An antibody binding a specific antigenic peptide of pemphigus vulgaris and use thereof

Abstract

The application belongs to the field of biological medicine, and provides an antibody combined with a specific antigen peptide of common pemphigus and application thereof, and the application uses the principle of phage display technology to perform in-vitro affinity screening on a constructed library, combines phages with target antigen Dsg3, elutes non-specific binders, retains phages combined with the antigen closely, and then amplifies the phages and performs multiple rounds of affinity enrichment; multiple rounds of panning are performed, and after 3-5 rounds of screening, phage clones with strong Dsg3 binding capacity and high specificity can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an antibody that binds to a pemphigus vulgaris-specific antigenic peptide and its application. Background Technology

[0002] Autoimmune bullous skin diseases (AIBDs) are a group of heterogeneous diseases characterized by organ specificity, triggered by autoantibodies targeting structural proteins of the skin and mucous membranes. This antigen-antibody reaction disrupts intercellular connections within the epidermis and between the epidermis and dermis, leading to loss of skin and mucous membrane adhesion function, resulting in the formation of blisters and erosions. Based on the location of blister formation, AIBDs can be divided into two main categories: pemphigus, characterized by intraepidermal blisters; and pemphigoid, characterized by subepidermal blisters. Pemphigus is a life-threatening chronic autoimmune bullous disease characterized by fissures within the epidermis and adjacent epithelium, accompanied by acantholysis. Pemphigus vulgaris (PV) is the most common type of pemphigus. The pathogenesis of pemphigus primarily involves autoantibodies attacking desmogleins (DSG) in the skin and mucous membranes. The main target antigen of pemphigus is desmosome core glycoprotein, especially Dsg3.

[0003] Dsg3, with a molecular weight of 64.95 KD, belongs to the cadherin superfamily and plays a crucial role in maintaining epidermal integrity. Disruption of its structure can lead to acantholysis, resulting in intraepidermal bullae. Dsg3 is a major target antigen of pemphigus, present both within and outside desmosomes. The intracellular components of Dsg3 interact with the cytoskeleton and form junctions via plaque-like proteins, while the extracellular region mediates intercellular connections. It is the extracellular region of Dsg3 that provides the antigenic epitopes recognized by PV antibodies. These key functional regions are called extracellular domains (ECs). The Dsg3 EC is divided into five domains (EC1-EC5), with EC-1 located at the N-terminus and EC-5 at the C-terminus. The extracellular domains of the desmosomal core glycoprotein contain four cadherin repeat sequences (EC1 to EC4) and an extracellular anchoring domain near the membrane. The N-terminal EC1 and EC2 domains contain residues crucial for adhesion interactions. Autoantibodies binding to DSG3 can directly interfere with cis or trans adhesion interactions, weakening desmosome adhesion. DSG3 clusters upon binding to pemphigus IgG and is internalized via a p38MAPK-dependent pathway. The native stereoconformation of the DSG3 molecule is important for eliciting autoimmune bullous eruptions.

[0004] Previous studies have obtained recombinant human Dsg3 protein, including the entire EC of Dsg3, through baculovirus expression systems and mammalian expression systems, and this has been confirmed by natural antibodies in the serum of human PV patients. Therefore, recombinant human Dsg3 protein can not only provide a reliable means for diagnosing PV, but also be used for further research on the pathogenesis of pemphigus. In pemphigus, the calcium signaling pathway is crucial for autoantibody-induced human epidermal bullae formation. PV IgG can induce PLC activation and Ca2+ signaling. 2+ Inflow, a process that requires the participation of Dsg1 (Desmoglein 1), while Dsg3 does not directly participate in Ca. 2+ The signaling pathway is initiated. However, the location and distribution of Dsg3 are affected by Ca. 2+ The influence of the signal pathway, when Ca 2+ When the signaling pathway is activated, the localization of Dsg3 at the cell edge becomes fragmented and partially translocates to the cytoplasm.

[0005] Phage display is a molecular biology method that fuses exogenous protein or peptide gene fragments into the capsid protein of a phage, allowing the expressed peptides or proteins to be displayed on the phage surface. First proposed by George P. Smith in 1985, this technology has been widely applied in antibody screening, vaccine development, drug target discovery, and protein-protein interaction research. Its core principle involves inserting a gene library to be screened into a phage vector, which then infects host cells to express and assemble into a complete phage, displaying peptide or protein epitopes. Next, affinity screening (biopanning) is performed to bind to specific target molecules, weeding out unbound phages and enriching specific binders. After multiple rounds of screening and sequencing analysis, peptides or antibodies with high affinity for the target molecule can be obtained. Compared to traditional methods, phage display technology offers advantages such as high throughput, low cost, and short cycle time, making it particularly suitable for situations where antibodies are difficult to obtain through in vivo immunization.

[0006] Pemphigus (PV) is a common autoimmune bullous disease that primarily affects the skin and mucous membranes. Patients possess autoantibodies against structural proteins in the intercellular spaces of keratinocytes, leading to impaired intercellular connections and the formation of characteristic lesions such as intraepidermal bullae. Early diagnosis is crucial for developing treatment strategies and predicting prognosis.

[0007] The diagnosis of PV includes the following aspects: 1) Clinical manifestations: Common features include ① the formation of loose, easily ruptured bullae on the skin; ② persistent erosions after rupture; ③ vesicles or erosions in mucous membranes; ④ a positive Nikolsky sign. 2) Histopathology: Microscopically, acantholysis is visible between epidermal or epithelial cells, accompanied by vesicle formation. 3) Immunological tests: Direct immunofluorescence (DIF) can observe IgG and / or complement deposition between cells in the lesion or surrounding normal tissue; indirect immunofluorescence (IIF) detects the presence of anti-epidermal intercellular antibodies in serum; enzyme-linked immunosorbent assay (ELISA) detects anti-Dsg3 antibodies. The diagnostic criteria are: meeting one of the clinical manifestations, combined with any one of the histopathological or immunological indicators; or meeting two of the clinical manifestations, combined with two positive immunological tests.

[0008] Currently, glucocorticoids remain the first-line treatment. For moderate to severe cases, early combination therapy with immunosuppressants is recommended, especially in cases with coexisting conditions such as diabetes, hypertension, and osteoporosis, where careful attention should be paid to the combined dose reduction strategy of hormones. While traditional hybridoma antibody preparation techniques can yield high-affinity antibodies, the process is cumbersome, time-consuming, and requires subsequent humanization, limiting the acquisition of human monoclonal antibodies and the accuracy of antibody quantification, thus affecting their targeting and efficacy in clinical treatment. Phage antibody library technology, on the other hand, does not require immunization and humanization steps. It relies on enrichment and screening in a fully controlled biochemical environment to achieve the rapid screening of high-affinity fully human antibody sequences.

[0009] Pemphigus is a potentially life-threatening autoimmune bullous skin disease. Approximately 60% of PV patients develop oral mucosal vesicles and bullae. These vesicles easily rupture spontaneously, forming erosions that are difficult to heal, severely impacting patients' eating and daily lives, and causing significant physical and psychological burden. In addition, some patients only present with persistent oral mucosal erosions without skin lesions. Therefore, rapid and accurate diagnostic methods are crucial for the treatment of this disease.

[0010] The pathogenicity of PV is mainly caused by IgG autoantibodies, with Dsg3 as the target antigen. After PV-IgG binds to Dsg3, it disrupts the intercellular junctions, leading to loss of cell adhesion function and ultimately the formation of blisters. Therefore, accurate and rapid detection of anti-Dsg3 antibody levels in the serum of PV patients is of great significance for in-depth research into the pathogenesis of this disease and for achieving precise diagnosis and treatment. Summary of the Invention

[0011] To address the shortcomings of existing technologies, the present invention aims to provide an antibody that binds to a specific antigenic peptide of pemphigus vulgaris (PV) and its application. This invention isolated PBMCs from 10 Dsg3 antibody-positive patients to construct a phage-based human antibody library. Through multiple rounds of screening, five human anti-Dsg3 monoclonal antibodies were identified. This antibody, by specifically binding to Dsg3, can quantitatively detect the level of anti-Dsg3 autoantibodies in PV patients, thereby monitoring disease activity and applying it to the clinical diagnosis of PV. This research provides theoretical and experimental basis for specific treatment of PV and also offers a new research direction for treating autoimmune diseases with autoantibodies as the main pathogenic mechanism.

[0012] To achieve this objective, the present invention adopts the following technical solution:

[0013] In a first aspect, the present invention provides an antibody that binds to a pemphigus vulgaris-specific antigenic peptide, wherein the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO:1; CDR2 is shown in SEQ ID NO:2; and CDR3 is shown in SEQ ID NO:3; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO:6; the amino acid sequence of CDR2 is AAS; and CDR3 is shown in SEQ ID NO:8.

[0014] Alternatively, the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO:11; CDR2 is shown in SEQ ID NO:12; and CDR3 is shown in SEQ ID NO:13; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO:16; the amino acid sequence of CDR2 is YDS; and CDR3 is shown in SEQ ID NO:18.

[0015] Alternatively, the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO:21; CDR2 is shown in SEQ ID NO:22; and CDR3 is shown in SEQ ID NO:23; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO:26, the amino acid sequence of CDR2 is AAS, and CDR3 is shown in SEQ ID NO:28.

[0016] Alternatively, the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO:31; CDR2 is shown in SEQ ID NO:32; and CDR3 is shown in SEQ ID NO:33; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO:36; the amino acid sequence of CDR2 is SNN; and CDR3 is shown in SEQ ID NO:38.

[0017] Alternatively, the heavy chain variable region CDR1 of the antibody is shown in SEQ ID NO:41; CDR2 is shown in SEQ ID NO:42; and CDR3 is shown in SEQ ID NO:43; the light chain variable region CDR1 of the antibody is shown in SEQ ID NO:46; the amino acid sequence of CDR2 is DNN; and CDR3 is shown in SEQ ID NO:48.

[0018] This invention screens monoclonal antibodies that bind to pemphigus vulgaris-specific antigenic peptides. Monoclonal antibodies are one of the most numerous and fastest-growing classes of therapeutic proteins, with over 500 therapeutic antibodies and their derivatives currently in clinical trials, primarily focused on the treatment of tumors and autoimmune diseases. Developing antibodies to detect serum levels of anti-Dsg3 antibodies is of great significance for understanding the pathogenesis of pemphigus vulgaris and achieving precise diagnosis and treatment.

[0019] This invention aims to screen for high-affinity human antibodies against the pathogenic antigen Dsg3 in pemphigus (PV). Peripheral blood cells (PBMCs) were isolated from peripheral blood of Dsg3 antibody-positive patients, and total RNA was extracted and quality-controlled. Qualified RNA samples were reverse transcribed into cDNA using reverse transcription PCR, and VH and VL (including VK and Vλ) gene fragments of the antibody variable region were amplified.

[0020] Subsequently, the amplified light chain variable region fragment was cloned into the phage display vector pATA-scFv-2 carrying the antibody framework, and assembled with the heavy chain variable region to form a single-chain antibody (scFv) structure, constructing a highly diverse antibody display library. The antibody genes in this library were further inserted upstream or N-terminally into the coding region of M13 phage capsid protein gene III (gIII) or gene VIII (gVIII) to ensure that the antibody fragment could be displayed on the phage surface as a fusion protein.

[0021] By infecting *E. coli* with helper phages, each bacterium produces phage particles carrying specific antibodies, ultimately forming a library containing billions of different antibody clones. Utilizing the principles of phage display technology, we perform in vitro affinity screening on the constructed library: phages are bound to the target antigen Dsg3, non-specific binders are eluted, and phages tightly bound to the antigen are retained. These are then amplified and subjected to multiple rounds of affinity enrichment. Multiple rounds of panning are performed under 1 mM CaCl2 conditions. After 3-5 rounds of screening, phage clones with strong binding affinity to Dsg3 and high specificity are obtained.

[0022] Finally, positive phage clones were screened and validated using ELISA, from which fully human antibodies with high affinity and high specificity were identified. This process not only avoids the limitations of animal immunization and humanization steps in the traditional hybridoma method, but also significantly improves screening efficiency and the clinical translation potential of the antibodies.

[0023] In this invention, a 1 mM Ca content is used. 2+ Buffer-treated DSG3 is more likely to maintain its native conformation and bind more readily to antibodies. The extracellular structure of DSG3 contains EC1-EC5 domains. Among these, Ca... 2+ Antibodies selected under causal conditions can bind to the EC1-EC2 domains, which are pathogenic epitopes of pemphigus; non-Ca... 2+ Antibodies screened under dependent conditions bind more to the EC2-EC5 domains, which are mostly non-pathogenic epitopes of pemphigus. This invention utilizes Ca... 2+ After screening, in the Ca-containing 2+ and without Ca 2+Under specific conditions, ELISA testing yielded antibodies with good binding ability and no pathogenicity, laying the foundation for the subsequent application of antibodies to the treatment of other diseases.

[0024] Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9.

[0025] Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:14; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0026] Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:24; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29;

[0027] Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:34; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:39;

[0028] Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:44; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:49.

[0029] Preferably, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:5; and the light chain amino acid sequence is shown in SEQ ID NO:10.

[0030] Alternatively, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:15; the light chain amino acid sequence is shown in SEQ ID NO:20;

[0031] Alternatively, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:25; the light chain amino acid sequence is shown in SEQ ID NO:30;

[0032] Alternatively, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:35; the light chain amino acid sequence is shown in SEQ ID NO:40;

[0033] Alternatively, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:45; the light chain amino acid sequence is shown in SEQ ID NO:50.

[0034] In a second aspect, the present invention provides a nucleic acid molecule that encodes the antibody described in the first aspect that binds to the pemphigus vulgaris-specific antigenic peptide.

[0035] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0036] Fourthly, the present invention provides a host cell containing at least one copy of the expression vector described in the third aspect; or the host cell has the nucleic acid molecule described in the second aspect integrated into its chromosome.

[0037] Fifthly, the present invention provides a composition for detecting Dsg3 protein in a sample, the composition comprising the antibody described in the first aspect that binds to the pemphigus vulgaris-specific antigenic peptide.

[0038] In a sixth aspect, the present invention provides a kit for detecting Dsg3 protein in a sample, the kit comprising the antibody described in the first aspect that binds to the pemphigus vulgaris-specific antigenic peptide.

[0039] In a seventh aspect, the present invention provides the use of the antibody that binds to the pemphigus vulgaris-specific antigenic peptide described in the first aspect, the composition for detecting Dsg3 protein in a sample described in the fifth aspect, and the kit for detecting Dsg3 protein in a sample described in the sixth aspect in the preparation of products for detecting pemphigus vulgaris.

[0040] Eighthly, the present invention provides a method for preparing the antibody that binds to the pemphigus vulgaris-specific antigenic peptide described in the first aspect, the method comprising:

[0041] (1) Isolate PBMCs from Dsg3 antibody-positive patients, extract total RNA, reverse transcribe it into cDNA by reverse transcription PCR, and amplify the VH and VL gene fragments of the antibody variable region to construct a phage display library;

[0042] (2) The phage is bound to the target antigen Dsg3, non-specific binders are eluted, and the phage that is tightly bound to the antigen is retained. Then, it is amplified and enriched for affinity. After rounds of screening, phage clones with strong binding ability to Dsg3 and high specificity can be obtained.

[0043] Preferably, the buffer solution used for screening contains 1 mM CaCl2.

[0044] In this invention, multiple rounds of panning are performed under 1 mM CaCl2 conditions. After 3-5 rounds of screening, phage clones with strong binding ability to Dsg3 and high specificity can be obtained. Compared with anti-DSG3 antibodies screened without CaCl2 conditions, those containing CaCl2... 2+ The selected antibodies bind more strongly to proteins on the surface of HacaT cells, resulting in better efficacy.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] This invention reverse-transcribes RNA from DSG3 antibody-positive patients into cDNA, and amplifies the VH and VL fragments of the DNA. The VK and Vλ gene fragments obtained in vitro are cloned into the pATA-scFv-2 vector using cloning technology to form VK and Vλ libraries. Plasmid vectors for the VK and Vλ libraries are extracted using a plasmid extraction kit, and the in vitro amplified VH gene fragment is inserted into these vectors to form KH and λH libraries. The artificially synthesized Dsg3 domain gene is recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector is transfected into DH10Bac competent cells and cultured, and the Dsg3 antigen protein is purified.

[0047] This invention involved three rounds of antibody phage library screening, identifying clones with an antigen group greater than three times that of the control group as positive clones. These single clones were then sequenced and analyzed. Erroneous and duplicate antibody sequences were eliminated, and combined with the antigen-antibody specific binding ability reflected in the ELISA experiment, five high-affinity antibodies were ultimately obtained, named DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), and DSG3-R3P1-B1 (ATGM1152). These antibodies exhibit high activity, good stability, and strong specificity, serving as a reference standard for qualitative detection of PV positivity and also for quantitative detection of anti-Dsg3 autoantibody levels in PV patients. Attached Figure Description

[0048] Figure 1 This is a PCR agarose gel electrophoresis image of a single clone of the VL phage library.

[0049] Figure 2 This is a PCR agarose gel electrophoresis image of a single clone of the KH phage library.

[0050] Figure 3 This is a PCR agarose gel electrophoresis image of a single clone of the λH phage library.

[0051] Figure 4 This is an SDS-PAGE electrophoresis image of the target protein Dsg3.

[0052] Figure 5 The results are from ELISA at different antibody dilutions.

[0053] Figure 6 The results of Western blot analysis show the binding of the antibody to the recombinant DSG3 protein.

[0054] Figure 7 Flow cytometry data showing the affinity of antibodies for HacaT cell surface antigens.

[0055] Figure 8 This is the IF data for antibody binding to human DSG3 protein. Detailed Implementation

[0056] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0057] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0058] Example 1

[0059] This embodiment provides a method for constructing a human ScFv phage display library.

[0060] Table 1 lists the main reagents used in this embodiment and their sources.

[0061] Table 1

[0062]

[0063] 1. Library Construction

[0064] 1.1 Assemble the heavy chain variable region (VH) and light chain variable region (VL). Table 2 shows the PCR reaction system, and Table 3 shows the PCR reaction steps.

[0065] Table 2

[0066]

[0067] Table 3

[0068]

[0069] The three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0070] The primer sequences are shown below:

[0071] upstream primer (F) for the heavy chain variable region:

[0072] 5' L-VH 1:ACAGGTGCCCACTCCCAGGTGCAG. (SEQ ID NO:47)

[0073] 5'L-VH 3:AAGGTGTCCAGTGTGARGTGCAG. (SEQ ID NO:51)

[0074] 5' L-VH 4 / 6: CCCAGATGGGTCCTGTCCCAGGTGCAG. (SEQ ID NO:52)

[0075] 5' L-VH 5 / 7: CAAGGAGTCTGTTCCGAGGTGCAG. (SEQ ID NO:53)

[0076] upstream primer (F) of the light chain variable region:

[0077] 5' L VK 1 / 2: ATGAGGSTCCCYGCTCAGCTGCTGG. (SEQ ID NO:54)

[0078] 5' L VK 3: CTCTTCCTCCTGCTACTCTGGCTCCCAG. (SEQ ID NO:55)

[0079] 5' L VK 4 / 5:ATTTCTCTGTTGCTCTGGATCTCTG. (SEQ ID NO:56)

[0080] 5' L Vλ1:GGTCCTGGGCCCAGTCTGTGCTG. (SEQ ID NO:57)

[0081] 5' L Vλ2: GGTCCTGGGCCCAGTCTGCCCTG. (SEQ ID NO:58)

[0082] 5' L Vλ3:GCTCTGTGACCTCCTATGAGCTG. (SEQ ID NO:59)

[0083] 5' L Vλ4 / 5: GGTCTCTCTCSCAGCYTGTGCTG. (SEQ ID NO:60)

[0084] 5' L Vλ6:GTTCTTGGGCCAATTTTATGCTG. (SEQ ID NO:61)

[0085] 5' L Vλ7: GGTCCAATTCYCAGGCTGTGGTG. (SEQ ID NO:62)

[0086] 5' L Vλ8 / 9 / 10:GAGTGGATTCTCAGACTGTGGTG. (SEQ ID NO:63)

[0087] Downstream primer (R) for the light chain variable region:

[0088] 3' CK: TGCGTCCTTGCTGTCCTGCT. (SEQ ID NO:64)

[0089] 3' Cλ: CACCAGTGTGGCCTTGTTGGCTTG. (SEQ ID NO:65)

[0090] Downstream primer (R) for the heavy chain variable region: 3'VH: ACTCGAGACGGTGACCAGGGTGCC. (SEQ ID NO:66)

[0091] 1.2 Construction of a light chain variable region phage display library

[0092] 1.2.1 Prepare the pATA-scFv-2 vector for library cloning.

[0093] 1.2.2 Digestion of vector and PCR products. Table 4 shows the reaction system for digesting vector and PCR products.

[0094] Table 4

[0095]

[0096] 1.2.3 After digestion, the mixture is ligated. Table 5 shows the ligation reaction system.

[0097] Table 5

[0098]

[0099] Incubate overnight at 16°C, then inactivate by heating at 65°C for 10 min.

[0100] 1.2.4 Electrical transfer.

[0101] Preparation of TG1 competent cells: Pre-warm 1 mL of SOC medium (Sigma, S1797) at 37°C. Place electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction). Remove electroporation competent cells from the -80°C freezer and place on ice until they are completely thawed (10–15 minutes). After thawing, gently mix the cells. Place 50 μL of cells into a frozen microcentrifuge tube on ice. Carefully add 3 μL of the DNA mixture to the frozen electroporation cuvette, avoiding air bubbles. Quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom. Electroporate at 600 Ω, 10 μF, and 1.8 kV. Immediately add 1 mL of pre-warmed SOC medium to each tube within 10 seconds of the pulse. Shake at 37°C and 250 rpm for 1 hour. Collect all electroporation medium. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / glucose plates. Incubate overnight at 37°C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the inoculum volume.

[0102] 1.3 Construction of VL-VH phage display library

[0103] 1.3.1 Digestion of vectors and PCR products.

[0104] Table 6 shows the digestion reaction system.

[0105] Table 6

[0106]

[0107] 1.3.2 Connection.

[0108] Table 7 shows the connection reaction system.

[0109] Table 7

[0110]

[0111] Incubate overnight at 16°C, then inactivate by heating at 65°C for 10 min.

[0112] 1.3.3 Electrical transfer.

[0113] Preparation of TG1 competent cells: Pre-warm 4 mL of SOC medium (Sigma, S1797) at 37°C. Place electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction). Remove electroporation competent cells from the -80°C freezer and place on ice until they are completely thawed (10–15 minutes). After thawing, gently mix the cells. Carefully add 6 μL of the DNA mixture to the frozen electroporation cuvette, avoiding air bubbles. Quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom. Electroporate at 600 Ω, 10 μF, and 2.5 kV. Immediately add 2 mL of pre-warmed SOC medium to each tube within 10 seconds of the pulse. Shake at 37°C and 250 rpm for 1 hour. Collect all electroporation medium. Serially dilute 10 μL of the culture to 90 μL of SOC medium and spread on LB / Amp / glucose. Incubate overnight at 37°C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the inoculation volume of the plate.

[0114] 1.4 Library Evaluation

[0115] 1.4.1 Colony PCR: PCR was performed using the constructed library as a template.

[0116] Table 8 shows the PCR reaction system, and Table 9 shows the PCR reaction conditions.

[0117] Table 8

[0118]

[0119] Table 9

[0120]

[0121] The three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0122] Primer sequences: Upstream primer (F) for pATA-scFv-2 vector identification: AGCGGATAACAATTTCACACAGGA. (SEQ ID NO:27) Downstream primer (R) for pATA-scFv-2 vector identification: GCCCCTTATTAGCGTTTGCCATC. (SEQ ID NO:37). Agarose gel electrophoresis results after PCR are shown below. Figures 1-3 As shown.

[0123] Figure 1 The results are the electrophoresis detection results of the constructed VL phage library, where lane M: DL2000, lanes 1-16: pATA-VK, and lanes 17-32: pATA-Vλ.

[0124] Figure 2 The results are the electrophoresis results of the constructed KH phage library, where lane M: DL2000, lanes 1-48: pATA-scFv-KH.

[0125] Figure 3 The results are the electrophoresis detection results of the constructed λH phage library, where lane M: DL2000, lanes 1-48: pATA-scFv-λH.

[0126] 1.4.2 Sequencing: Selected positive clones were sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing.

[0127] 1.5 Expression of Dsg3 protein

[0128] The Dsg3 gene sequence was artificially synthesized and recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector; the cloning site is BamH1 / Xho1.

[0129] The amino acid sequence of Dsg3 is SEQ ID NO:7.

[0130] The gene sequence of Dsg3 is SEQ ID NO:17.

[0131] The Dsg3-pFastBac1 expression vector was transfected into DH10Bac competent cells and cultured. The precipitate was collected and subjected to GST tag affinity chromatography to obtain Dsg3 protein. The purified Dsg3 was then subjected to SDS-PAGE (polyacrylamide gel electrophoresis) to verify its purity. The SDS-PAGE electrophoresis image of the purified Dsg3 is shown below. Figure 4 As shown, the purity is greater than 90%. Figure 4 The image shows the target protein Dsg3 SDS-PAGE electrophoresis result. The protein size is 64.95 kDa and the purity is greater than 90%.

[0132] Example 2

[0133] Preparation of monoclonal antibodies that specifically bind to DSG3

[0134] Table 10 lists the main reagents used in this embodiment.

[0135] Table 10

[0136]

[0137] 1. Experimental Procedure

[0138] 1.1 Selection

[0139] (1) Coating of immunotubes: DSG3 50 μg / mL, PBS buffer (1 mM CaCl2), 1 mL per tube, overnight at 4°C.

[0140] (2) Wash the immunotubes three times with 5 mL PBST.

[0141] (3) 5 mL 5% milk / PBST (1 mM CaCl2) blocked at 30℃ for 1 h.

[0142] (4) Wash once with 5 mL PBS.

[0143] (5) Negative screening of library phage: Add 0.5% skim milk / PBST (1 mM CaCl2) or 0.1% casein / PBST (1 Mm CaCl2) to the library phage (phage amplified in the previous round), add PBS to the total volume of 1 mL, and incubate at room temperature for 0.5 h.

[0144] (6) Add the phage after negative sieving to each tube and incubate at 30°C for 2 h.

[0145] (7) Wash 6 times with 5 mL PBST.

[0146] (8) Add 1 mL of Gly-HCl (pH=2.2) to the tube to elute the phage, and incubate with shaking at room temperature for about 6-8 min. Add 120-130 μL of Tris-HCl (pH=9.6) to neutralize the solution to pH=7.0-8.0.

[0147] (9) After eluting, the phage was diluted and used to infect logarithmic Escherichia coli TG1. The titer was determined by plating.

[0148] 1.2 Amplification of eluted bacteriophages

[0149] (1) Take the eluted phage and add it to the logarithmic phase Escherichia coli TG1 bacterial culture. After standing at 37°C for 30 min, incubate at 220 rpm for 30 min-1 h.

[0150] (2) Add antibiotic Amp to the culture medium and incubate at 37℃ and 220 rpm until the bacterial OD is about 0.4-0.6.

[0151] (3) Add helper phages to the bacterial culture. After standing at 37℃ for 30 min, incubate at 220 rpm for 45 min-1 h.

[0152] (4) Centrifuge the bacterial culture at 3000-5000 rpm and discard the supernatant. Resuspend the bacterial cells in an equal volume of 2YT-Amp-Kan medium. Incubate overnight at 30℃ and 220 rpm.

[0153] (5) The next day, centrifuge the bacterial culture at 8000 rpm, 4℃, for 20 min, and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of PEG / NaCl solution. Mix thoroughly and place on ice or at 4℃ for 1-2 h.

[0154] (6) Centrifuge at 8000 rpm, 4℃ for 30 min, and discard the supernatant. Resuspend the precipitate in about 1 mL of PBS. Centrifuge at 8000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube.

[0155] (7) Dilute the amplified phage, infect TG1 in the logarithmic phase, and determine the titer by plating.

[0156] 1.3 Selection

[0157] Table 11 shows the selection criteria for each round.

[0158] Table 11

[0159]

[0160] 1.4 Polyclonal phage ELISA detection

[0161] Experimental group (Ag), control group 1 (NC1), and control group 2 (NC2).

[0162] (1) Coating of immunosuppressed plates (experimental group: 4 μg / mL DSG3; control group 1: 4 μg / mL NC-His; control group 2: PBS (1 mM CaCl2), incubated overnight at 4℃).

[0163] (2) Wash three times with 300 μL PBST.

[0164] (3) Add 300 μL of 5% skim milk (1 mM CaCl2) and seal at 37℃ for 2 hours.

[0165] (4) Dilute the phages after each round of amplification with PBS, increasing the dilution factor by 3-fold. The initial concentration is 1×10⁻⁶. 12 pfu / mL. Add 100 μL of diluted amplified phage to each well. Incubate at 32°C for 1 h, then wash three times with 300 μL PBST.

[0166] (5) Add 100 μL of secondary antibody (anti-phage M13-HRP) dilution solution, incubate at 30℃ for 1 h, and wash 3 times with 300 μL PBST.

[0167] (6) Add 100 μL of TMB colorimetric solution and develop color in the dark for 3-8 min. Add 50 μL of 2 M HCl to terminate the reaction. Read the microplate reader (450 nm-620 nm).

[0168] 1.5 Monoclonal phage ELISA screening

[0169] (1) Select R3 eluted phage, dilute it to an appropriate concentration, infect TG1 in the logarithmic phase, and plate it.

[0170] (2) The next day, 96 single clones were picked from the plate and inoculated into a 96-well plate and cultured at 37°C and 250 rpm until the bacterial OD=0.4-0.6.

[0171] (3) Add helper phages to the 96-well plate medium. Incubate at 37°C for 30 min, then shake at 250 rpm at 37°C for 45 min-1 h.

[0172] (4) Centrifuge the 96-well plate at 4000 rpm for 5 min and discard the supernatant. Resuspend the bacterial culture in 2YT-Amp-Kan medium in each well and incubate overnight at 30℃ and 250 rpm with shaking.

[0173] (5) The next day, centrifuge the 96-well plate at 4000 rpm for 10-15 min and take the supernatant for ELISA experiment.

[0174] (6) Coating of immunosuppressed plates: antigen protein (experimental group: 4 μg / mL DSG3; control group: 4 μg / mL NC-His, buffer PBS (1 mM CaCl2)), 100 μL, overnight at 4℃.

[0175] (7) Wash 3 times with 300 μL PBST.

[0176] (8) Block 300 μL of 5% skim milk / PBST (1 mM CaCl2) at 37℃ for 1 h. Wash 3 times with 300 μL of PBST.

[0177] (9) Add 100 μL of phage supernatant to each well. Incubate at 32°C for 1 h. Wash three times with 300 μL of PBST.

[0178] (10) Add 100 μL of secondary antibody (anti-phage M13) dilution solution, incubate at 32℃ for 1 h, and wash 3 times with 300 μL PBST.

[0179] (11) Add 100 μL of TMB colorimetric solution and develop color in the dark for 3-8 min. Add 50 μL of 2 M HCl to terminate the reaction. Read the microplate reader (450 nm-620 nm).

[0180] 1.6 Secondary Validation of Positive Clones

[0181] (1) Take 50 μL of positive clones and inoculate them into 2 mL of 2YT-AG medium, and culture until OD. 600 =0.4-0.6.

[0182] (2) Add helper phages to the culture medium. Incubate at 37°C for 30 min, then shake at 250 rpm at 37°C for 45 min-1 h.

[0183] (3) Centrifuge the bacterial culture at 4000 rpm for 5 min and discard the supernatant. Resuspend the bacterial culture in 2 mL of 2YT-Amp-Kan medium in each well and incubate overnight at 30℃ and 250 rpm with shaking.

[0184] (4) Centrifuge the overnight cultured bacterial solution at 5000 rpm for 5 min, and take the supernatant for ELISA experiment.

[0185] (5) Coat the immunosuppressed plates (experimental group: 4 μg / mL DSG3; control group 1: 4 μg / mL NC-His, control group 2: PBS (1 mM CaCl2)) and incubate at 32℃ for 2 hours. Wash 3 times with 300 μL PBST.

[0186] (6) Add 300 μL of 5% skim milk (1 mM CaCl2) and seal at 37°C for 2 hours.

[0187] (7) Add 100 μL of phage supernatant stock solution (5-fold serial dilution) to each well. Incubate at 32°C for 2 hours. Wash 3 times with 300 μL PBST.

[0188] (8) Add 100 μL of secondary antibody (anti-phage M13-HRP) dilution solution, incubate at 32℃ for 1 h, and wash 3 times with 300 μL PBST.

[0189] (9) Add 100 μL of TMB colorimetric solution and develop color in the dark for 3-8 min. Add 50 μL of 2 M HCl to terminate the reaction. Read the microplate reader (450 nm-620 nm).

[0190] 2. Experimental Results

[0191] (1) Selection results

[0192] Table 12 shows the selection results.

[0193] Table 12

[0194]

[0195] (2) Results of polyclonal phage ELISA

[0196] Experimental group (Ag): 4 μg / mL DSG3; Control group 1 (NC1): 4 μg / mL NC-His; Control group 2 (NC2): PBS (1 mM CaCl2).

[0197] Table 13 shows the results of polyclonal phage ELISA.

[0198] Table 13

[0199]

[0200] Based on the polyclonal results, we selected a third round of monoclonal screening and chose 96 clones for monoclonal ELISA testing of the antigen group and the control group.

[0201] (3) Results of monoclonal phage ELISA

[0202] Table 14 shows the ELISA results of R3P1 monoclonal phage (antigen group), Ag: 4 μg / mL DSG3.

[0203] Table 14

[0204]

[0205] Table 15 shows the ELISA results of R3P1 monoclonal phage (control group) NC: 4 μg / mL NC-His.

[0206] Table 15

[0207]

[0208] Based on the experimental results, clones were selected and sent for sequencing analysis, resulting in 12 different sequences. These 12 clones were then used for secondary verification.

[0209] (4) Results of the second ELISA test

[0210] Experimental group (Ag): 4 μg / mL DSG3.

[0211] Control group 1 (NC1) NC1: 4 μg / mL NC-His.

[0212] Control group 2 (NC2): NC2: PBS.

[0213] Table 16 shows the results of the second ELISA test (1 mM CaCl2 was not added during the experiment).

[0214] Table 16

[0215]

[0216] Table 17 shows the results of the second ELISA test (1 mM CaCl2 was added during the experiment).

[0217] Table 17

[0218]

[0219] 3. Conclusion

[0220] After this screening process, five positive clones were finally selected, which could bind to the antigen but not to the control label.

[0221] 4. Sequencing of antibody sequences

[0222] Five phage-positive clones obtained through screening—DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), and DSG3-R3P1-B1 (ATGM1152)—were subjected to full-sequence sequencing. The corresponding antibody heavy and light chains, as well as their full sequences, are shown in Tables 18 and 19 below.

[0223] Table 18

[0224]

[0225] Table 19

[0226]

[0227] Example 3

[0228] OD values ​​of antibodies at different dilution concentrations detected by ELISA

[0229] Table 20 lists the main reagents used in this embodiment.

[0230] Table 20

[0231]

[0232] 1. Experimental Procedure

[0233] (1) Coating: Coat the microplate with 100 μL / well of Dsg3 protein (4 μg / mL) (1 mM CaCl2) and incubate overnight at 4°C.

[0234] (2) Washing: Discard the liquid in the microplate, wash each well twice with 300 μL of 0.05% PBST, and wash once with PBS.

[0235] (3) Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0236] (4) Incubation of positive antibodies: DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), and DSG3-R3P1-B1 (ATGM1152) antibodies were serially diluted, and 100 μL of the diluted antibody solution was added to each well. The mixture was incubated at 30°C for 2 hours.

[0237] (5) Washing: Same as step 2.

[0238] (6) Secondary antibody incubation: Dilute the goat anti-human IgG (H+L) antibody 10,000 times with blocking buffer, add 100 μL of diluted secondary antibody to each well, and incubate at 37°C for 1 hour.

[0239] (7) Washing: Same as step (2).

[0240] (8) Color development: Add 100 μL TMB to each well, incubate at 37°C for 10 minutes, and then add 50 μL 2 M HCl to each well to terminate the reaction.

[0241] (9) Plate reading: Use an ELISA reader to read values ​​at 450 nm-630 nm, such as... Figure 5 As shown.

[0242] Figure 5 ELISA results for antibody diluted at different concentrations. From Figure 5 It is known that DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), and DSG3-R3P1-B1 (ATGM1152) can all strongly bind to recombinant DSG3 protein, while DSG3-R3P1-C1 (ATGM1151) can weakly bind to recombinant DSG3 protein.

[0243] Example 4

[0244] Western blot detection of the binding of monoclonal antibody to the target antigen DSG3

[0245] Table 21 lists the main reagents used in this embodiment.

[0246] Table 21

[0247]

[0248] 1. Experimental Procedure

[0249] 1.1 Protein Sample Preparation

[0250] (1) Wash the collected HacaT cells with 1 mL PBS, centrifuge at 3000 rpm for 5 min, and add 0.9 g.

[0251] (2) Discard the supernatant and resuspend in 120 μL of lysis buffer (strong RIPA + phosphatase inhibitor).

[0252] (3) On ice, pyrolysis for 20 min; after pyrolysis, 12000 rpm for 30 min at 4℃.

[0253] (4) After centrifugation, take 110 μL of supernatant into a new label tube.

[0254] (5) BCA method for detecting protein concentration:

[0255] Prepare BCA working solution: Solution A: Solution B = 50:1 200 μL / well.

[0256] Prepare Pr sample wells: 3 μL of Pr stock solution diluted 10 times + 27 μL of ddw = 30 μL.

[0257] Add 200 μL / well of working solution to the 96-well plate.

[0258] Add 25 μL of pore protein sample and BSA standard.

[0259] Incubate at 37°C for 30 minutes.

[0260] After incubation, the embryos are placed on the machine.

[0261] (6) Calculate the protein concentration and loading volume, control the protein loading amount to 30 μg, and add the total protein extracted by HacaT and ATGE5228 (recombinant DSG3 extracellular protein fragment).

[0262] 1.2 Electrophoresis and membrane transfer

[0263] (1) Add total protein extracted by HacaT and ATGE5228 (recombinant DSG3 extracellular protein fragment) to the gel wells.

[0264] (2) Pour in electrophoresis buffer: concentrate protein voltage 80 V, 20 min, separate protein voltage 120 V, 40 min.

[0265] (3) Activate the PVDF membrane with methanol in advance, cut the upper left corner to mark it, and prepare the transfer sandwich.

[0266] (4) Clamp the transfer clamp, pour new transfer solution into the tank, transfer on ice, 100 V, 120 min.

[0267] 1.3 Blocking, antibody incubation, and color development

[0268] (1) After the transfer is completed, add 5% skim milk, seal at room temperature for 1 h, and rotate at 0.

[0269] (2) After sealing, rinse off the surface sealing liquid with 1× TBST;

[0270] (3) Prepare primary antibody solutions of DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), and DSG3-R3P1-B1 (ATGM1152) at a concentration of 1 μg / mL using antibody dilution buffer, and incubate overnight at 4°C on a shaker.

[0271] (4) 1× TBST cleaning, 15 min, three times, speed=80.

[0272] (5) Prepare HRP-labeled anti-human IgG Fcγ antibody (Anti-HumanIgG, Fc gamma Fragment Specific, HRP-Linked Antibody) secondary antibody solution at a concentration of 1:10000 using antibody dilution buffer, and shake slowly at room temperature for 1 h with speed = 0.

[0273] (6) 1× TBST cleaning, 15 min, three times, speed=80.

[0274] (7) Prepare the colorimetric solution, A solution: B solution = 1:1.

[0275] (8) Exposure to the machine. Experimental results are as follows: Figure 6 As shown.

[0276] from Figure 6 It is evident that human anti-Dsg3 monoclonal antibodies exhibit strong specific binding ability to the extracellular domain of recombinant Dsg3 protein. ATGE5228 represents the extracellular domain of recombinant DSG3 protein. Specifically, DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-A2 (ATGM1150), and DSG3-R3P1-B1 (ATGM1152) bind strongly to the recombinant DSG3 protein, DSG3-R3P1-E1 (ATGM1149) binds weakly, and DSG3-R3P1-C1 (ATGM1151) does not bind (suggesting that its binding site is a spatial epitope rather than a linear epitope).

[0277] Example 5

[0278] Flow cytometry detection of affinity between monoclonal antibodies and HacaT cell surface antigens

[0279] Table 22 lists the main reagents used in this embodiment.

[0280] Table 22

[0281]

[0282] 1. Experimental Procedure

[0283] (1) Collect 10 6 HacaT cells were transferred to 1.5 mL EP tubes containing PBS with 2% FBS, 50 μL / tube.

[0284] (2) Blocking: Add 2.5 μL of human Fc blocking solution (Human TruStain FcXTM) to the EP tube and incubate at room temperature for 10 min.

[0285] (3) Add primary antibody solution directly: 4 μg / mL DSG3-R3P1-D1(ATGM1148), DSG3-R3P1-E1(ATGM1149), DSG3-R3P1-A2(ATGM1150), DSG3-R3P1-C1(ATGM1151), DSG3-R3P1-B1(ATGM1152) and hIgG1-ISOtype, PBS containing 2% FBS, and incubate at 4℃ for 30 min.

[0286] (4) Centrifuge at 4℃, 500 g for 6 min.

[0287] (5) Washing off unbound primary antibody: PBS containing 2% FBS, centrifuged at 500 g for 6 min at 4°C.

[0288] (6) Add fluorescently labeled secondary antibody: Resuspend cells in 100 μL of PBS containing 2% FBS. At this point, avoid light and add 10... 6 Prepare an antibody mixture with 1 μL of antibody corresponding to 1 μL of cell volume, add 1 μL of PE-labeled goat anti-human IgG Fc terminal secondary antibody (PE-anti human IgG Fc), and incubate at 4°C in the dark for 30 min.

[0289] (7) Centrifuge at 4℃, 500 g for 6 min.

[0290] (8) Washing off unbound secondary antibody: PBS containing 2% FBS, centrifuged at 500 g for 6 min at 4°C.

[0291] (9) Resuspend cells in 200 μL PBS, pass through a 40 μm sieve into flow cytometry tubes, and perform analysis; experimental results are as follows. Figure 7 As shown.

[0292] from Figure 7 It was found that human anti-Dsg3 monoclonal antibody has a strong specific binding ability to HacaT surface antigen. Blank: fluorescence negative group, excluding fluorescence background interference; hIgG: isotype control group (ISOtype control group), excluding non-specific antibody binding; ATGM1148, ATGM1149, ATGM1150, ATGM1151, and ATGM1152 were experimental groups. Among them, DSG3-R3P1-A2 (ATGM1150) has the strongest binding ability to HacaT surface antigen, followed by DSG3-R3P1-E1 (ATGM1149) and DSG3-R3P1-B1 (ATGM1152), while DSG3-R3P1-D1 (ATGM1148) and DSG3-R3P1-C1 (ATGM1151) have relatively weak binding abilities to HacaT surface antigen.

[0293] Example 6

[0294] IF method for detecting the binding of monoclonal antibodies to human DSG3

[0295] Table 23 lists the main reagents used in this embodiment.

[0296] Table 23

[0297]

[0298] 1. Experimental Procedure

[0299] 1.1 Plasmid transfection

[0300] (1) 293T cells were plated in a confocal dish and transfected with 2 μg of DSG3-pTT5 plasmid when the cell density was 70-80%.

[0301] Plasmid transfection MIX system:

[0302] Add 2 μg of DSG3-pTT5 plasmid to Opti-MEM, vortex thoroughly, and let stand at room temperature for 5 min.

[0303] b. Add PEI to the settled Opti-MEM mixture at a ratio of 1:2 for all plasmids:PEI, mix well, vortex thoroughly, and let stand for 20 min at RT.

[0304] c. Add the prepared plasmid suspension directly to the culture dish.

[0305] After 5 hours, change the medium and continue culturing for another 24 hours.

[0306] (2) After 24 hours, when the cell density in the confocal dish is appropriate, discard the culture medium and wash three times with PBS. Washing conditions: stand at room temperature / shake gently for 5 minutes.

[0307] (3) Add 1 mL of 4% paraformaldehyde and let it stand at room temperature for 15 minutes to fix.

[0308] (4) Discard the 4% paraformaldehyde and wash three times with PBS.

[0309] (5) Add 500 μL of PBS containing 10% FBS and block for 1 hour.

[0310] (6) After discarding the blocking solution, wash three times with PBS.

[0311] (7) Add 4 μg / mL of DSG3-R3P1-D1 (ATGM1148), DSG3-R3P1-E1 (ATGM1149), DSG3-R3P1-A2 (ATGM1150), DSG3-R3P1-C1 (ATGM1151), DSG3-R3P1-B1 (ATGM1152) and hIgG1-ISOtype solution, and let stand overnight at 4℃.

[0312] (8) Take it out the next day and wash it three times with PBS.

[0313] (9) Add the prepared AF568 fluorescently labeled anti-human secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568) (1:1000 PBS dilution), and incubate at room temperature in the dark for 1 hour.

[0314] (10) Take the secondary antibody, wash three times with PBS, add 1 mL of prepared diphenylimine (Hoechst) (diluted with PBS at 1:10000) to stain the cell nuclei, and incubate at room temperature in the dark for 15 minutes.

[0315] (11) Discard the diphenylimine (Hoechst), wash three times with PBS, and then take pictures under a Leica laser confocal microscope. The experimental results are as follows: Figure 8 As shown.

[0316] from Figure 8It was found that the human anti-Dsg3 monoclonal antibody specifically bound to 293t cells overexpressing human DSG3 protein. hIgG: isotype control group, excluding non-specific antibody binding; Flag: protein tag, showing protein expression; ATGM1148, ATGM1149, ATGM1150, ATGM1151, and ATGM1152 were the experimental groups. Among them, ATGM1148, ATGM1150, and ATGM1152 showed stronger binding affinity to human DSG3 protein, while ATGM1149 and ATGM1151 showed relatively weaker binding affinity.

[0317] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-Dsg3 monoclonal antibody, characterized in that, The heavy chain variable region CDR1 of the antibody is shown as SEQ ID NO: 21, CDR2 is shown as SEQ ID NO: 22, and CDR3 is shown as SEQ ID NO: 23; the light chain variable region CDR1 of the antibody is shown as SEQ ID NO: 26, the amino acid sequence of CDR2 is AAS, and CDR3 is shown as SEQ ID NO:

28.

2. The anti-Dsg3 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 24; and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

29.

3. The anti-Dsg3 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO: 25; and the amino acid sequence of the light chain is shown as SEQ ID NO:

30.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-Dsg3 monoclonal antibody of any one of claims 1-3.

5. An expression vector, characterized by, The expression vector contains the nucleic acid molecule of claim 4.

6. A host cell, characterized in that, The host cell contains at least one copy of the expression vector of claim 5; or the nucleic acid molecule of claim 4 is integrated into the chromosome of the host cell.

7. A composition for detecting Dsg3 protein in a sample, characterized in that, The composition comprises the anti-Dsg3 monoclonal antibody of any one of claims 1-3.

8. A kit for detecting Dsg3 protein in a sample, characterized by, The kit comprises the anti-Dsg3 monoclonal antibody of any one of claims 1-3.

9. Use of the anti-Dsg3 monoclonal antibody of any one of claims 1-3, the composition for detecting Dsg3 protein in a sample of claim 7, or the kit for detecting Dsg3 protein in a sample of claim 8 in the preparation of a product for detecting pemphigus vulgaris.

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