models were used to analyze risk factors for no remission after treatment, and Cox regression models were used to assess risk factors for renal insufficiency. After univariate regression analysis, variables with P < 0.10 were included in the multivariate regression model. Results are expressed as odds ratios (OR) or hazard ratios (HR) with a 95% confidence interval (CI). P < 0.05 was considered statistically significant.

[0225] 2. Results

[0226] 2.1 Relationship between anti-PLA2R antibodies and clinicopathology and prognosis in Chinese pMN patients

[0227] 2.1.1 Clinicopathological characteristics and prognosis of Chinese pMN patients

[0228] This study collected general information, clinical manifestations, pathological features, and prognosis of pMN patients at our center from 2008 to 2016, and also measured the levels of anti-THSD7A and anti-PLA2R antibodies in the patients' circulation.

[0229] In 371 patients diagnosed with pMN by renal biopsy, circulating anti-PLA2R antibodies were qualitatively and quantitatively detected in all patients. 243 patients (65.5%) were positive for circulating anti-PLA2R antibodies, with a median anti-PLA2R antibody titer of 77.8 (34.9–200.0) RU / ml. Qualitative detection of circulating anti-THSD7A antibodies was performed in 295 of the 371 pMN patients, revealing that 4 patients (1.4%) were positive for circulating anti-THSD7A antibodies.

[0230] 371 patients diagnosed with pMN by renal biopsy were followed up for at least six months, with a median follow-up time of 26.9 (range 13.9–45.4) months. Of these, 168 (45.3%) achieved complete clinical remission, 157 (42.3%) achieved partial clinical remission, and 46 (12.4%) did not achieve clinical remission. Eight (2.2%) patients progressed to end-stage renal disease, and 67 (18.1%) developed renal insufficiency.

[0231] Table 2: Clinicopathological features and prognosis of pMN patients

[0232]

[0233] 2.2 Relationship between anti-PLA2R antibodies and clinicopathological features in Chinese pMN patients

[0234] Of the 371 patients with pMN, 243 (65.5%) were positive for circulating anti-PLA2R antibodies, and 128 (34.5%) were negative. Comparison revealed that, at the onset of the disease, patients with positive anti-PLA2R antibodies exhibited more severe clinical and pathological manifestations: higher levels of proteinuria, lower levels of albumin, more severe chronic tubulointerstitial damage, and a higher proportion of acute tubulointerstitial damage.

[0235] Long-term follow-up revealed that patients with positive anti-PLA2R antibodies were less likely to achieve clinical remission after treatment and were more likely to develop renal insufficiency. (Table 3)

[0236] Table 3. Clinicopathological features of pMN patients who are positive and negative for circulating anti-PLA2R antibodies.

[0237]

[0238] 2.3 Positive anti-PLA2R antibody in plasma is an independent risk factor for clinical non-remission in patients.

[0239] Logistic regression analysis revealed risk factors for clinical non-remission in pMN patients. Univariate analysis showed that urinary protein level, albumin level, eGFR level, anti-PLA2R antibody positivity, and anti-PLA2R antibody titer were risk factors for clinical non-remission in pMN patients (P<0.10). Multivariate regression analysis of these variables showed that anti-PLA2R antibody positivity was an independent risk factor for clinical non-remission, and the odds ratio for clinical non-remission in patients with positive anti-PLA2R antibodies was 4.626 times higher than that in patients with negative anti-PLA2R antibodies (P=0.006).

[0240] 3. Anti-PLA2R antibodies in Chinese pMN patients recognize PLA2R linear peptides.

[0241] 3.1 Clinicopathological characteristics of pMN patients

[0242] This study included 24 patients with PLA2R-related pMN. The inclusion criteria for these 24 patients were: a renal biopsy diagnosis of membranous nephropathy at our hospital; positive PLA2R staining on the glomeruli and positive serum anti-PLA2R antibodies (>20 RU / ml). Exclusion criteria included: secondary factors such as hepatitis B, lupus, poisoning, etc.; and prior use of hormones and immunosuppressants. These 24 patients were newly enrolled in the clinical trial, newly diagnosed and untreated, with no overlap with the 371 cases in the retrospective study mentioned above. All patients were positive for anti-PLA2R antibodies, with a median level of 126.5 (interquartile range, 66.0–187.8) RU / ml.

[0243] 3.2 Patient plasma recognizes PLA2R peptide

[0244] In this study, the inventors decomposed PLA2R into 123 peptides, each overlapping by 8-10 amino acids. At least one peptide was recognized in 18 / 24 (75.0%) patients. The recognition rate of each peptide in the plasma of pMN patients is shown in [the table below]. Figure 1 Table 4 shows that pMN patients had a positive recognition rate >20% for 6 / 123 (4.9%) peptides. These 6 peptides are distributed in the N-terminal head, middle region, and C-terminal tail of the PLA2R structure, namely: CysR10 (positive recognition rate 20.8%), CTLD3-12 (positive recognition rate 20.8%), CTLD6-6-1 (positive recognition rate 25.0%), CTLD8-8 (positive recognition rate 25.0%), CTLD8-10 (positive recognition rate 20.8%), and CTLD8-12 (positive recognition rate 20.8%). Among them, CTLD8-8, CTLD8-10, and CTLD8-12 are a continuous 34-amino acid sequence with a positive recognition rate of 37.5%.

[0245] The positive recognition rate of 23 / 123 (18.7%) peptides in pMN patients ranged from 10% to 20%, and they were also distributed in various regions of PLA2R, but were most common in the N-terminal head, namely the CysR region (6 peptides) and the C-terminal tail, namely the CTLD7 and CTLD8 regions (8 peptides). As shown in Table 5, among the 23 peptides, CysR2, CysR4, and CysR6 are a continuous sequence of 43 amino acids, with a positive recognition rate of 20.8%; FnII-1 and FnII-3 are a continuous sequence of 40 amino acids, with a positive recognition rate of 25.0%; CTLD2-10 and CTLD2-11 are a continuous sequence of 28 amino acids, with a positive recognition rate of 29.2%; CTLD7-3, CTLD7-4, CTLD7-6, and CTLD7-8 are a continuous sequence of 72 amino acids, with a positive recognition rate of 20.8%; and CTLD8-9 and CTLD8-11 are a continuous sequence of 40 amino acids, with a positive recognition rate of 16.7%. Patients showed a positive recognition rate between 0% and 10% for 63 / 123 (51.2%) peptides, and a positive recognition rate of 0% for 31 / 123 (25.2%) peptides.

[0246] Table 4. Positive recognition rate of 6 peptides in plasma from pMN patients.

[0247]

[0248] Table 5. Positive recognition rate of 5 consecutive amino acid sequence peptides in pMN patient plasma.

[0249]

[0250] 3.3 Clinical Relevance Analysis of Anti-PLA2R Linear Peptide Antibody and pMN Patients

[0251] The inventors further analyzed the correlation between plasma antibody recognition of each polypeptide or continuous amino acid sequence and the clinicopathological severity of pMN patients. The included clinicopathological indicators included general information such as age and gender; laboratory indicators such as serum albumin, serum creatinine, plasma anti-PLA2R antibody titer, eGFR, and urinary protein; and pathological characteristics such as pathological stage, glomerular PLA2R antigen deposition, IgG subtype, and C3 deposition. Among them, patients who could identify CysR2 / 4 / 6 had higher plasma albumin levels [27.2 (25.1-45.5) vs. 22.7 (19.8-26.1), P=0.015], patients who could identify CTLD6-6-1 had higher plasma anti-PLA2R antibody titers [294.0 (147.3-936.3) vs. 117.0 (59.8-148.3), P=0.018], and patients who could identify CTLD7-11 had higher plasma anti-PLA2R antibody titers [272.5 (168.5-983.3) vs. 117.0 (59.3-148.8), P=0.01] and higher albumin levels [27.2 (25.6, 41.9) vs. 23.2 (19.9-26.1), P=0.029]. See the results below. Figure 2 The remaining indicators were not correlated with peptide recognition.

[0252] 4. Discussion

[0253] Compared to antibody-negative patients, patients with positive anti-PLA2R antibodies had more severe disease at baseline, with higher levels of proteinuria, lower serum albumin levels, and more severe tubulointerstitial damage. In this longitudinal study, after long-term treatment and follow-up, patients with positive PLA2R antibodies had more difficulty achieving clinical remission and were more likely to develop renal insufficiency. Notably, positive anti-PLA2R antibodies were an independent predictor of clinical prognosis. This indicates that circulating anti-PLA2R antibodies are associated with disease severity and prognosis, a conclusion consistent with other previous studies.

[0254] The inventors' research revealed that antibody recognition of peptides targeting linear epitopes, particularly CysR2 / 4 / 6, CTLD6-6-1, and CTLD7-11, is related to the level of antibodies against the intact anti-PLA2R antigen and the severity of the disease in patients. CTLD6-6-1 showed the highest recognition rate, which was associated with higher anti-PLA2R antibody titers at diagnosis; the latter is a prognostic indicator of clinical non-remission and renal insufficiency. In autoimmune responses, autoreactive B cells targeting CTLD6-6-1 may continuously stimulate autoreactive T cells, leading to a persistent autoimmune response and continuous production of autoantibodies, thus causing more severe kidney damage. Therefore, CTLD6-6-1 may be a linear epitope associated with disease progression. This study found that the positive rate of anti-PLA2R antibodies in pMN patients in northern China was approximately 65%. Anti-PLA2R antibodies are closely related to clinical manifestations; patients with positive antibodies have more severe conditions and are less likely to achieve clinical remission. Antibodies targeting linear epitopes of PLA2R autoantigens can be detected in patient plasma. The recognized continuous amino acid sequence CTLD8-8, CTLD8-10, and CTLD8-12 may be a conformational structure and are associated with the pathogenesis. The recognition of linear peptides CysR2 / 4 / 6, CTLD6-6-1, and CTLD7-11 is related to the severity of the disease and may be epitopes associated with disease progression.

[0255] Example 2: Study on the binding and presentation of PLA2R linear peptide by pMN-susceptible HLA molecules

[0256] This study is the first to experimentally explore the ability of pMN-related susceptible HLA molecules to present PLA2R antigen, confirming that pMN susceptibility is related to the presentation between DRB1 molecules and PLA2R antigen epitopes.

[0257] 1. Materials and Methods

[0258] 1.1 Experimental reagents and materials

[0259] RPMI 1640 medium: Gibco

[0260] Fetal bovine serum: Gibco

[0261] BSA: Sigma Corporation

[0262] DMSO: Sigma-Aldrich

[0263] Fc receptor blockers: Biolegend

[0264] Mouse anti-human DRB1 IgG: MaxPab

[0265] Mouse anti-human DQA IgG: MaxPab

[0266] Normal mouse IgG: Thermo Fisher Scientific

[0267] PE-labeled goat anti-mouse IgG: Biolegend

[0268] APC-labeled streptavidin: Biolegend

[0269] 1.2 Synthetic peptides

[0270] A set of full-length peptides covering 10 domains of the extracellular region of human PLA2R were designed, each containing approximately 15-20 amino acids, with an overlap of 8-10 amino acids between each peptide, totaling 123 linear peptides. These peptides were named and numbered according to their respective regions. See Table 1 for details. Peptide synthesis was performed using the F-moc method on an automated peptide synthesizer (Beijing Zhongke Yaguang Co., Ltd.). Biotin was labeled at the N-terminus of the sequence. Purification was performed by HPLC, and mass spectrometry analysis confirmed the correct sequence. The purity of all peptides used in the experiments was above 98%. All peptides were dissolved in 10% DMSO and 90% ultrapure water, then aliquoted (concentration 500 μg / ml) and stored at -40℃ until use.

[0271] 1.3 Detection of the binding ability of peptides to HLA molecules

[0272] (1) Culture EB virus-treated B lymphocytes at 37°C and 5% CO2. Once the cells are in good condition, collect them, wash them with buffer, resuspend them, and count them.

[0273] (2) Dilute the Fc receptor blocker at a ratio of 1:20, for every 5 × 10 5 Add 100 μl of blocking solution to each cell and incubate at room temperature for 10 min.

[0274] (3) 5×10 5 Cells were incubated with 10 μg / ml peptides and mouse anti-human DRB1 (1:40) IgG at room temperature for 20 min. Additionally, an isotype control was included for each peptide, and normal mouse IgG was used as the primary antibody to eliminate non-specific binding.

[0275] (4) Wash the cells with washing buffer, 800 μl per tube, wash twice, centrifuge and discard the supernatant.

[0276] (5) Add 1:100 PE-labeled goat anti-mouse IgG and 1:2000 APC-labeled streptavidin to the cells and incubate at room temperature for 20 min.

[0277] (6) Wash cells with washing buffer, 800 μl per tube, wash twice, centrifuge and discard the supernatant.

[0278] (7) Resuspend the cells in 500 μl PBS and mix well by pipetting.

[0279] (8) Within 2 hours, the fluorescence signal on the cell surface was detected by flow cytometry to reflect whether the peptide binds to the HLA-DRB1 molecule and the degree of binding. The data was processed using the flow cytometry data processing software FlowJo. The affinity of the peptide to the HLA-DRB1 molecule was expressed as the percentage of PE and APC double-positive cells minus the percentage of non-specific binding.

[0280] The above experiment was repeated 3 times.

[0281] 1.4 Co-localization of peptides with HLA DR / DQ molecules

[0282] (1) Culture EB virus-treated B lymphocytes at 37℃ and 5% CO2. Once the cells are in good condition, collect them, wash them with buffer, resuspend them, and count them.

[0283] (2) Dilute the Fc receptor blocker at a ratio of 1:20, for every 5 × 10 5 Add 100 μl of blocking solution to each cell and incubate at room temperature for 10 min.

[0284] (3) 10 5 One cell was incubated with 10 μg / ml peptide and mouse anti-human DRB1 (1:40) IgG at room temperature for 20 min. Another 10 5 One cell was incubated with 10 μg / ml peptide and mouse anti-human DQA (1:40) IgG at room temperature for 20 min.

[0285] (4) Wash the cells with washing buffer, 800 μl per tube, wash twice, centrifuge and discard the supernatant.

[0286] (5) Add 1:100 PE-labeled goat anti-mouse IgG and 1:1000 APC-labeled streptavidin to the cells and incubate at room temperature for 20 min.

[0287] (6) Wash cells with washing buffer, 800 μl per tube, wash twice, centrifuge and discard the supernatant.

[0288] (7) Resuspend the cells in 100 μl PBS, clamp them on a spin plate, and centrifuge at 1000 rpm for 5 min using a spin plate machine.

[0289] (8) Drop 50 μl of 4% paraformaldehyde onto the cells adhering to the glass slide and fix at 4°C for 10 min.

[0290] (9) Mount the slide with mounting medium containing DAPI.

[0291] (10) Observe the fluorescence localization on the cell surface under a confocal fluorescence microscope.

[0292] 1.5 Statistical Methods

[0293] Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Normally distributed data were expressed as mean ± standard deviation, and t-tests were used for comparisons between groups. Non-normally distributed data were expressed as interquartile ranges, and nonparametric rank-sum tests were used for comparisons between groups. Qualitative data were expressed as absolute values ​​and percentages, and chi-square tests, Fisher's exact test, or Pearson's chi-square test were used for statistical analysis.

[0294] 2. Binding of HLA molecules to PLA2R peptide

[0295] The inventors synthesized a set of 123 peptides based on the full-length amino acid sequence of PLA2R. They utilized two cell lines, DRB1 and DRB2, homozygous for susceptible HLA alleles. "1501 homozygote" and "DRB1" "0301 homozygote", two cell lines "DRB1" that are homozygous for protective HLA alleles "0901 homozygote" and "DRB1" "0701 homozygote", two heterozygotes "DRB1" 1501 / DRB1 "0901" and "DRB1" 0301 / DRB1 "0701" is used to detect the binding ability of different peptides to different HLA molecules. The names and HLA genotypes of the above six cell lines are shown in Table 6.

[0296] Table 6: Names and HLA genotypes of the 6 cell lines

[0297]

[0298] These cell lines express both the HLA DR molecule and the corresponding HLA DQ molecule. Through confocal immunofluorescence analysis, the inventors discovered that on the cell surface, the PLA2R peptide co-localizes with the DR molecule, but not with the DQ molecule. Figure 3 As shown, CTLD7-1, CTLD7-2 and DRB1 Taking the 1501 homozygote as an example, both peptides were able to co-localize with HLA-DRB1 molecules on the cell surface, but not with HLA-DQ molecules. This indicates that the peptides bind to and are presented with HLA-DRB1 molecules on the cell surface, rather than HLA-DQ molecules.

[0299] Referencing methods from published literature (Cao Y, Schmitz JL, Yang J, et al. DRB1) 15allele is a risk factor for PR3-ANCA disease in African Americans. J Am SocNephrol. 2011, 22(6):1161-1167; Gu QH, Jia XY, Li JN, et al. The criticalamino acids of a nephritogenic epitope on human Goodpasture autoantigen forbinding to HLA-DRB1 (1501. Mol Immunol. 2017, 88:1-9.) The binding affinity of each PLA2R linear peptide to different HLA molecules was detected using a biotin-avidin system and flow cytometry. The binding affinity of the linear peptide to DRB1 was also investigated. Taking molecule 1501 as an example, the specific method is as follows: biotin-labeled PLA2R linear peptide, mouse anti-DRB1 antibody and DRB1 1501 homozygous cell line (expressing DRB1 on cell surface) The peptide was co-incubated with 1501 molecules, followed by incubation with APC-avidin and PE-anti-mouse IgG. The proportion of APC and PE double-positive cells to the total cell count was analyzed by flow cytometry, indicating the affinity of the peptide for DRB1. The binding affinity A (%) of 1501 molecules; in addition, biotin-labeled PLA2R linear peptide, normal mouse IgG and DRB1 were compared. 1501 homozygous cell line (expressing DRB1 on cell surface) The peptide was co-incubated with 1501 molecules, followed by incubation with APC-avidin and PE-anti-mouse IgG. The proportion of APC and PE double-positive cells to the total cell count was analyzed by flow cytometry, indicating the affinity of the peptide for DRB1. The non-specific binding affinity (B%) of 1501 homozygous cells; the difference between binding affinity A (%) and non-specific binding affinity B (%) represents the peptide's affinity for DRB1. Specific binding affinity of molecule 1501 (%).

[0300] according to Figure 4 123 peptides bind to homozygous DRB1 with varying abilities. 1501 molecules and homozygous DRB1 On molecule 0301, 31 peptides showed high binding affinity (first quartile, DRB1). 1501: 64.0%-98.0%, DRB1 0301: 77.5%-97.8%), 31 peptides showed moderate binding affinity (second quartile, DRB1) 1501: 10.8%-62.1%, DRB1 0301: 16.5%-75.2%), 61 peptides showed low binding or no binding (third and fourth quartiles, DRB1) 1501: 0.2%-9.9%, DRB1 0301: 0.1%-12.9%). There are 17 peptides associated with DRB1. 1501 and DRB1 0301 showed high binding affinity (Table 7), consisting of CysR1, CysR3, CysR10, CysR12, FnII3, CTLD2-9, CTLD3-4, CTLD3-9, CTLD3-10, CTLD3-11, CTLD4-10, CTLD5-2-1, CTLD6-5, CTLD7-1, CTLD7-2, CTLD8-1-1, and CTLD8-5-2. Six peptides bound to DRB1. 1501 exhibits high bonding strength with DRB1 0301 exhibits low / no binding, including CTLD2-5, CTLD4-11, CTLD6-4-2, CTLD6-6-1, CTLD6-11, and CTLD7-4. It also contains three peptides: FnII2, CTLD2-2, and CTLD7-9, and DRB1. 0301 has high bonding strength with DRB1 1501 shows low / no binding.

[0301] Table 7: 17 types and DRB1 1501 and DRB1 0301 all exhibit high binding affinity of PLA2R peptide segments.

[0302]

[0303] according to Figure 4 123 peptides bind to homozygous DRB1 with varying abilities. 1501 molecules and homozygous DRB1 On molecule 0301. 31 peptides show high binding (first quartile, DRB1). 1501: 64.0%-98.0%, DRB1 0301: 77.5%-97.8%), 31 peptides showed moderate binding (second quartile, DRB1) 1501: 10.8%-62.1%, DRB1 0301: 16.5%-75.2%), 61 peptides showed low or no binding (third and fourth quartiles, DRB1) 1501: 0.2%-9.9%, DRB1 0301: 0.1%-12.9%). There are 17 higher-order DRB1 binding agents. 1501 and DRB1 The peptides of 0301 (Table 7) include CysR1, CysR3, CysR10, CysR12, FnII-3, CTLD2-9, CTLD3-4, CTLD3-9, CTLD3-10, CTLD3-11, CTLD4-10, CTLD5-2-1, CTLD6-5, CTLD7-1, CTLD7-2, CTLD8-1-1, and CTLD8-5-2. Six of these are related to DRB1. 1501 higher combined with DRB1 0301 contains low-level / unbound peptides, including CTLD2-5, CTLD4-11, CTLD6-4-2, CTLD6-6-1, CTLD6-11, and CTLD7-4. Three peptides, FnII-2, CTLD2-2, and CTLD7-9, are highly bound to DRB1. 1501 Lower grade / Not combined.

[0304] Compared to susceptible HLA molecules, protective HLA homozygous DRB1 0901 molecules and homozygous DRB1 The 0701 molecule has a slightly lower ability to bind peptides. Excluding peptides with low / no binding, the PLA2R peptide binds to DRB1. The bonding capability of 0901 is much lower than that of DRB1. Binding capacity of 1501 [36.4% (17.5%, 70.2%) vs. 63.1% (36.6%, 92.4%), P=0.001]. PLA2R peptide and DRB1 The bonding capability of 0701 is much lower than that of DRB1. Binding ability of 0301 [58.9% (33.2%, 79.8%) vs. 76.3% (50.0%, 92.2%), P = 0.017].

[0305] With DRB1 Of the 31 peptides that exhibit high binding affinity to DRB1, 11 peptides are associated with DRB1. 0901 exhibits high binding affinity, while the six peptides bind to DRB1. 0901 shows low / no binding. These 6 peptides are associated with DRB1. The binding ability of the 1501 / 0901 heterozygote was also significantly reduced.

[0306] With DRB1 Of the 31 peptides that exhibit high binding affinity to 0301, 16 peptides are bound to DRB1. 0701 exhibits high binding affinity, while the three peptides bind to DRB1. 0701 shows low / no binding. These three peptides are associated with DRB1. The binding ability of the 0301 / 0701 heterozygote was also significantly reduced.

[0307] 3. Discussion

[0308] Since the discovery of the pMN-specific antigen PLA2R in 2009, research on this antigen has mainly focused on B-cell conformational epitopes and epitope expansion. As an autoimmune disease, T-cell epitopes are the initiating link of the entire autoimmune response; therefore, research on the T-cell epitopes of PLA2R is crucial for understanding its pathogenesis.

[0309] The foundation for research on T-cell epitopes is identifying the susceptible HLA genotypes for pMN. In 2017, a previous study (Cui Z, Xie LJ, Chen FJ, et al. MHC Class II Risk Alleles and Amino Acid Residues in Idiopathic Membranous Nephropathy. J Am Soc Nephrol. 2017, 28(5):1651-1664) discovered DRB1 by detecting HLA II genotypes in patients with membranous nephropathy and healthy controls. 1501 and DRB1 0301 represents two independent risk genotypes of pMN. Based on the physiological functions of the HLA molecules encoded by this gene, it is hypothesized that these two pMN susceptibility HLA molecules play a decisive role in the presentation of the PLA2R linear antigen peptide. In other autoimmune diseases such as multiple sclerosis, similar linear peptide profiles of different disease-associated susceptibility HLA molecules have been found to be presented on whole antigens. Therefore, the inventors hypothesize that DRB1... 1501 and DRB1 0301, representing two different pMN genotypes, is associated with disease, possibly because their linear peptide profiles presented on PLA2R are similar. Furthermore, earlier (Le WB, Shi JS, Zhang T, et al. HLA-DRB1) 15:01 and HLA-DRB3 02:02 in PLA2R-Related Membranous Nephropathy. J Am Soc Nephrol. 2017, 28(5):1642-1650) In a study of risk genes for membranous nephropathy, it was shown that co-carrying DRB1 1501 and DRB3 0202 (DRB1) Individuals with the strongly linked genotype 0301 did not show a significant difference in disease severity compared to those carrying only one susceptibility genotype. Therefore, the inventors hypothesized that DRB1... 1501 and DRB1 The susceptibility effects of 0301 are overlapping rather than additive, meaning that the two susceptibility mechanisms for the presented PLA2R antigenic peptide are likely to overlap. The inventors experimentally demonstrated that for the 123 linear peptides spanning the entire amino acid sequence of PLA2R, DRB1... 1501 and DRB1 The HLA molecules encoded by 0301 have very similar presentation capabilities and can highly bind to DRB1. Of the 1501 peptide molecules, more than half are also related to DRB1. The 0301 molecule exhibits high binding, and vice versa. The key pockets for amino acid binding on the HLA molecule are 1, 4, 6, 7, and 9. The inventors focused on DRB1... 1501 and DRB1 Sequence alignment of the 20 amino acids in the key pocket of 0301 revealed DRB1. 1501 and DRB1 0301 shows 55% similarity, which may explain the similarity in their binding spectra on PLA2R. Additionally, compared to other HLA molecules, DRB1... 1501 and DRB1 0301 exhibits amino acid specificity at certain sites, namely V86, Q70, and F47 on pockets 1, 4, and 7. This specificity at these sites may be due to DRB1. 1501 and DRB1 The reason why 0301, rather than other HLA types, is the pMN susceptibility genotype.

[0310] Regarding susceptibility of the HLA-DQA gene to disease, intergenetic interaction analysis revealed that it does not interact with PLA2R1. The inventors hypothesized that in membranous nephropathy, the HLA-DQA molecule may not play a role in the presentation of PLA2R. The result of indirect immunofluorescence in this experiment, which showed that the antigenic peptide could not co-localize with DQ molecules on the cell surface, to some extent confirms the inventors' hypothesis.

[0311] In the HLA molecule binding experiments of this study, the inventors not only determined the binding between the PLA2R linear peptide and susceptible HLA molecules, but also detected the binding between the linear peptide and DRB1 encoded by the protective HLA allele. 0901 and DRB1 The combination of 0701. The inventors discovered that DRB1 0901 and DRB1 The linear peptide profiles of 0701-bound PLA2R are similar, possibly because they share 50% similarity in the amino acid sequence of the key pocket. Some linear peptides of PLA2R can be bound to both protective and susceptible HLA molecules, but protective HLA molecules have a weaker binding ability to linear peptides and are less likely to present antigenic peptides, thus less likely to induce T cell activation. This may explain their protective effect (Singh T, Fakiola M, Oommen J, et al. Epitope-Binding Characteristics for Risk versus Protective DRB1 Alleles for Visceral Leishmaniasis. J Immunol. 2018, 200(8):2727-2737). In addition, some peptides can be bound to susceptible HLA molecules but not to protective HLA molecules. The binding ability of these peptides to heterozygotes is significantly reduced, suggesting that protective HLA molecules may reduce disease risk by inhibiting the presentation of certain antigenic peptides. Some peptides can only be bound by protective HLA molecules but not by susceptible HLA molecules. Their protective effect may be due to the protective HLA molecules presenting antigenic peptides, which activate anti-inflammatory T cells and prevent disease development. In studies on GBM disease (Ooi JD, Petersen J, Tan YH, et al. Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells. Nature. 2017, 545(7653):243-247), protective DR1 primarily induces the expression of tolerance cytokines by CD4+. + FoxP3 + Regulatory T cells, but risk DR15 induces a T cell phenotype that secretes pro-inflammatory cytokines. Ultimately, DR15 / DR1 heterozygous transgenic mice exhibit Treg-dominant T cell activation, thereby avoiding disease.

[0312] This study found HLA-DRB1 1501 and DRB1 The 0301 molecule can bind to a portion of the linear peptides in PLA2R, which is largely consistent with the results predicted by computer modeling in previous literature, particularly with the linear peptide CTLD7-2. In this study, the inventors experimentally demonstrated for the first time that CTLD7-2 has the ability to bind highly to two susceptible HLA molecules, inferring that the binding is due to the compatibility of the HLA molecular pocket with the physicochemical properties, size, and structure of key amino acids in CTLD7-2.

[0313] Studies on epitope expansion (Seitz-Polski B, Dolla G, Payré C, et al. Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy. J Am Soc Nephrol. 2016, 27(5):1517-1533) have found that a higher proportion of patients recognize the CysR region compared to those recognizing other regions, suggesting that the CysR region may be the starting point of an autoimmune response. With epitope expansion, patients can recognize more epitopes, producing more antibodies and experiencing disease severity. Recognition of regions outside of CysR is an independent risk factor for poor renal prognosis. Previous literature (Fresquet M, Joet TA, Gummadova J, et al. Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy. J Am Soc Nephrol. 2015, 26(2):302-313) confirmed the importance of the CysR region in PLA2R, and the 31-mer conformation can directly bind to anti-PLA2R antibodies under non-denaturing conditions. The 31-mer consists of two linear peptides, peptide 1 (GIFVIQSESLKK) and peptide 2 (SVLTLENCK), which can significantly inhibit antibody binding to PLA2R. In this study, PLA2R... 38-52 (CysR1) contains peptide 1, PLA2R 52-66 (CysR3) contains peptide 2, and the inventors experimentally demonstrated that both CysR1 and CysR3 can bind to the susceptible HLA molecule DRB1. 1501 and DRB1 The high binding of 0301 suggests that they may induce T cell activation.

[0314] Example 3: Study on PLA2R linear peptide-induced T cell proliferation in peripheral blood of pMN patients

[0315] This study included patients who underwent renal biopsy at our hospital between 2018 and 2019. Inclusion criteria were: 1. Membranous nephropathy diagnosed by light and / or electron microscopy in renal biopsy pathology; 2. Circulating anti-PLA2R antibody >20 RU / ml. Exclusion criteria were: 1. Secondary factors such as hepatitis B, hepatitis C, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, heavy metal or drug poisoning; 2. Prior treatment with glucocorticoids / immunosuppressants / rituximab / plasma exchange. General information, clinical manifestations, laboratory results, and pathological characteristics were collected. Venous blood was collected from patients before they started immunosuppressant therapy, and regular follow-up was conducted after discharge, with clinical laboratory results recorded and venous blood collected. Age- and sex-matched healthy individuals were included as healthy controls; venous blood was collected from these controls, and general information was collected. Plasma from both patients and controls was collected, aliquoted, and stored at -80°C to avoid repeated freeze-thaw cycles.

[0316] This study was approved by our hospital's ethics committee and complies with the Helsinki Declaration. Informed consent was obtained from both patients and healthy controls.

[0317] 1. Materials and Methods

[0318] 1.1 Experimental reagents and materials

[0319] RPMI 1640 medium: Gibco

[0320] Fetal bovine serum: Gibco

[0321] BSA: Sigma Corporation

[0322] CFSE: Thermo Fisher Scientific

[0323] Ficoll cell separation medium: Biolegend

[0324] Lymphocyte-specific culture medium: DAKEWE BIOTECH

[0325] 96-well cell culture plate: Nunc

[0326] Zombie NIR Cell Viability Assay Kit: Biolegend

[0327] APC-labeled mouse anti-human CD4 IgG: Biolegend

[0328] PE-labeled mouse anti-human CD3 IgG: Biolegend

[0329] Human Th reagent kit: Biolegend

[0330] 1.2 Synthesis of polypeptides

[0331] The inventors designed 17 peptides contained in PLA2R, each containing approximately 15-20 amino acids. The specific peptide names and amino acid sequences are shown in Table 1. Peptide synthesis was performed using the F-moc method on an automated peptide synthesizer (Beijing Zhongke Yaguang Co., Ltd.). Sequence accuracy was confirmed by HPLC purification and mass spectrometry analysis. The purity of all peptides used in the experiments was above 98%. Under aseptic conditions, all peptides were dissolved in 5% DMSO and 95% membrane-passed ultrapure water, then aliquoted (concentration 5000 μg / ml) and stored at -40℃ for later use.

[0332] 1.3 Isolation of peripheral blood mononuclear cells

[0333] 20 ml of fresh peripheral blood samples were collected from patients and healthy individuals, and PBMCs were separated using Ficoll density gradient centrifugation.

[0334] 1.4 Carboxyfluorescein succinimide (CFSE) staining

[0335] (1) PBMCs were washed twice in PBS, resuspended, and counted. The cell density was adjusted to 1×10⁶ cells / year using serum-free, pre-warmed RPMI 1640 medium. 6 After staining with 1.25 μM CFSE at 37°C for 20 min in the dark, the staining was completed.

[0336] (2) Add 5 times the volume of RPMI 1640 medium containing 10% fetal bovine serum and incubate at room temperature for 10 minutes to terminate the CFSE staining reaction.

[0337] (3) Wash twice with PBS.

[0338] (4) The cells were placed at 10 6 Resuspended in lymphocyte-specific culture medium at a density of / ml and cultured in 96-well cell culture plates at 2×10⁶ / ml. 5 Inoculation quantity per well.

[0339] (5) Place the cell culture plate in a cell culture incubator for 20 min to allow the CFSE to be hydrolyzed with acetate, thus stabilizing the staining effect.

[0340] (6) To detect the ability of peptides to stimulate T cell proliferation, the dissolved peptides were added to the cells, with three wells for each peptide and a peptide concentration of 50 μg / ml per well. Hemocyanin and ultrapure water containing 5% DMSO were used as positive and blank controls, respectively.

[0341] 1.5 Flow Cytometry Analysis

[0342] (1) PBMCs were harvested after 6 days of cell culture.

[0343] (2) Wash the PBMC once with 500 μl PBS buffer, centrifuge and discard the supernatant.

[0344] (3) The cells were stained with a 1:100 Zombie NIR cell viability assay kit and incubated at room temperature in the dark for 15 min.

[0345] (4) Wash the cells once with 500 μl of PBS buffer containing 1% BSA, and discard the supernatant after centrifugation.

[0346] (5) Incubate the cells with 1:20 APC-labeled mouse anti-human CD4 IgG and 1:20 PE-labeled mouse anti-human CD3 IgG at room temperature in the dark for 20 min.

[0347] (6) Wash the cells twice with 500 μl of PBS buffer containing 1% BSA, and discard the supernatant after centrifugation.

[0348] (7) Resuspend the cells in 500 μl PBS and mix well by pipetting.

[0349] (8) Detect the fluorescence signal on the cell surface using flow cytometry within 2 hours. Analyze the data using FlowJo software: calculate the percentage of CD4+ T cell proliferation by dividing the number of live CD4+ T cells with low CFSE by the total number of live CD4+ T cells. Calculate the stimulation index (SI): SI = percentage of CD4+ T cell proliferation stimulated with peptide / percentage of CD4+ T cell proliferation stimulated with the carrier (ultrapure water containing 5% DMSO). The average of the measurements from 3 wells was used to calculate the SI for each peptide. SI ≥ 2 was considered a positive effect.

[0350] 1.6 Cytokine profile analysis

[0351] (1) After stimulating cells with PLA2R peptide for 6 days, the cell culture supernatant of PBMCs was collected.

[0352] (2) Cytokines, including IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α, were measured using the Human Th kit via a cell counting bead array assay.

[0353] (3) Add 25 μl of cell culture supernatant to the well of a V-plate and add 25 μl of a mixture of 13 magnetic beads of different sizes and APC fluorescence intensities that are bound to specific antibodies against the 13 analytes.

[0354] (4) Shake the plate at 500 rpm for 2 hours in the dark at room temperature.

[0355] (5) Wash the cells twice with washing buffer.

[0356] (6) Incubate the cells with 25 μL of a mixture containing 13 different biotin-labeled detection antibodies for 1 h, then add 25 μL of PE-labeled streptavidin and shake at 500 rpm for 30 min at room temperature.

[0357] (7) Wash the cells twice with washing buffer.

[0358] (8) Resuspend the magnetic beads in 150 μL of buffer solution.

[0359] (9) Fluorescence signals on the cell surface were detected by flow cytometry within 2 hours. Analysis was performed using LEGENDplex software. The mean value of DMSO-stimulated samples +2SD was considered the cutoff value; cytokine concentrations above the cutoff value were defined as positive reactions.

[0360] 1.7 HLA genotyping

[0361] The HLA class II DRB loci were sequenced using an HLA detection kit, a step of which was outsourced to Beijing Siercheng Biotechnology Co., Ltd.

[0362] 1.8 Statistical Methods

[0363] Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Normally distributed data were expressed as mean ± standard deviation, and t-tests were used for comparisons between groups. Non-normally distributed data were expressed as interquartile ranges, and nonparametric rank-sum tests were used for comparisons between groups. Qualitative data were expressed as absolute values ​​and percentages, and chi-square tests, Fisher's exact test, or Pearson's chi-square test were used for statistical analysis. Linear regression analysis (r-correlation coefficient) was used to identify correlations. Kaplan-Meier curves were used to analyze the cumulative probability of anti-PLA2R antibody seroconversion, and the log-rank test was used to assess statistical significance. After univariate regression analysis, variables with p < 0.10 were included in the multiple regression model. Results are expressed as hazard ratios (HR) with a 95% confidence interval (CI). P < 0.05 was considered statistically significant.

[0364] 2. Results

[0365] 2.1 Patient clinicopathological characteristics and genotype distribution

[0366] This study included 36 patients with PLA2R-related pMN. All patients were positive for anti-PLA2R antibodies, with a median level of 124.5 (interquartile range, 59.3–192.5) RU / ml.

[0367] HLA genotyping of patients and healthy controls revealed that 100% of patients carried at least one susceptibility allele, with 6 patients (16.7%) carrying DRB1. 1501 + 0301 + 28 patients (77.8%) had DRB1. 1501 + 0301 - Two patients (5.5%) had DRB1. 1501 - 0301 + Among the 32 healthy controls, 1 individual (3.1%) had DRB1. 1501 + 0301 + 10 individuals (31.3%) were DRB1. 1501 + 0301 - Two individuals (6.2%) were DRB1. 1501 - 0301 + All other individuals did not carry any risk alleles. Regarding the carrier status of the protective gene, the inventors found that 2 patients (5.5%) carried DRB1. 0901+ 0701 - Six patients (16.7%) carried DRB1. 0901 - 0701 + The remaining patients did not carry the protective HLA gene; while 13 (40.6%) healthy individuals had DRB1. 0901 + 0701 - And 5 healthy individuals (21.9%) had DRB1. 0901 - 0701 + .

[0368] 2.2 PLA2R peptide induces T cell proliferation in pMN patients

[0369] Previous experiments have found that 17 PLA2R peptides are related to HLA DRB1. 1501 and DRB1 The 0301 molecules exhibited high binding affinity, and the inventors further evaluated their ability to induce CD4+ T cell proliferation in peripheral blood of PLA2R-associated pMN patients and healthy controls (Table 8). Additionally, the inventors evaluated a molecule found in HLA molecular binding assays that does not bind to HLA DRB1. 1501 and DRB1 The antigenic peptide CTLD7-7-1, which binds to molecule 0301, is linked to HLA DRB1. 1501 and DRB1 The ability of the moderately bound antigenic peptide CTLD7-11 to induce CD4+ T cell proliferation in patients and healthy controls was assessed. The proliferation index of CD4+ T cells stimulated by different PLA2R peptides was analyzed by flow cytometry using CFSE staining.

[0370]

[0371] like Figure 5As shown, among the 17 PLA2R peptides, 15 peptides, except for CysR3 and CTLD2-9, were found to induce CD4+ T cell proliferation in patients. Two peptides, CTLD7-1 and CTLD7-2, induced a proliferative response in healthy controls. The difference in SI values ​​between patients carrying the risk gene and the control group was compared, and the results showed that 10 PLA2R peptides induced higher levels of CD4+ T cell proliferation in the patient group compared to the control group (P<0.05): CysR1, CysR10, CysR12, FnII-3, CTLD3-9, CTLD3-10, CTLD3-11, CTLD5-2-1, CTLD7-1, and CTLD7-2. Two peptides found in HLA molecular binding assays did not bind to HLA DRB1. 1501 and DRB1 The antigenic peptide CTLD7-7-1 bound to molecule 0301 binds to HLA DRB1. 1501 and DRB1 The moderately bound antigenic peptide CTLD7-11 of molecule 0301 could not induce CD4+ T cell proliferation in patients (SI<2). Figure 5 ).

[0372] Because the study subjects carried different HLA genotypes, the inventors conducted subgroup analysis on different HLA carriers, including the patient group carrying two risk genes (DRB1). 1501 + 1501 + 0701 - 0901 - DRB1 1501 + 0301 + 0701 - 0901) and the patient group carrying a risk gene (DRB1) 1501 + 0301 - 0701 - 0901 and DRB1 1501 - 0301 + 0701 - 0901 - The study examined the T-cell proliferation capacity of CysR10, CTLD3-4, CTLD3-9, CTLD3-10, and CTLD3-11, finding that they caused differences in T-cell proliferation between the two patient groups. Figure 5 ).

[0373] 2.3 The index of PLA2R peptide-induced T cell proliferation is correlated with patients' baseline clinical characteristics and prognosis.

[0374] By comparing the relationship between the SI (Self-Induced Proliferation Index) of CD4+ T cells and baseline clinical characteristics and laboratory tests after stimulation with different peptides, the inventors found that 13 / 21 (61.9%) patients became negative for anti-PLA2R antibodies after treatment. The inventors compared the SI (Self-Induced Proliferation Index) and circulating antibody seroconversion after stimulation with each peptide, finding that the SI of CD4+ T cells stimulated by CTLD7-1 was an independent risk factor affecting circulating antibody seroconversion. Kaplan-Meier curve analysis showed that, dividing the SI of T cells after CTLD7-1 stimulation into three levels—SI<2, 2≤SI<4, and SI≥4—patients with higher SI levels induced by CTLD7-1 experienced slower circulating antibody seroconversion than patients with lower SI levels. Figure 5 ).

[0375] 2.4 The cytokine profile induced by PLA2R peptide from Th cells

[0376] The inventors obtained 10 PLA2R peptides that could induce differences in the T cell proliferation index between patients and healthy individuals. To determine the type of Th response after stimulation of patient T cells by these 10 peptides, the inventors preliminarily inferred the activated Th subtype by comprehensively detecting the concentrations of cytokines in the patient cell supernatant. The cytokines detected included IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFN-γ, and TNF-α. Figure 6 , Figure 7 ).

[0377] The results showed that stimulation with the 10 peptides of PLA2R generally induced high levels of pro-inflammatory cytokines IL-6, TNF-α, IL-10, IL-17F, and IL-9 in PBMCs of pMN patients. Most peptide stimulation also induced IL-4 secretion in PBMCs of patients carrying two risk genes. CTLD7-1 stimulation resulted in higher levels of IL-4, IL-6, IL-5, IL-9, IL-22, and IL-17A secretion in PBMCs of patients carrying two risk genes. While there was no statistically significant difference compared to the solute control, most of these 10 peptides tended to inhibit IL-2 and IFN-γ secretion. Figure 6 , Figure 7 )

[0378] While the cytokine profiles induced by stimulation of 10 PLA2R peptides were generally similar, significant differences remained. The inventors defined the average cytokine secretion of patients stimulated by the solvent control group plus 2SD as the critical value. If the concentration of a particular cytokine in a patient after peptide stimulation exceeded the critical value, a positive reaction was defined as such. The positive rate of each cytokine after stimulation of the patient's PBMCs by each peptide was calculated accordingly. Figure 6 (Figure g). CysR1 and CysR10 induced Th1 (TNF-α) activation in 90% of patients and Treg (IL-10) activation in 75% and 85% of patients, respectively. CysR12, FnII-3, and CTLD7-2 not only induced Th1 (TNF-α) and Treg activation in more than 80% of patients, but also induced Th17 and Th22 cytokine secretion in more than half of patients, and also induced Th2 (IL-9) activation in some patients. CTLD3-9 / 10 / 11 also induced Th1, Treg, Th2, Th17, and Tfh activation in patients, but the proportion of patients who showed a positive response to these three peptides was significantly lower than that of the five peptides mentioned above. In addition, CTLD5-2-1 and CTLD7-1 induced a Treg response in 55% of patients.

[0379] Not with HLA DRB1 1501 and DRB1 The antigenic peptide CTLD7-7-1 bound to molecule 0301 binds to HLA DRB1. 1501 and DRB1 The 0301 molecule moderately bound antigenic peptide CTLD7-11 failed to induce significant secretion of cytokines from patients. Figure 6 , Figure 7 ).

[0380] 3. Discussion

[0381] This is the first study on T-cell epitopes of PLA2R, the major specific antigen of membranous nephropathy. The inventors have identified 10 linear peptides of PLA2R: PLA2R38-52 (CysR1), PLA2R101-120 (CysR10), PLA2R113-129 (CysR12), PLA2R193-212 (FnII-3), PLA2R602-621 (CTLD3-9), PLA2R612-631 (CTLD3-10), PLA2R622-641 (CTLD3-11), PLA2R829-838 (CTLD5-2-1), PLA2R1121-1140 (CTLD7-1), and PLA2R1129-1150 (CTLD7-2) as potential T-cell epitopes of PLA2R. These molecules can bind to susceptible HLA DRB1 molecules, induce the proliferation of CD4+ T cells in pMN patients, and cause activated PBMCs to produce cytokines. The discovery of T cell epitopes lays the foundation for exploring the immune response mechanism of PLA2R-related pMN and identifying targets for immunotherapy.

[0382] Based on experimental results, the inventors discovered specific T cells targeting CTLD7-1 and CTLD7-2 in the peripheral blood of healthy individuals. The inventors hypothesize that under physiological conditions, healthy individuals primarily exhibit peripheral immune tolerance to CTLD7-1 and CTLD7-2; the specific T cells in the peripheral blood of healthy individuals only come into contact with extremely low concentrations of antigenic peptides and do not proliferate. However, in patients, due to increased antigen levels or exposure of cryptic epitopes, peripheral tolerance to CTLD7-1 and CTLD7-2 is disrupted, leading to disease.

[0383] The inventors' earlier research found that both CysR1 and CysR3 can highly bind to DRB1. 1501 and DRB1 The 0301 molecule, CysR1, can stimulate the proliferation of CD4+ T cells in patients, while CysR3 cannot. The inventors initially believed that CysR1 is a T-cell epitope of PLA2R. Combined with literature on B-cell conformational epitopes, the initial antibodies produced by B cells are likely to recognize the structure containing CysR1. Therefore, this peptide may be a common epitope for both T cells and B cells. Studies on GBM disease and ANCA-associated small vessel vasculitis have also found that T-cell epitopes and B-cell epitopes can partially or completely overlap, confirming that B-cell epitopes can overlap with pathogenic T-cell epitopes. Generally, common dominant epitopes play a crucial role in the mechanisms of autoimmune diseases because they may be the initial epitopes in the entire immune process. CysR1 is located on the inner surface of the β-cloverwise region of the CysR domain and is deeply embedded in the 31-mer conformation. Therefore, under normal circumstances, CysR1 is not exposed, and CysR1-specific T cells are not present in the peripheral blood of healthy individuals. However, triggered by environmental factors, epitope exposure stimulates the production of CysR1-specific T cells, leading to pathogenesis. Furthermore, subgroup analysis revealed that DRB1 carriers... In patients with DRB1 1501 but without carrying any other susceptibility or protective genes, CysR1 was still able to stimulate significant proliferation of CD4+ T cells, indicating that this peptide is involved in DRB1. The pathogenesis of pMN in 1501 carriers.

[0384] This study also identified a continuous linear peptide (PLA2R602-641) along the CTLD3 domain that likely contains a pMN T-cell epitope. CTLD3-9, CTLD3-10, and CTLD3-11 can all bind to susceptible HLA molecules, induce CD4+ T-cell proliferation, and elicit similar cytokine profiles. These are three consecutive overlapping peptides, therefore, two epitopes may exist within this long region: one located in the common sequence of CTLD3-9 and CTLD3-10 (PLA2R612-621), and the other in the common sequence of CTLD3-10 and CTLD3-11 (PLA2R622-631).

[0385] The inventors also discovered other T cell epitopes: CysR10 (PLA2R101-120), CysR12 (PLA2R113-129), FnII-3 (PLA2R193-212) and CTLD5-2-1 (PLA2R829-838). Among them, CysR10 (PLA2R101-120) is also a linear B cell epitope that the inventors previously discovered that can be recognized by patient plasma. By recognizing the corresponding self-antigen polypeptide CysR10, T cells can not only induce the secretion of inflammatory factors, but also promote the maturation of B cells and the production of autoantibodies by plasma cells. This peptide CysR10 can serve as a common antigenic epitope for T cells and B cells. PLA2R198-209 (EDDLLWCATTSR) is located in the FnII-3 region, where the amino acid D at position 200 can be mutated to Y

[135] . Point mutations may cause the exposure of occult epitopes, thereby causing disease. CTLD5-2-1 primarily induces the secretion of the cytokine IL-10, an anti-inflammatory cytokine. Generally, Treg cells maintain immune tolerance by producing IL-10, inhibiting B cell maturation and antibody production, suppressing the secretion of pro-inflammatory factors (Ooi JD, Petersen J, Tan YH, et al. Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells. Nature. 2017,545(7653):243-247; Couper KN, Blount DG, Riley EM. IL-10: the master regulator of immunity to infection. J Immunol. 2008, 180(9):5771-5777).In pMN, Treg cells are known to play an important role. Patients with pMN show a significantly reduced percentage of Treg cells at baseline, which increases after rituximab treatment. [Ruggenenti P, Debiec H, Ruggiero B, et al. Anti-Phospholipase A2Receptor Antibody Titer Predicts Post-Rituximab Outcome of Membranous Nephropathy. J Am Soc Nephrol. 2015, 26(10):2545-2558, Fervenza FC, Cosio FG, Erickson SB, et al. Rituximab treatment of idiopathic membranous nephropathy. Kidney Int. 2008 Jan;73(1):117-125, Fervenza FC, Abraham RS, Erickson SB, et al. Rituximab therapy in idiopathic membranous nephropathy: a 2-year study. Clin J Am Soc Nephrol. 2010, 5(12):2188-2198].

[0386] The inventors performed pairwise alignments of the amino acid sequences of the discovered T-cell epitopes and found common motifs among some epitopes. Specifically, CysR1 and CysR10 share the motif SLXXC; CysR1 and CTLD5-2-1 share the motif SEXL; CsR10 and FnII-3 share the motif SXYE; FnII-3 and CTLD3-9 share the motif WXXXXXRY; and CTLD3-11 and CTLD7-2 share the motif KXCXXXKA. These common motifs may be key sequences for future design of biological agents.

[0387] The inventors' research has certain limitations. First, after testing the ability of the PLA2R linear peptide to bind to HLA molecules, the inventors artificially selected peptides with high binding capacity. Generally, these peptides are more likely to be presented and stimulate T cell proliferation, but it cannot be ruled out that some peptides with moderate binding capacity may also be T cell epitopes. Second, regarding the selection of the research population, since the Han Chinese population carries DRB1... The proportion of 1501 is relatively high, while DRB1 The carrier rate of 0301 is low, therefore it carries DRB1. The response of individuals with pMN (such as Caucasians from Europe and America) to the PLA2R peptide requires further investigation. Finally, although the concentrations of cytokines differed after peptide stimulation, the distribution of cytokine profiles was very similar. This may be because all 10 peptides exhibit high binding affinity to HLA molecules and can induce significant T cell proliferation, resulting in a high degree of similarity in their cytokine profiles. Further research is needed to understand the overall cytokine profile of pMN patients.

[0388] The inventors are among the 17 individuals who can be identified by HLA-DRB1. 1501 and DRB1 Ten potential T-cell epitopes of PLA2R were further identified within the highly bound peptides of the 0301 molecule. These epitopes can stimulate the proliferation of CD4+ T cells in PLA2R-associated pMN patients and the secretion of inflammatory cytokines, primarily pro-inflammatory. Identifying these T-cell epitopes will contribute to understanding the pathogenesis of autoimmune dysregulation associated with PLA2R and will aid in the development of immunotherapies targeting these epitopes.

[0389] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide, the amino acid sequence of which is SEQ ID NO:

38.

2. A nucleic acid comprising a polynucleotide sequence encoding the polypeptide according to claim 1.

3. A vector comprising the nucleic acid according to claim 2.

4. A host cell comprising the nucleic acid of claim 2 or the vector of claim 3.

5. A composition comprising the polypeptide according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, and / or the host cell according to claim 4.

6. Use of a polypeptide having the amino acid sequence SEQ ID NO: 38, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a host cell containing the vector, or a composition containing one or more of the above in the preparation of a kit for (1) predicting clinical remission or renal function in patients with primary membranous nephropathy after treatment; or (2) determining the severity of primary membranous nephropathy.

7. The use according to claim 6, wherein the severity includes one or more of the following: higher levels of proteinuria, more severe chronic tubulointerstitial damage, or more severe acute tubulointerstitial damage compared to patients with primary membranous nephropathy who are negative for anti-PLA2R antibodies; or the treatment includes treatment with angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, immunosuppressants, calcineurin inhibitors, cyclophosphamide, and / or rituximab.

8. The use according to claim 6, wherein the patient expresses HLA DRB1 1501 molecules and / or HLA DRB1 0301 molecule.

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