Monoclonal antibody binding to ds g3 and uses thereof

Abstract

The application provides a monoclonal antibody binding to Dsg3 and application thereof. The heavy chain CDR3 of the monoclonal antibody comprises an amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33 or SEQ ID NO: 43; and the light chain CDR3 of the monoclonal antibody comprises an amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 36 or SEQ ID NO: 46. The monoclonal antibody can specifically bind to Dsg3, has high affinity, can quantitatively detect the level of anti-Dsg3 autoantibody in a PV patient, monitor the activity of the disease, and can be used for clinical diagnosis of the PV patient.

Description

[0001] This application is a divisional application of patent application number 2023102434061 (the original application was filed on March 14, 2023, and the invention was entitled "A Monoclonal Antibody that Binds Dsg3 and Its Application"). Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody that binds to Dsg3 and its applications. Background Technology

[0003] Pemphigus is an autoimmune disease with four subtypes: pemphigus vulgaris, pemphigus vesicularis, pemphigus foliaceus, and pemphigus erythematosus. Pemphigus vulgaris (PV) is a bullous skin disease primarily affecting the skin and mucous membranes, and it is relatively common clinically. Patients have autoantibodies circulating in their serum targeting the intercellular protein structure of keratinocytes, disrupting intercellular adhesion and resulting in clinical manifestations primarily consisting of intraepidermal bullae. Approximately 60% of PV patients develop vesicles and bullae in the oral mucosa, which spontaneously rupture and are difficult to heal, severely impacting their eating and daily life, causing significant physical and psychological suffering. Furthermore, some patients only present with persistent oral mucosal erosions without any skin lesions. Therefore, a rapid and accurate diagnostic method for PV is needed.

[0004] Early diagnosis of PV is crucial for treatment and prognosis. PV diagnosis includes clinical manifestations, histopathology, and immunodiagnostic indicators. Clinical manifestations: ① flaccid vesicles and bullae appear on the skin, easily ruptured; ② Ruptured vesicles and bullae form persistent erosions; ③ Vesicles or erosions appear on mucous membranes; ④ Nikolsky's sign is positive. Histopathology: Spina dissection between epidermal or epithelial cells forms vesicles and bullae. Immunodiagnostic indicators: ① Direct immunofluorescence (DIF) of the lesion area or surrounding normal skin shows IgG and / or complement deposition between epidermal (or epithelial) cells; ② Indirect immunofluorescence (IIF) detects anti-epidermal intercellular antibodies in serum; ③ ELISA detects anti-Dsg antibodies in serum. A diagnosis is confirmed if at least one of the clinical manifestations is met, along with at least one of the histopathological and immunodiagnostic indicators. A diagnosis is also confirmed if at least two of the clinical manifestations are met, along with two of the immunodiagnostic indicators.

[0005] The diagnosis of PV requires high-affinity anti-Dsg3 monoclonal antibodies. The classic hybridoma technique requires a lot of time and effort to obtain high-affinity antibodies, and subsequent humanization modification is also required, which makes it difficult to obtain human monoclonal antibodies through hybridoma technology.

[0006] Phage display technology has been widely applied in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigen epitope research, and cell signal transduction research. Phage antibody library technology involves preparing a human antibody library by expressing Fab fragments or single-chain antibodies (ScFv) on the surface of bacteriophages, thereby screening and enriching specific antibodies. Almost all recombinant human monoclonal antibodies that specifically react with antigens can be screened from the single-pot antibody library system. Therefore, when using phage antibody technology, various antibody fragments (Fab or ScFv) applicable to in vivo diagnosis or treatment can be obtained. Phage antibody library technology does not require immunization or humanization steps; it relies on enrichment and screening in a completely controlled biochemical environment to achieve the rapid screening of high-affinity fully human antibody sequences.

[0007] The pathological features of pemphigus vulgaris include intraepidermal bullae and acantholysis. The direct cause of this pathological change is anti-acantholysis antibodies, which are present in the serum of almost all patients with active pemphigus. The target antigen of PV antibodies is located on desmosomes, the intercellular junction structures of epidermal cells, specifically the transmembrane desmoglein core protein (desmoglein 3, Dsg3). Dsg3 has a molecular weight of 64.95 kDa and belongs to the cadherin superfamily. It plays an important role in maintaining the integrity of the epidermis, and its structural disruption can lead to acantholysis and cause intraepidermal bullae. The intracellular components of Dsg3 interact with the cytoskeleton through plaque-like proteins and form junctions, while the extracellular region mediates intercellular junctions. The extracellular region of Dsg3 provides the antigenic epitopes recognized by PV antibodies. The region in Dsg3 that plays a major role is called the extracellular domain (EC). The Dsg3 EC is divided into five domains (EC1-EC5), with EC-1 located at the amino terminus and EC-5 located at the carboxyl terminus. The native stereoconformation of the Dsg3 molecule is important for eliciting autoimmune bullous lesions. Existing studies have obtained recombinant human Dsg3 protein, including the entire Dsg3 EC, through baculovirus expression systems and mammalian expression systems. The obtained recombinant human Dsg3 protein can specifically bind to the natural antibodies in the serum of human PV patients. The 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.

[0008] Therefore, screening for a human antibody that binds to a pemphigus vulgaris-specific antigenic peptide using phage antibody library technology has important application value in the diagnosis and / or treatment of pemphigus. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a monoclonal antibody that binds to Dsg3 and its applications. In this invention, a phage-derived antibody library is constructed using peripheral blood mononuclear cells (PBMCs) from Dsg3 antibody-positive patients. Monoclonal antibodies that specifically bind to the Dsg3 protein are screened for their ability to bind to the Dsg3 antigen. These monoclonal antibodies exhibit high affinity and specificity, enabling quantitative detection of anti-Dsg3 autoantibody levels in PV patients, monitoring disease activity, and facilitating 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 primary pathogenic mechanism.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a monoclonal antibody that binds to Dsg3, wherein the heavy chain CDR3 of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33 or SEQ ID NO:43;

[0012] The light chain CDR3 of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36 or SEQ ID NO:46.

[0013] This invention screens monoclonal antibodies that bind to Dsg3 with high specificity and strong affinity from a phage library. These monoclonal antibodies are fully human antibodies, which have lower immunogenicity and have great application potential in the development of antibody drugs and diagnostic drugs.

[0014] Preferably, the heavy chain CDR1 of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31 or SEQ ID NO:41.

[0015] Preferably, the heavy chain CDR2 of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32 or SEQ ID NO:42.

[0016] Preferably, the light chain CDR1 of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:34 or SEQ ID NO:44.

[0017] Preferably, the light chain CDR2 of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:35 or SEQ ID NO:45.

[0018] Preferably, the heavy chain variable region of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37 or SEQ ID NO:47.

[0019] Preferably, the light chain variable region of the monoclonal antibody includes the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38 or SEQ ID NO:48.

[0020] Preferably, the heavy chain of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:29, SEQ ID NO:39 or SEQ ID NO:49.

[0021] Preferably, the light chain of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:40 or SEQ ID NO:50.

[0022] This invention isolated PBMCs from Dsg3 antibody-positive patients, extracted RNA, and performed quality control on the RNA. RT-PCR was used to reverse transcribe the quality-controlled RNA into cDNA, amplifying all VH and VL antibody gene fragments. The amplified Vκ and Vλ gene fragments were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library. This antibody gene combinatorial library was inserted immediately downstream of the leader sequence of phage-encoded membrane protein gene III (g3) or gene VIII (g8). Through helper phage superinfection, the polypeptide expressed by the exogenous antibody gene could be displayed as a fusion protein at the N-terminus of the phage coat protein pIII or pVIII. Each phage particle encodes and presents a different antibody, containing billions of individual clones. In these antibody libraries, the genes encoding antibodies that can bind to antigens are selected through an in vitro affinity enrichment-mild elution-phage amplification process. This enrichment and screening process is repeated several times until a phage library with high specificity and affinity is obtained. Positive clones are then screened from this library. Positive clones are identified using ELISA, and finally, fully human antibodies with high specificity and affinity are selected.

[0023] After screening the antibody phage library, five high-affinity monoclonal antibodies were finally obtained, named 76F-DSG3-IC-R2P1-G1, 76F-DSG3-IC-R2P1-F2, 76F-DSG3-IC-R2P1-E6, 76F-DSG3-IC-R2P1-C9, and 76F-DSG3-IC-R2P1-H9. These monoclonal antibodies exhibit high activity, good stability, and strong specificity, making them suitable as a reference standard for qualitative detection of PV positivity and also for quantitative detection of anti-Dsg3 autoantibody levels in PV patients.

[0024] Monoclonal antibody 76F-DSG3-IC-R2P1-G1:

[0025] SEQ ID NO:1 (heavy chain CDR1): GFTLANST.

[0026] SEQ ID NO:2 (heavy chain CDR2): SVVGNDKT.

[0027] SEQ ID NO:3 (heavy chain CDR3): AASSHFWSGSLDI.

[0028] SEQ ID NO:4 (light chain CDR1): QSVLYSSNNKNY.

[0029] SEQ ID NO:5 (light chain CDR2): WAS.

[0030] SEQ ID NO: 6 (Light chain CDR3): QQYYSTPLT.

[0031] SEQ ID NO: 7 (Heavy chain variable region):

[0032] EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSS.

[0033] SEQ ID NO: 8 (Light chain variable region):

[0034] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKLEIK.

[0035] SEQ ID NO: 9 (Amino acid sequence of the heavy chain of 76F-DSG3-IC-R2P1-G1 antibody):

[0036] MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0037] SEQ ID NO:10 (Light chain amino acid sequence of 76F-DSG3-IC-R2P1-G1 antibody):

[0038] MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST PLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0039] Monoclonal antibody 76F-DSG3-IC-R2P1-F2:

[0040] SEQ ID NO:11 (heavy chain CDR1): GFTLANST.

[0041] SEQ ID NO:12 (heavy chain CDR2): SVVGNDKT.

[0042] SEQ ID NO:13 (heavy chain CDR3): AASSHFWSGSLDI.

[0043] SEQ ID NO:14 (light chain CDR1): QSVLYSSNNKNY.

[0044] SEQ ID NO:15 (light chain CDR2): WAS.

[0045] SEQ ID NO:16 (light chain CDR3): QQYYSTPIT.

[0046] SEQ ID NO:17 (Heavy chain variable region):

[0047] EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS.

[0048] SEQ ID NO:18 (Light chain variable region):

[0049] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGQGTRLEIK.

[0050] SEQ ID NO:19 (Amino acid sequence of the heavy chain of 76F-DSG3-IC-R2P1-F2 antibody):

[0051] MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0052] SEQ ID NO:20 (Amino acid sequence of the light chain of 76F-DSG3-IC-R2P1-F2 antibody):

[0053] MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST PITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0054] Monoclonal antibody 76F-DSG3-IC-R2P1-E6:

[0055] SEQ ID NO:21 (heavy chain CDR1): GFTLANST.

[0056] SEQ ID NO:22 (heavy chain CDR2): SVVGNDKT.

[0057] SEQ ID NO:23 (heavy chain CDR3): AASSHFWSGSLDV.

[0058] SEQ ID NO:24 (light chain CDR1): QSVLYNSNNKNY.

[0059] SEQ ID NO:25 (light chain CDR2): WAS.

[0060] SEQ ID NO:26 (light chain CDR3): HQYFGTPYT.

[0061] SEQ ID NO:27 (Heavy chain variable region):

[0062] QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGN DKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLV TVSS.

[0063] SEQ ID NO:28 (Light chain variable region):

[0064] EIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGTPYTFGQGTKVEIK.

[0065] SEQ ID NO:29 (Amino acid sequence of the heavy chain of 76F-DSG3-IC-R2P1-E6 antibody):

[0066] MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGNDKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0067] SEQ ID NO:30 (Amino acid sequence of the light chain of 76F-DSG3-IC-R2P1-E6 antibody):

[0068] MVLQTQVFISLLLWISGAYGEIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGT PYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0069] Monoclonal antibody 76F-DSG3-IC-R2P1-C9:

[0070] SEQ ID NO:31 (heavy chain CDR1): GGSFSGYY.

[0071] SEQ ID NO:32 (heavy chain CDR2): INHSGST.

[0072] SEQ ID NO:33 (heavy chain CDR3): ARGRYLATVRHYYYYYMDV.

[0073] SEQ ID NO:34 (light chain CDR1): QSLENSAGNTY.

[0074] SEQ ID NO:35 (light chain CDR2): KVS.

[0075] SEQ ID NO:36 (light chain CDR3): MQGSHWPPYT.

[0076] SEQ ID NO:37 (Heavy chain variable region):

[0077] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTN YNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKG TLVTVSS.

[0078] SEQ ID NO:38 (Light chain variable region):

[0079] EIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWPPYTFGQGTKVEIK.

[0080] SEQ ID NO:39 (Amino acid sequence of the heavy chain of 76F-DSG3-IC-R2P1-C9 antibody):

[0081] MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0082] SEQ ID NO:40 (Amino acid sequence of the light chain of 76F-DSG3-IC-R2P1-C9 antibody):

[0083] MVLQTQVFISLLLWISGAYGEIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWP PYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0084] Monoclonal antibody 76F-DSG3-IC-R2P1-H9:

[0085] SEQ ID NO:41 (heavy chain CDR1): GFTLANST.

[0086] SEQ ID NO:42 (heavy chain CDR2): SVVGNDKT.

[0087] SEQ ID NO:43 (heavy chain CDR3): AASSHFWSGSLDI.

[0088] SEQ ID NO:44 (light chain CDR1): QSVLYSSNNKNY.

[0089] SEQ ID NO:45 (light chain CDR2): WAS.

[0090] SEQ ID NO:46 (light chain CDR3): QQYYSTPFT.

[0091] SEQ ID NO:47 (Heavy chain variable region):

[0092] QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS.

[0093] SEQ ID NO:48 (Light chain variable region):

[0094] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK.

[0095] SEQ ID NO:49 (Amino acid sequence of the heavy chain of 76F-DSG3-IC-R2P1-H9 antibody):

[0096] MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0097] SEQ ID NO:50 (Amino acid sequence of the light chain of 76F-DSG3-IC-R2P1-H9 antibody):

[0098] MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST PFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0099] In a second aspect, the present invention provides a nucleic acid molecule that encodes the monoclonal antibody that binds Dsg3 as described in the first aspect.

[0100] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0101] Fourthly, the present invention provides a host cell containing at least one copy of the expression vector described in the third aspect.

[0102] Fifthly, the present invention provides a pharmaceutical composition comprising the monoclonal antibody binding Dsg3 as described in the first aspect, and a pharmaceutically acceptable carrier and / or diluent.

[0103] In a sixth aspect, the present invention provides a kit for detecting Dsg3 protein in a sample, the kit comprising the monoclonal antibody that binds Dsg3 as described in the first aspect.

[0104] In a seventh aspect, the present invention provides the use of any one or a combination of at least two of the monoclonal antibody binding Dsg3 described in the first aspect, the host cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament for the diagnosis and / or treatment of pemphigus.

[0105] Compared with the prior art, the present invention has the following beneficial effects:

[0106] Through screening of an antibody phage library, this invention obtained five high-affinity monoclonal antibodies that bind to Dsg3. These monoclonal antibodies have high activity, good stability, and strong specificity, and can be used as a reference standard for qualitative detection of PV positivity, as well as for quantitative detection of anti-Dsg3 autoantibody levels in PV patients. Attached Figure Description

[0107] Figure 1 This is a PCR agarose gel electrophoresis image of a single clone of a VL phage library;

[0108] Figure 2 This is a PCR agarose gel electrophoresis image of a single clone of a κH phage library;

[0109] Figure 3 This is a PCR agarose gel electrophoresis image of a single clone of the λH phage library;

[0110] Figure 4A These are the sequencing quality control results for the light chain library;

[0111] Figure 4B This is the sequencing quality control result of the heavy chain library;

[0112] Figure 5 This is an SDS-PAGE electrophoresis image of purified Dsg3;

[0113] Figure 6A The results are from an ELISA test of 76F-DSG3-IC-R2P1-G1 antibody and Dsg3.

[0114] Figure 6B The results are from an ELISA test of 76F-DSG3-IC-R2P1-F2 antibody and Dsg3.

[0115] Figure 6C The results are from an ELISA test of 76F-DSG3-IC-R2P1-E6 antibody and Dsg3.

[0116] Figure 6D The results are from an ELISA test of 76F-DSG3-IC-R2P1-C9 antibody and Dsg3.

[0117] Figure 6E The results are from an ELISA test of 76F-DSG3-IC-R2P1-H9 antibody and Dsg3. Detailed Implementation

[0118] 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.

[0119] 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. Detailed description of the invention:

[0121] Monoclonal antibodies constitute the largest and fastest-growing group of therapeutic proteins, with over 500 therapeutic antibodies and their derivatives currently undergoing clinical trials, primarily focusing on cancer treatment and autoimmune diseases. This invention isolated PBMCs from Dsg3 antibody-positive patients, extracted RNA, and performed quality control on the RNA. RT-PCR was used to reverse transcribe the quality-controlled RNA into cDNA, amplifying all VH and VL antibody gene fragments. The amplified Vκ and Vλ gene fragments were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library.

[0122] Antibody gene conjugate libraries are inserted immediately downstream of the leader sequence of gene III (g3) or gene VIII (g8) encoding membrane proteins of phages. Through helper phage superinfection, the polypeptides expressed by the exogenous antibody genes can be displayed as fusion proteins at the N-terminus of the phage coat protein pIII or pVIII. Each phage particle encodes and presents a different antibody, containing billions of individual clones. In these antibody libraries, genes encoding antibodies that bind to antigens are selected through affinity enrichment-mild elution-phage amplification in vitro. This enrichment and screening process is repeated several times to obtain a phage library with high specificity and affinity. Positive clones are then screened from this library. Positive clones are identified using ELISA, and finally, fully human antibodies with high specificity and affinity are selected.

[0123] This invention uses The III 1st Strand cDNA Synthesis Kit (+gDNAwiper) reverse transcription kit reverse transcribed RNA from Dsg3 antibody-positive patients into cDNA, amplifying the VH and VL fragments of the DNA. The Vκ and Vλ gene fragments obtained in vitro were cloned into the pATA-scFv-2 vector using cloning technology to form Vκ and Vλ libraries. Plasmid vectors for the Vκ and Vλ libraries were extracted using a plasmid extraction kit, and the in vitro amplified VH gene fragment was inserted into the plasmid vectors of the Vκ and Vλ libraries to construct κH and λH libraries, respectively.

[0124] The artificially synthesized Dsg3 domain gene was recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector was transfected into DH10Bac competent cells and cultured, and the Dsg3 antigen protein was purified.

[0125] After three rounds of screening of antibody phage libraries, clones with an antigen group greater than 3 times that of the control group were defined as positive clones, and these monoclonal antibodies were sequenced and analyzed. Erroneous and duplicate antibody sequences were excluded, and combined with the antigen-antibody specific binding ability reflected in the ELISA experiment, five high-affinity antibodies were finally obtained, named 76F-DSG3-IC-R2P1-G1, 76F-DSG3-IC-R2P1-F2, 76F-DSG3-IC-R2P1-E6, 76F-DSG3-IC-R2P1-C9, and 76F-DSG3-IC-R2P1-H9. These monoclonal antibodies exhibit high activity, good stability, and strong specificity, and can serve as a reference standard for qualitative detection of PV positivity, as well as for quantitative detection of anti-Dsg3 autoantibody levels in PV patients.

[0126] Example 1: Method for constructing a human ScFv phage display library

[0127] In this embodiment, PBMCs from Dsg3 antibody-positive patients were isolated, RNA was extracted, and the RNA underwent quality control. The quality-controlled RNA was reverse transcribed into cDNA using RT-PCR, and all VH and VL antibody gene fragments were amplified. The amplified Vκ and Vλ gene fragments were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library.

[0128] The main reagents used in this embodiment are shown in Table 1:

[0129] Table 1

[0130]

[0131]

[0132] 1. Library Construction

[0133] 1.1 Assemble the heavy chain variable region (VH) and light chain variable region (VL). The experimental steps are as follows: PCR reaction conditions and steps are shown in Table 2.

[0134] Table 2

[0135]

[0136] The denaturation, annealing, and extension (1) steps were repeated 30 times; the primer sequences are shown below:

[0137] Upstream primer (F):

[0138] 5'L-VH 1 (SEQ ID NO:51): acaggtgcccactcccaggtgcag.

[0139] 5'L-VH 3 (SEQ ID NO: 52): aaggtgtccagtgtgargtgcag.

[0140] 5'L-VH 4 / 6 (SEQ ID NO: 53): cccagatgggtcctgtcccaggtgcag.

[0141] 5'L-VH 5 / 7 (SEQ ID NO: 54): caaggagtctgttccgaggtgcag.

[0142] 5'L VK 1 / 2 (SEQ ID NO: 55): atgaggstcccygctcagctgctgg.

[0143] 5'L VK 3 (SEQ ID NO: 56): ctcttcctcctgctactctggctcccag.

[0144] 5'L VK 4 / 5 (SEQ ID NO: 57): atttctctgttgctctggatctctg.

[0145] 5'L Vλ1 (SEQ ID NO: 58): ggtcctgggcccagtctgtgctg.

[0146] 5'L Vλ2 (SEQ ID NO: 59): ggtcctgggcccagtctgccctg.

[0147] 5'L Vλ3 (SEQ ID NO: 60): gctctgtgacctcctatgagctg.

[0148] 5'L Vλ4 / 5 (SEQ ID NO: 61): ggtctctctcscagcytgtgctg.

[0149] 5'L Vλ6 (SEQ ID NO: 62): gttcttgggccaattttatgctg.

[0150] 5'L Vλ7 (SEQ ID NO: 63): ggtccaattcycaggctgtggtg.

[0151] 5'L Vλ8 / 9 / 10 (SEQ ID NO: 64): gagtggattctcagactgtggtg.

[0152] Downstream primer (R):

[0153] 3'CK (SEQ ID NO:65): tgctgtccttgctgtcctgct.

[0154] 3'Cλ (SEQ ID NO:66): caccagtgtggccttgttggcttg.

[0155] 1.2 Construction of a light chain variable region phage display library

[0156] 1.2.1 Prepare the pATA-scFv-2 vector for library cloning.

[0157] 1.2.2 Digestion of vector and PCR product. The reaction system for digesting vector and PCR product is shown in Table 3.

[0158] Table 3

[0159]

[0160]

[0161] 1.2.3 The digested vector and PCR product were ligated. The ligation reaction system is shown in Table 4. The prepared ligation reaction system was incubated at 16℃ overnight and the reaction was terminated by heating at 65℃ for 10 min to inactivate the PCR product.

[0162] Table 4

[0163]

[0164] 1.2.4 The ligation product was electroporated into competent cells. The electroporation steps are as follows:

[0165] 1.2.4.1 Preparation of TG1 competent cells.

[0166] 1.2.4.2 Preheat 1 mL of SOC medium (Sigma, S1797) to 37℃; place electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).

[0167] 1.2.4.3 Remove electrocompetent cells from the -80°C freezer and place them on ice until they are completely thawed (10-15 min); after the cells are thawed, mix them gently; place 50 μL of cells into a frozen microcentrifuge tube on ice.

[0168] 1.2.4.4 Carefully add 3 μL of the DNA mixture to a frozen electroporation cuvette, being careful not to create air bubbles; quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom.

[0169] 1.2.4.5 Electroporation was performed at 600Ω, 10μF and 1.8kV; within 10s of the pulse, 1mL of preheated SOC medium was added to each test tube immediately; shaken at 37℃ and 250rpm for 1h.

[0170] 1.2.4.6 Collect all electroporation media; serially dilute 10 μL of culture into 90 μL of SOC medium and spread it on LB / Amp / Glucose medium. 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.

[0171] 1.3 Construction of VL-VH phage display library

[0172] 1.3.1 Digestion of vector and PCR product, the digestion reaction system is shown in Table 5.

[0173] Table 5

[0174]

[0175]

[0176] 1.3.2 The ligation reaction system is shown in Table 6. The prepared ligation reaction system was incubated at 16℃ overnight, and the reaction was terminated by heating at 65℃ for 10 min to inactivate it.

[0177] Table 6

[0178]

[0179] 1.3.3 The ligation product was electroporated into competent cells. The electroporation steps are as follows:

[0180] 1.3.3.1 Preparation of TG1 competent cells.

[0181] 1.3.3.2 Preheat 4 mL of SOC medium (Sigma, S1797) to 37 °C. Place the electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).

[0182] 1.3.3.3 Remove the electrocompetent cells from the -80°C freezer and place them on ice until they are completely thawed (10-15 min). After thawing, gently mix the cells.

[0183] 1.3.3.4 Carefully add 6 μL of the DNA mixture to a frozen electroporation cuvette, being careful not to create air bubbles. Quickly flick the tube downwards with your wrist to allow the cells to settle at the bottom.

[0184] 1.3.3.5 Electroporation at 600 Ω, 10 μF, and 2.5 kV. Immediately within 10 seconds of the pulse, add 2 mL of preheated SOC medium to each tube. Incubate at 37°C with shaking at 250 rpm for 1 h.

[0185] 1.3.3.6 Collect all electroporation media. Serially dilute 10 μL of culture into 90 μL of SOC medium and spread it onto LB / Amp / Glucose medium. 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.

[0186] 1.4 Library Evaluation

[0187] 1.4.1 Colony PCR: PCR was performed using the constructed library as a template; the PCR reaction conditions are shown in Table 7.

[0188] Table 7

[0189]

[0190] The denaturation, annealing, and extension (1) steps were repeated 30 times. The primer sequences for the PCR reaction are shown below:

[0191] Upstream primer (F) (SEQ ID NO:67): agcggataacaatttcacacagga.

[0192] Downstream primer (R) (SEQ ID NO:68): gcccccttattagcgtttgccatc.

[0193] The results of agarose gel electrophoresis after PCR are as follows: Figures 1-3 As shown. Agarose gel electrophoresis image of a single clone of the VL phage library for PCR is shown below. Figure 1 As shown, Figure 1 In the image, lane M: DL2000; lanes 1-16: 21000076F pATA-Vκ; lanes 17-32: 21000076F pATA-Vλ. The PCR agarose gel electrophoresis image of a single clone of the κH phage library is shown below. Figure 2 As shown, Figure 2 In the image, lane M: DL2000, lanes 1-48: 21000076F pATA-scFv-κH. The PCR agarose gel electrophoresis image of a single clone of the λH phage library is shown below. Figure 3 As shown, Figure 3 In the middle, lane M: DL2000, lanes 1-48: 21000076FpATA-scFv-λH.

[0194] 1.4.2 Sequencing

[0195] Phage display libraries were sequenced, and positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing quality control results of the phage display libraries are as follows: Figure 4A and Figure 4B As shown. Figure 4A The sequencing quality control results for the light chain library Figure 4B These are the sequencing quality control results for the heavy chain library. From Figure 4A and Figure 4B As can be seen from the data, the library sequence covers most of the strains of the heavy and light chain variable regions of human antibodies, and the library diversity meets the expected requirements.

[0196] 1.5 Expression of Dsg3 protein

[0197] The artificially synthesized Dsg3 domain gene was recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector was transfected into DH10Bac competent cells and cultured, and the Dsg3 antigen protein was purified. The artificially synthesized Dsg3 gene sequence was recombined into the expression vector plasmid pFastBac1 to obtain the Dsg3-pFastBac1 expression vector; the cloning site is BamHⅠ / XhoⅠ, and the amino acid sequence of Dsg3 is shown in SEQ ID NO:69.

[0198] .

[0199] 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 further subjected to SDS-PAGE (polyacrylamide gel electrophoresis) to verify its purity. The SDS-PAGE electrophoresis image of the purified Dsg3 is shown below. Figure 5 As shown, the protein size is 64.95 kDa and the purity is greater than 90%.

[0200] Example 2: Screening for monoclonal antibodies that specifically bind to Dsg3

[0201] The desired specific antibody clones were enriched from the phage antibody library using a suitable panning strategy. The main reagents used in this example are shown in Table 8.

[0202] Table 8

[0203] 96-well plate 42592 Costar Tween 20 P2287 Sigma Tris (tris(hydroxymethyl)aminomethane) RES3098T-B7 Sigma Glycine G8200 Solarbio PEG (Polyethylene Glycol) 181986 Sigma PBS (phosphate buffered saline) C10010500BT Life Sodium casein salt S12003-100g shyuanye Skim milk 6342932 BD HRP-labeled M13 antibody MVV05401H AntibodySystem

[0204] 1. First round of selection

[0205] 1.1 Biological Screening

[0206] 1.1.1 Coating: Coat centrifuge tubes and incubate overnight at 4°C. Antigen group: 1 mL Dsg3 transfection buffer (50 μg / mL), control group: 500 μL transfection buffer (0 μg / mL).

[0207] 1.1.2 Washing: Discard the liquid in the centrifuge tube and wash three times with 5 mL of 0.05% PBST.

[0208] 1.1.3 Blocking: Add 5 mL of 5% skim milk or 1% casein (dissolved in PBST) to the tube and incubate at 37°C for 2 h.

[0209] 1.1.4 Washing: Discard the liquid in the centrifuge tube and wash once with 5 mL of 0.05% PBST.

[0210] 1.1.5 Incubation: Dilute the phage library with 1% skim milk or 0.2% casein (dissolved in PBST), add 1 mL to a centrifuge tube, and incubate at 32°C for 2 h.

[0211] 1.1.6 Washing: Discard the liquid in the centrifuge tube, wash three times with 5 mL of 0.05% PBST, and wash twice with PBS.

[0212] 1.1.7 Elution: Elute the phages bound to Dsg3 with 1 mL of glycine-hydrochloric acid (pH 2.2), and then neutralize to pH 7.0 with Tris-HCl.

[0213] 1.2 Determination of the titer of diluted bacteriophages

[0214] 1.2.1 Coat centrifuge tubes with 1 mL of LDsg3 transfection buffer (50 μg / mL) and incubate overnight at 4°C.

[0215] 1.2.2 Discard the liquid in the centrifuge tube and wash three times with 5 mL of 0.05% PBST.

[0216] 1.2.3 Add 5 mL of 5% skim milk (dissolved in PBST) to the tube and incubate at 37°C for 2 h.

[0217] 1.2.4 Discard the liquid in the centrifuge tube and wash once with 5 mL of 0.05% PBST.

[0218] 1.2.5 Transfer the phage from 1.1.7 to a centrifuge tube and incubate at 32°C for 2 hours.

[0219] 1.2.6 Culture Escherichia coli TG1 until OD 600 =0.4-0.6.

[0220] 1.2.7 Mix 10 μL of diluted eluted phage with 180 μL of Escherichia coli TG1.

[0221] 1.2.8 Incubate the mixture at 37℃ for 30 min, then pour it into 2×YT-A (Amp 100μg / mL) medium and incubate the medium upside down at 37℃ overnight.

[0222] 1.3 Phage Library Amplification

[0223] 1.3.1 Add 10 μL of LE.coli TG1 to 800 μL of 2YT culture medium and incubate at 37℃ until OD. 600 =0.4-0.6.

[0224] 1.3.2 Transfer TG1 cultured to the logarithmic growth phase into 10 mL of 2YT-G medium (final concentration 2% glucose), and incubate on a shaker at 37°C until OD500. 600 =0.4-0.6.

[0225] 1.3.3 Add the eluted product, incubate at 37°C for 30 min, and then culture on a shaker at 37°C for 30 min.

[0226] 1.3.4 Add 30 mL of 2YT-AG culture medium (final concentration 0.1% Amp, 2% glucose) and incubate at 37°C in a shaker for 1 h.

[0227] 1.3.5 Add M13KO7 (M13KO7:TG1 = 20:1), incubate at 37℃ for 30 min, and then culture in a shaker at 37℃ for 30 min.

[0228] 1.3.6 The bacterial culture was centrifuged at 5000 rpm for 5 min, resuspended in 40 mL of 2YT-AK (final concentrations of Amp 100 μg / mL and Kan 100 μg / mL), and incubated overnight in a shaker at 30 °C.

[0229] 1.3.7 Centrifuge at 8000 rpm for 10 min and collect the supernatant. Add 1 / 5 (v / v) PEG / NaCl to the supernatant, mix, and incubate on ice for 2 h.

[0230] 1.3.8 Resuspend in 1 mL PBS, centrifuge at 12000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0231] 1.4 Determination of the titer of the amplified phage library, following the same procedure as in 1.2.

[0232] 2. Second and third rounds of selection

[0233] 2.1 Biological Screening

[0234] Step 1 was repeated twice, with each phage library being eluted from the previous round of amplification. The results of the biopanning are shown in Table 9.

[0235] Table 9

[0236]

[0237] As shown in Table 9, after three rounds of screening, the ratio of phages obtained from the antigen group to the input phages increased in each round, indicating that the phages had been enriched to a certain extent.

[0238] 3. Polyclonal phage ELISA

[0239] 3.1 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL / well of Dsg3 protein (4 μg / mL), control group: 100 μL / well of protein dilution buffer (0 μg / mL).

[0240] 3.2 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0241] 3.3 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 h.

[0242] 3.4 Phage incubation: Add 100 μL of diluted phage to each well as shown in Table 10, and incubate at 32℃ for 2 h.

[0243] 3.5 Washing: Same as step 3.2.

[0244] 3.6 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (HRP-labeled M13 antibody) diluted with blocking buffer (1:6000) to each well and incubate at 32°C for 1 h.

[0245] 3.7 Washing: Same as step 3.2.

[0246] 3.8 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 100 μL 2M HCl to each well to terminate the reaction.

[0247] 3.9 Plate reading: Read values ​​at 450-630 nm using an ELISA reader. The results of polyclonal phage ELISA are shown in Table 10.

[0248] Table 10

[0249]

[0250] As shown in Table 10, the ELISA results of the antigen group increased with each round, indicating that the positive phages were enriched in each round. The difference between the antigen group and the control group was relatively large in the second round. Therefore, the elution products of the second group were selected for monoclonal phage ELISA screening.

[0251] 4. Monoclonal phage ELISA (select the second round of elution products for monoclonal ELISA based on polyclonal results)

[0252] 4.1 Ninety-six clones were selected from the culture dish for elution titer determination; these clones were incubated at 37°C and 250 rpm until OD... 600nm =0.4-0.6.

[0253] 4.2 M13KO7 infection culture (MOI = 20:1) was incubated at 37°C for 30 min and then cultured on a shaker at 37°C for 30 min. The bacterial culture was centrifuged and the precipitate was resuspended with an equal volume of 2×YT-AK (final concentrations Amp 100 μg / mL and Kan 100 μg / mL) and cultured overnight at 30°C.

[0254] 4.3 Centrifuge the culture and use the supernatant for ELISA.

[0255] 4.4 Coating: Coat the microplate and incubate overnight at 4°C; Antigen group: 100 μL / well of Dsg3 protein (4 μg / mL), control group: 100 μL / well of protein dilution buffer (0 μg / mL).

[0256] 4.5 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0257] 4.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 h.

[0258] 4.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 h.

[0259] 4.8 Washing: Same as step 4.5.

[0260] 4.9 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (HRP-labeled M13 antibody) diluted with blocking buffer (1:6000) to each well and incubate at 32°C for 1 h.

[0261] 4.10 Washing: Same as step 4.5.

[0262] 4.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 100 μL 2M HCl to each well to terminate the reaction.

[0263] 4.12 Plate reading: Values ​​were read using an ELISA reader at 450-630 nm, and highly specific clones were sequenced. The results of the antigen group monoclonal phage ELISA are shown in Table 11.

[0264] Table 11

[0265] A 0.02 0.05 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 B 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.02 C 0.02 0.02 0.02 0.02 0.02 0.04 0.02 0.02 0.14 0.02 0.02 0.02 D 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 E 0.02 0.02 0.02 0.02 0.02 0.44 0.02 0.02 0.51 0.02 0.02 0.03 F 0.02 0.27 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 G 0.40 0.04 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 H 0.02 0.02 0.22 0.02 0.03 0.02 0.02 0.02 0.29 0.02 0.02 0.02

[0266] The results of the control group monoclonal phage ELISA are shown in Table 12.

[0267] Table 12

[0268] A 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 B 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 C 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.03 D 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 E 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 F 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 G 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.04 0.03 H 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02

[0269] The results in Tables 11 and 12 show that the antigen group of clones G1, F2, E6, C9, and F9 differed from the control group by more than 0.1. Therefore, these five clones were temporarily identified as positive clones and were subjected to secondary verification.

[0270] 5. ELISA verification of positive clones

[0271] 5.1 Add 50 μL of positive clones to 2 mL of 2YT-AG medium (final concentration 0.1% Amp, 2% glucose) and incubate until OD600. 600 =0.4-0.6.

[0272] 5.2 M13KO7 infection culture (MOI = 20:1) was incubated at 37°C for 30 min, followed by shaking culture at 37°C for 30 min. The bacterial culture was centrifuged and the pellet was resuspended with an equal volume of 2×YT-AK (final concentrations of Amp 100 μg / mL and Kan 100 μg / mL), and cultured overnight at 30°C.

[0273] 5.3 Centrifuge the culture and use the supernatant for ELISA.

[0274] 5.4 Coating: Coat the microplate and incubate overnight at 4°C. Antigen group: 100 μL / well of Dsg3 protein (4 μg / mL), control group: 100 μL / well of protein dilution buffer (0 μg / mL).

[0275] 5.5 Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0276] 5.6 Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 h.

[0277] 5.7 Phage incubation: Add 100 μL of phage supernatant to each well and incubate at 32°C for 2 h.

[0278] 5.8 Washing: Same as step 4.5.

[0279] 5.9 Secondary antibody incubation: Add 100 μL of anti-M13-HRP antibody (HRP-labeled M13 antibody) diluted with blocking buffer (1:6000) to each well and incubate at 32°C for 1 h.

[0280] 5.10 Washing: Same as step 4.5.

[0281] 5.11 Color development: Add 100 μL TMB to each well, incubate at room temperature, and then add 100 μL 2M HCl to each well to terminate the reaction.

[0282] 5.12 Plate reading: Values ​​were read using an ELISA reader at 450-630 nm, and highly specific clones were sequenced. The results of the positive monoclonal phage ELISA are shown in Table 13.

[0283] Table 13

[0284]

[0285] As shown in Table 13, the secondary validation results for all five positive clones were positive. The positive clones were then sent for sequencing, yielding the sequences of highly specific antibodies.

[0286] Example 3: Affinity determination of highly specific antibodies

[0287] This embodiment measures the affinity of the antibodies screened in Example 2 by using enzyme-linked immunosorbent assay (ELISA) to detect OD values ​​under different dilution conditions. The ELISA experimental steps are as follows:

[0288] 1. Coating: Coat the microplate with 100 μL of Dsg3 protein (4 μg / mL) per well and incubate overnight at 4°C.

[0289] 2. Washing: Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST.

[0290] 3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 h.

[0291] 4. Incubation of positive antibodies: 76F-DSG3-IC-R2P1-G1, 76F-DSG3-IC-R2P1-F2, 76F-DSG3-IC-R2P1-E6, 76F-DSG3-IC-R2P1-C9, and 76F-DSG3-IC-R2P1-H9 antibodies were serially diluted to 4000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.6 ng / mL, 7.8 ng / mL, 3.9 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, and 0.25 ng / mL. 100 μL of the diluted antibody solution was added to each well, and the mixture was incubated at 37°C for 1 h.

[0292] 5. Washing: Same as step 4.5.

[0293] 6. Secondary antibody incubation: Dilute Goat Anti-Human IgG (H+L) antibody (Jackson, code: 109-035-088) 10000 times with blocking buffer, add 100 μL of diluted secondary antibody to each well, and incubate at 37°C for 30 min.

[0294] 7. Washing: Same as step 4.5.

[0295] 8. Color development: Add 100 μL TMB to each well, incubate at 37°C for 10 min, and then add 50 μL 2M HCl to each well to terminate the reaction.

[0296] 9. Reading the plate: Use an ELISA reader to read the values ​​at 450-630nm.

[0297] The results of enzyme-linked immunosorbent assay (ELISA) are as follows: Figures 6A-6E As shown. Figure 6A The results are from an ELISA assay of 76F-DSG3-IC-R2P1-G1 antibody and Dsg3. Figure 6B The results are from an ELISA assay of 76F-DSG3-IC-R2P1-F2 antibody and Dsg3. Figure 6C The results are from an ELISA assay of 76F-DSG3-IC-R2P1-E6 antibody and Dsg3. Figure 6D The results are from an ELISA assay of 76F-DSG3-IC-R2P1-C9 antibody and Dsg3. Figure 6EThe results show the ELISA detection of 76F-DSG3-IC-R2P1-H9 antibody and Dsg3. As shown in 6A-6E, the five screened human anti-Dsg3 monoclonal antibodies all exhibit strong specific binding ability to the Dsg3 protein. These antibodies can serve as a reference standard for qualitative detection of PV positivity and can also be used for quantitative detection of anti-Dsg3 autoantibody levels in PV patients.

[0298] In summary, this invention constructs a phage-based human antibody library using PBMCs from Dsg3 antibody-positive patients. By specifically binding to Dsg3, monoclonal antibodies that specifically bind to the Dsg3 antigen are screened. These monoclonal antibodies exhibit high activity, good stability, and strong specificity, serving as a reference standard for qualitative detection of PV positivity and also quantitatively detecting anti-Dsg3 autoantibody levels in PV patients, thereby monitoring disease activity and applicable 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 primary pathogenic mechanism.

[0299] The applicant declares that 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. A monoclonal antibody that binds to Dsg3, characterized in that, The monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region and the light chain includes a light chain variable region; The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is GGSFSGYY; The amino acid sequence of heavy chain CDR2 is INHSGST; The amino acid sequence of the heavy chain CDR3 is ARGRYLATVRHYYYYYMDV; The amino acid sequence of the light chain CDR1 of the monoclonal antibody is QSLENSAGNTY. The amino acid sequence of the light chain CDR2 is KVS; The amino acid sequence of the light chain CDR3 is MQGSHWPPYT.

2. The monoclonal antibody binding to Dsg3 according to claim 1, characterized in that, The heavy chain variable region of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:37; the light chain variable region of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:

38.

3. The monoclonal antibody binding to Dsg3 according to claim 1, characterized in that, The heavy chain of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:39; the light chain of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:

40.

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

5. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains at least one copy of the expression vector of claim 5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal antibody binding Dsg3 as described in any one of claims 1-3, and a pharmaceutically acceptable carrier and / or diluent.

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

9. Use of any one or a combination of at least two of the monoclonal antibody binding Dsg3 according to any one of claims 1-3, the host cell according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for the diagnosis and / or treatment of pemphigus.

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