CN116496395B - Monoclonal antibody combined with Dsg3 and application thereof - Google Patents
Monoclonal antibody combined with Dsg3 and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a monoclonal antibody combined with Dsg3 and application thereof. The heavy chain CDR3 of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 3, SEQ ID NO. 13, SEQ ID NO. 23, SEQ ID NO. 33 or SEQ ID NO. 43; the light chain CDR3 of the monoclonal antibody comprises an amino acid sequence shown as 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 be specifically combined with Dsg3, has high affinity, can quantitatively detect the level of the anti-Dsg 3 autoantibody in a PV patient, monitors the disease activity, and can be used for clinical diagnosis of the PV patient.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a monoclonal antibody combined with Dsg3 and application thereof.
Background
Pemphigus is an autoimmune disease with four subtypes, the vulgaris, the proliferative, the deciduous and the erythroid. Pemphigus vulgaris (pemphigus vulgaris, PV) is a bullous skin disorder that involves mainly the skin and mucous membranes, PV being more common clinically. The presence of autoantibodies to the structure of keratinocyte intercellular proteins in the patient's serum circulation disrupts the intercellular adhesion function, leading to clinical manifestations of predominantly intraepidermal blisters. About 60% of PV patients can have blisters and bullae of oral mucosa, and spontaneously break, the erosion surface is difficult to heal, the feeding and daily life of the patients are seriously affected, and great pain is brought to the body and the mind of the patients. In addition, a part of patients only showed persistent oral mucosal erosion without any skin injury. Therefore, there is a need for effective detection means for rapid and accurate diagnosis of PV.
Early diagnosis of PV has an extremely important role in therapy and prognosis, and diagnosis of PV includes clinical manifestations, histopathology, and immunodiagnostic indicators. Clinical manifestations: (1) the skin is subject to loose blisters and bullae and is easy to break; (2) forming intractable erosion after blister and bulla are broken; (3) the mucous membrane is blister or erosion; (4) positive for NIDDM. Histopathology: the epidermis or the intercellular layer of the epithelium opens to form blisters and bullae. Immunodiagnosis index: (1) normal skin Direct Immunofluorescence (DIF) at or around the lesion shows IgG and/or complement deposition between epidermal (or epithelial) cells; (2) indirect Immunofluorescence (IIF) detected the presence of anti-epidermal intercellular antibodies in serum; (3) ELISA detected the presence of anti-Dsg antibodies in serum. At least 1 of the clinical manifestations plus at least 1 of the histopathological and immunodiagnostic indices are satisfied. At least 2 of the clinical manifestations are met plus 2 of the immunodiagnostic indicators.
High affinity anti-Dsg 3 monoclonal antibodies are required in the diagnosis of PV, and classical hybridoma technology requires a lot of time and effort to obtain high affinity antibodies, and requires subsequent humanized engineering, which makes it difficult to obtain human monoclonal antibodies by hybridoma technology.
Phage display technology has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, epitope research, cell signaling research, and the like. Phage antibody library technology is to screen and enrich specific antibodies by preparing a human antibody library (library) and expressing Fab fragments or single chain antibodies (ScFv) on the surface of phage. Almost all recombinant human monoclonal antibodies that specifically react with antigens are screened from a single spot antibody library system, and thus, when phage antibody technology is used, various antibody fragments (Fab or ScFv) that can be used for in vivo diagnosis or treatment can be obtained. Phage antibody library technology does not require immunization and humanization transformation steps, relies on enrichment screening in a fully controllable biochemical environment, and achieves screening of higher affinity fully human antibody sequences in a short period of time.
The pathological features of pemphigus vulgaris are epidermoid blisters and acanthocellular relaxation, the direct cause of this pathological change is anti-acanthocellular interstitial antibodies, which are present in the serum of almost all active pemphigus patients, and the target antigen acted by the PV antibodies is located on the desmosome, the junction structure between epidermoid desmosomes, which is the transmembrane desmosome core protein (desmoglein 3, dsg 3). Dsg3 has a molecular weight of 64.95KD, and is one of the superfamily members of cadherins (cadherins), playing an important role in maintaining the integrity of the epidermis, and disruption of its structure can lead to acanthosis, causing blisters in the epidermis. The intracellular components of Dsg3 interact with the cytoskeleton via desmosomal discotic proteins and form links, while the extracellular domains mediate intercellular links, with the extracellular domains of Dsg3 providing epitopes recognized by PV antibodies. The region of Dsg3 that plays the main role is called the extracellular domain (extracellular domain, EC), and the EC of Dsg3 is divided into 5 domains (EC 1-EC 5), with EC-1 at the amino-terminus and EC-5 at the carboxy-terminus. The natural steric conformation of the Dsg3 molecule is important for the triggering of autoimmune bullae. Previous studies have obtained human Dsg3 recombinant proteins, including whole ECs of Dsg3, via baculovirus expression systems and mammalian expression systems, and the resulting human Dsg3 recombinant proteins can specifically bind to natural antibodies in human PV patient serum. The human Dsg3 recombinant protein not only can provide a reliable means for diagnosing PV, but also can be used for further researching the pathogenesis of pemphigus.
Therefore, the phage antibody library technology is utilized to screen a human antibody which binds to pemphigus vulgaris specific antigen peptide, and has important application value in diagnosis and/or treatment of pemphigus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a monoclonal antibody combined with Dsg3 and application thereof. In the invention, a phage humanized antibody library is constructed by peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) of a Dsg3 antibody positive patient, and monoclonal antibodies capable of specifically binding to a Dsg3 antigen are screened by specifically binding to a Dsg3 protein. The monoclonal antibody has high affinity and strong specificity, can quantitatively detect the level of the anti-Dsg 3 autoantibody in a PV patient, monitors the disease activity, and is used for clinical diagnosis of the PV patient. The research of the invention provides theoretical and experimental basis for specific treatment of PV, and simultaneously provides a new research direction for treating autoimmune diseases with autoantibodies as main pathogenic mechanisms.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a monoclonal antibody that binds Dsg3, the heavy chain CDR3 of which comprises the amino acid sequence of SEQ ID No. 3, SEQ ID No. 13, SEQ ID No. 23, SEQ ID No. 33 or SEQ ID No. 43;
the light chain CDR3 of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 6, SEQ ID NO. 16, SEQ ID NO. 26, SEQ ID NO. 36 or SEQ ID NO. 46.
The invention screens out monoclonal antibody with good specificity and strong affinity for combining Dsg3 from phage library, the monoclonal antibody is fully human antibody, the fully human antibody has lower immunogenicity, and has great application potential in antibody medicine and diagnostic medicine development.
Preferably, the heavy chain CDR1 of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 21, SEQ ID NO. 31 or SEQ ID NO. 41.
Preferably, the heavy chain CDR2 of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 2, SEQ ID NO. 12, SEQ ID NO. 22, SEQ ID NO. 32 or SEQ ID NO. 42.
Preferably, the light chain CDR1 of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 4, SEQ ID NO. 14, SEQ ID NO. 24, SEQ ID NO. 34 or SEQ ID NO. 44.
Preferably, the amino acid sequence of the light chain CDR2 of the monoclonal antibody comprises WAS or KVS.
Preferably, the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 7, SEQ ID NO. 17, SEQ ID NO. 27, SEQ ID NO. 37 or SEQ ID NO. 47.
Preferably, the light chain variable region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 8, SEQ ID NO. 18, SEQ ID NO. 28, SEQ ID NO. 38 or SEQ ID NO. 48.
Preferably, the heavy chain of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 29, SEQ ID NO. 39 or SEQ ID NO. 49.
Preferably, the light chain of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 10, SEQ ID NO. 20, SEQ ID NO. 30, SEQ ID NO. 40 or SEQ ID NO. 50.
The invention separates PBMC of Dsg3 antibody positive patient, extracts RNA and carries out quality inspection on RNA. Reverse transcribing the qualified RNA into cDNA by RT-PCR technology, and amplifying all the VH and VL gene fragments of the antibody. Cloning in vitro amplified V kappa and V lambda gene fragments into pATA-scFv-2 vector to construct the antibody combinatorial library. The antibody gene combinatorial library is inserted into the immediate downstream of the leader sequence of the phage-encoded membrane protein gene III (g 3) or gene VIII (g 8), and the polypeptide expressed by the exogenous antibody gene can be displayed at the N-terminal of phage coat protein pIII or pVIII in the form of fusion protein by assisting in the super-infection of phage. Each phage particle encodes and presents a different antibody, which contains billions of individual clones. In these antibody libraries, genes encoding those antibodies that bind to the antigen are amplified by affinity enrichment-mild elution-phage amplification of the antigen in vitro, and the enrichment screening process is repeated until a specific, strong affinity antibody phage library is obtained after several cycles, from which positive clones are screened. And (3) identifying positive clones by an ELISA method, and finally screening the fully-humanized antibodies with good specificity and strong affinity from the positive clones.
Through the screening of the antibody phage library, 5 monoclonal antibodies with high affinity are finally obtained, which are 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. The monoclonal antibody has high activity, good stability and stronger specificity, can be used as a reference standard for qualitatively detecting the positive of the PV, and can also quantitatively detect the level of the anti-Dsg 3 autoantibody in the PV patient.
Monoclonal antibody 76F-DSG3-IC-R2P1-G1:
SEQ ID NO:1 (heavy chain CDR 1): GFTLANST.
SEQ ID NO. 2 (heavy chain CDR 2): SVVGNDKT.
SEQ ID NO:3 (heavy chain CDR 3): AASSHFWSGSLDI.
SEQ ID NO. 4 (light chain CDR 1): QSVLYSSNNKNY.
Light chain CDR2: WAS.
SEQ ID NO. 6 (light chain CDR 3): QQYYSTPLT.
SEQ ID NO. 7 (heavy chain variable region):
EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS。
SEQ ID NO. 8 (light chain variable region):
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKLEIK。
SEQ ID NO. 9 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-G1 antibody):
MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
SEQ ID NO. 10 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-G1 antibody):
MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
monoclonal antibody 76F-DSG3-IC-R2P1-F2:
SEQ ID NO. 11 (heavy chain CDR 1): GFTLANST.
SEQ ID NO. 12 (heavy chain CDR 2): SVVGNDKT.
SEQ ID NO:13 (heavy chain CDR 3): AASSHFWSGSLDI.
SEQ ID NO. 14 (light chain CDR 1): QSVLYSSNNKNY.
Light chain CDR2: WAS.
SEQ ID NO. 16 (light chain CDR 3): QQYYSTPIT.
SEQ ID NO. 17 (heavy chain variable region):
EVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS。
SEQ ID NO. 18 (light chain variable region):
DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGQGTRLEIK。
SEQ ID NO. 19 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-F2 antibody):
MKHLWFFLLLVAAPRWVLSEVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
SEQ ID NO. 20 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-F2 antibody):
MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
monoclonal antibody 76F-DSG3-IC-R2P1-E6:
SEQ ID NO. 21 (heavy chain CDR 1): GFTLANST.
SEQ ID NO. 22 (heavy chain CDR 2): SVVGNDKT.
SEQ ID NO. 23 (heavy chain CDR 3): AASSHFWSGSLDV.
SEQ ID NO. 24 (light chain CDR 1): QSVLYNSNNKNY.
Light chain CDR2: WAS.
SEQ ID NO. 26 (light chain CDR 3): HQYFGTPYT.
SEQ ID NO. 27 (heavy chain variable region):
QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGN DKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLV TVSS。
SEQ ID NO. 28 (light chain variable region):
EIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWAST RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGTPYTFGQGTKVEIK。
SEQ ID NO. 29 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-E6 antibody):
MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGHRLEWMGWSVVGNDKTDYPQNLQERVTFTRDLSTGTASMTLSSLTSEDTAIYYCAASSHFWSGSLDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
SEQ ID NO. 30 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-E6 antibody):
MVLQTQVFISLLLWISGAYGEIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYFGTPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
monoclonal antibody 76F-DSG3-IC-R2P1-C9:
SEQ ID NO. 31 (heavy chain CDR 1): GGSFSGYY.
SEQ ID NO. 32 (heavy chain CDR 2): INHSGST.
SEQ ID NO: SEQ ID NO (heavy chain CDR 3): ARGRYLATVRHYYYYYMDV.
SEQ ID NO:34 (light chain CDR 1): QSLENSAGNTY.
Light chain CDR2: and (5) KVS.
SEQ ID NO:36 (light chain CDR 3): MQGSHWPPYT.
SEQ ID NO. 37 (heavy chain variable region):
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTN YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKG TLVTVSS。
SEQ ID NO. 38 (light chain variable region):
EIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWPPYTFGQGTKVEIK。
SEQ ID NO. 39 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-C9 antibody):
MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRYLATVRHYYYYYMDVWGKGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
SEQ ID NO. 40 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-C9 antibody):
MVLQTQVFISLLLWISGAYGEIVLTQSPLFLPVTLGQPASISCRSSQSLENSAGNTYLHWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
monoclonal antibody 76F-DSG3-IC-R2P1-H9:
SEQ ID NO. 41 (heavy chain CDR 1): GFTLANST.
SEQ ID NO. 42 (heavy chain CDR 2): SVVGNDKT.
SEQ ID NO:43 (heavy chain CDR 3): AASSHFWSGSLDI.
SEQ ID NO. 44 (light chain CDR 1): QSVLYSSNNKNY.
Light chain CDR2: WAS.
SEQ ID NO:46 (light chain CDR 3): QQYYSTPFT.
SEQ ID NO. 47 (heavy chain variable region):
QVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGN DKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVT VSS。
SEQ ID NO. 48 (light chain variable region):
DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK。
SEQ ID NO. 49 (heavy chain amino acid sequence of 76F-DSG3-IC-R2P1-H9 antibody):
MKHLWFFLLLVAAPRWVLSQVQLVQSGPEVKKPGTSVEVSCRASGFTLANSTVQWVRQARGQRLEWMGWSVVGNDKTDYPQKFQERVTFTRDLSTGTASMTLSSLTSEDTAFYYCAASSHFWSGSLDIWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
SEQ ID NO. 50 (light chain amino acid sequence of 76F-DSG3-IC-R2P1-H9 antibody):
MVLQTQVFISLLLWISGAYGDIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
in a second aspect, the invention provides a nucleic acid molecule encoding a monoclonal antibody according to the first aspect that binds Dsg 3.
In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the second aspect.
In a fourth aspect, the invention provides a host cell comprising at least one copy of the expression vector of the third aspect.
In a fifth aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody that binds Dsg3 according to the first aspect, and a pharmaceutically acceptable carrier and/or diluent.
In a sixth aspect, the invention provides a kit for detecting Dsg3 protein in a sample, the kit comprising a monoclonal antibody according to the first aspect that binds Dsg 3.
In a seventh aspect, the invention provides the use of any one or a combination of at least two of a monoclonal antibody that binds Dsg3 according to the first aspect, a host cell according to the fourth aspect or a pharmaceutical composition according to the fifth aspect in the manufacture of a medicament for the diagnosis and/or treatment of pemphigus.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, 5 monoclonal antibodies with high affinity and capable of binding with Dsg3 are obtained through screening of an antibody phage library, and the monoclonal antibodies are high in activity, good in stability and strong in specificity, can be used as a reference standard for qualitatively detecting PV positive, and can also quantitatively detect the level of the anti-Dsg 3 autoantibody in a PV patient.
Drawings
FIG. 1 is a schematic of a monoclonal PCR agarose gel electrophoresis of a VL phage library;
FIG. 2 is a schematic diagram of a monoclonal PCR agarose gel electrophoresis of a kappa H phage library;
FIG. 3 is a diagram of a monoclonal PCR agarose gel electrophoresis of a lambda H phage library;
FIG. 4A is a sequencing quality control result of a light chain library;
FIG. 4B is a sequencing quality control result of the heavy chain library;
FIG. 5 is an SDS-PAGE electrophoresis of purified Dsg 3;
FIG. 6A is the ELISA detection results of 76F-DSG3-IC-R2P1-G1 antibody and Dsg 3;
FIG. 6B is the ELISA assay result for 76F-DSG3-IC-R2P1-F2 antibody and Dsg 3;
FIG. 6C is the ELISA assay results for 76F-DSG3-IC-R2P1-E6 antibodies with Dsg 3;
FIG. 6D is the ELISA detection results of 76F-DSG3-IC-R2P1-C9 antibodies with Dsg 3;
FIG. 6E shows ELISA detection results of 76F-DSG3-IC-R2P1-H9 antibody and Dsg 3.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Detailed description of the invention:
monoclonal antibodies are the largest and fastest growing group of therapeutic proteins, and currently more than 500 therapeutic antibodies and their derivatives are being tested in clinical trials, focusing on cancer treatment and autoimmune diseases. The invention separates PBMC of Dsg3 antibody positive patient, extracts RNA and carries out quality inspection on RNA. Reverse transcribing the qualified RNA into cDNA by RT-PCR technology, and amplifying all the VH and VL gene fragments of the antibody. Cloning in vitro amplified V kappa and V lambda gene fragments into pATA-scFv-2 vector to construct the antibody combinatorial library.
The antibody gene combinatorial library is inserted into the immediate downstream of the leader sequence of the phage-encoded membrane protein gene III (g 3) or gene VIII (g 8), and the polypeptide expressed by the exogenous antibody gene can be displayed at the N-terminal of phage coat protein pIII or pVIII in the form of fusion protein by assisting in the super-infection of phage. Each phage particle encodes and presents a different antibody, which contains billions of individual clones. In these antibody libraries, genes encoding those antibodies that bind to the antigen are amplified by affinity enrichment-mild elution-phage amplification of the antigen in vitro, and the enrichment screening process is repeated until a specific, strong affinity antibody phage library is obtained after several cycles, from which positive clones are screened. And (3) identifying positive clones by an ELISA method, and finally screening the fully-humanized antibodies with good specificity and strong affinity from the positive clones.
The invention usesIII 1st Strand cDNA Synthesis Kit (+gDNA wind) reverse transcription kit RNA from Dsg3 antibody positive patients was reverse transcribed into cDNA, and VH and VL fragments of DNA were amplified. The vk and vλ gene fragments obtained by in vitro amplification are cloned into pATA-scFv-2 vector by cloning technology to form vk library and vλ library. Extracting plasmid vectors of the V kappa library and the V lambda library by using a plasmid extraction kit, and inserting the in-vitro amplified VH gene fragments into the plasmid vectors of the V kappa library and the V lambda library to form a kappa H library and a lambda H library.
The artificially synthesized gene of the Dsg3 domain is recombined into an expression vector plasmid pFastBac1 to obtain a Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector is transfected into DH10Bac competence to be cultured, and the Dsg3 antigen protein is obtained by purification.
Clones with an antigen group greater than 3 times that of the control group were determined to be positive clones by three rounds of antibody phage library screening, and sequencing analysis was performed on these monoclonal clones. The error antibody sequence and the repeated antibody sequence are removed, and the specific binding capacity of antigen and antibody reflected by ELISA experiments is combined, so that 5 high-affinity antibodies are finally obtained, which are 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. The monoclonal antibody has high activity, good stability and stronger specificity, can be used as a reference standard for qualitatively detecting the positive of the PV, and can also quantitatively detect the level of the anti-Dsg 3 autoantibody in the PV patient.
Example 1 construction method of human ScFv phage display library
In this example PBMC of Dsg3 antibody positive patients were isolated, RNA was extracted and quality checked. Reverse transcribing the qualified RNA into cDNA by RT-PCR technology, and amplifying all the VH and VL gene fragments of the antibody. Cloning in vitro amplified V kappa and V lambda gene fragments into pATA-scFv-2 vector to construct the antibody combinatorial library.
The main reagents used in this example are shown in table 1:
TABLE 1
1. Library construction
1.1 assembling heavy chain variable region (VH) and light chain variable region (VL), experimental steps are as follows: the PCR reaction conditions and procedures are shown in Table 2.
TABLE 2
Wherein, three steps of denaturation, annealing and extension (1) are repeated for 30 times; the primer sequences are shown below:
upstream primer (F):
5′L-VH 1(SEQ ID NO:51)：acaggtgcccactcccaggtgcag。
5′L-VH 3(SEQ ID NO:52)：aaggtgtccagtgtgargtgcag。
5′L-VH 4/6(SEQ ID NO:53)：cccagatgggtcctgtcccaggtgcag。
5′L-VH 5/7(SEQ ID NO:54)：caaggagtctgttccgaggtgcag。
5′L VK 1/2(SEQ ID NO:55)：atgaggstcccygctcagctgctgg。
5′L VK 3(SEQ ID NO:56)：ctcttcctcctgctactctggctcccag。
5′L VK 4/5(SEQ ID NO:57)：atttctctgttgctctggatctctg。
5′L Vλ1(SEQ ID NO:58)：ggtcctgggcccagtctgtgctg。
5′L Vλ2(SEQ ID NO:59)：ggtcctgggcccagtctgccctg。
5′L Vλ3(SEQ ID NO:60)：gctctgtgacctcctatgagctg。
5′L Vλ4/5(SEQ ID NO:61)：ggtctctctcscagcytgtgctg。
5′L Vλ6(SEQ ID NO:62)：gttcttgggccaattttatgctg。
5′L Vλ7(SEQ ID NO:63)：ggtccaattcycaggctgtggtg。
5′L Vλ8/9/10(SEQ ID NO:64)：gagtggattctcagactgtggtg。
downstream primer (R):
3′CK(SEQ ID NO:65)：tgctgtccttgctgtcctgct。
3′Cλ(SEQ ID NO:66)：caccagtgtggccttgttggcttg。
1.2 construction of light chain variable region phage display library
1.2.1 preparation of pATA-scFv-2 vector for library cloning.
1.2.2 digestion of vector and PCR products the reaction system for digestion of vector and PCR products is shown in Table 3.
TABLE 3 Table 3
1.2.3 ligation of digested vector and PCR product, ligation reaction system as shown in Table 4; the prepared ligation reaction system was incubated at 16℃overnight and heat-inactivated at 65℃for 10min to terminate the reaction.
TABLE 4 Table 4
| / | pATA-Vκ | pATA-Vλ |
| T4 DNA ligase (Thermo) | 3μL | 3μL |
| 10×T4 DNA ligase buffer | 8μL | 8μL |
| Cloning vector (NheI/NotI) | 1μg | 1μg |
| V kappa or V lambda fragment (NheI/NotI) | 0.3μg | 0.3μg |
| H 2 O | Added to the reaction system in total 80. Mu.L | Added to the reaction system in total 80. Mu.L |
1.2.4 electrotransformation of ligation products into competent cells, the steps of electrotransformation are as follows:
1.2.4.1TG1 competent cells were prepared.
1.2.4.2 1mL of SOC medium (Sigma, S1797) was pre-warmed at 37 ℃; electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes were placed on ice (one cuvette and one microcentrifuge tube per conversion reaction).
1.2.4.3 the electrocompetent cells were removed from the freezer at-80℃and placed on ice until they were completely thawed (10-15 min); after thawing the cells, gently mixing; mu.L of cells were placed in a frozen microcentrifuge tube on ice.
1.2.4.4 carefully add 3. Mu.L of DNA mixture to a frozen electroporation cuvette without generating air bubbles; the tube was flicked down quickly with the wrist to deposit cells on the bottom.
1.2.4.5 electroporation at 600Ω,10 μF and 1.8 kV; immediately 1mL of pre-warmed SOC medium was added to each tube within 10 seconds of the pulse; shaking at 37℃and 250rpm for 1h.
1.2.4.6 all electrotransformation media was collected; mu.L of the culture was serially diluted into 90. Mu.L of SOC medium and plated on LB/Amp/Glucose (LB/ampicillin/Glucose medium). Incubating overnight at 37 ℃; the total number of transformants was calculated by counting the number of colonies, multiplying the culture volume by the plating volume.
1.3 construction of VL-VH phage display libraries
1.3.1 digestion of vector and PCR products, digestion reaction system is shown in Table 5.
TABLE 5
1.3.2 ligation reaction System As shown in Table 6, the prepared ligation reaction system was incubated at 16℃overnight and heat-inactivated at 65℃for 10min to terminate the reaction.
TABLE 6
| / | pATA-scFv-κH | pATA-scFv-λH |
| T4 DNA ligase (Thermo) | 10μL | 10μL |
| 10×T4 DNA ligase buffer | 14μL | 14μL |
| Cloning vector (SfiI/XhoI/NcoI) | 2μg | 2μg |
| VH fragment (SfiI/XhoI) | 0.6μg | 0.6μg |
| H 2 O | Added to the reaction system in total 140. Mu.L | Added to the reaction system in total 140. Mu.L |
1.3.3 electrotransformation of ligation products into competent cells, the steps of electrotransformation are as follows:
1.3.3.1TG1 competent cells were prepared.
1.3.3.2 4mL of SOC medium (Sigma, S1797) was pre-warmed at 37 ℃. Electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes were placed on ice (one cuvette and one microcentrifuge tube per conversion reaction).
1.3.3.3 the electrokinetic competent cells were removed from the-80℃refrigerator and placed on ice until they were completely thawed (10-15 min). After thawing the cells, mix gently.
1.3.3.4 6. Mu.L of the DNA mixture was carefully added to a frozen electroporation cuvette without generating air bubbles. The tube was flicked down quickly with your wrist, depositing cells on the bottom.
1.3.3.5 electroporation at 600Ω,10 μF and 2.5 kV. Within 10 seconds of the pulse, 2mL of pre-warmed SOC medium was immediately added to each tube. Shaking at 37℃and 250rpm for 1h.
1.3.3.6 all electrotransformation media was collected. mu.L of the culture was serially diluted into 90. Mu.L of SOC medium and plated on LB/Amp/Glucose (LB/ampicillin/Glucose medium). Incubate overnight at 37 ℃. The total number of transformants was calculated by counting the number of colonies, multiplying the culture volume by the plating volume.
1.4 library evaluation
1.4.1 colony PCR: performing PCR by taking the constructed library as a template; the PCR reaction conditions are shown in Table 7.
TABLE 7
Wherein, three steps of denaturation, annealing and extension (1) are repeated 30 times. The primer sequences for the PCR reactions are shown below:
upstream primer (F) (SEQ ID NO: 67): agcggataacaatttcacacagga.
Downstream primer (R) (SEQ ID NO: 68): gcccccttattagcgtttgccatc.
The agarose gel electrophoresis detection results after PCR are shown in FIGS. 1-3. The monoclonal bacterial PCR agarose gel electrophoresis of the VL phage library is shown in FIG. 1, lane M in FIG. 1: DL2000; lanes 1-16:21000076F pATA-V kappa, lanes 17-32:21000076F pATA-V lambda. The monoclonal bacterial PCR agarose gel electrophoresis of the kappa H phage library is shown in FIG. 2, lane M in FIG. 2: DL2000, lanes 1-48:21000076F pATA-scFv- κH. The PCR agarose gel electrophoresis of the monoclonal bacteria of the phage library λh is shown in fig. 3, lane M in fig. 3: DL2000, lanes 1-48:21000076F pATA-scFv- λH.
1.4.2 sequencing
Sequencing the phage display library, and selecting positive clones to be sent to the Wohan engineering Co., ltd for sequencing, wherein the sequencing quality control result of the phage display library is shown in FIG. 4A and FIG. 4B. FIG. 4A shows the results of the sequencing quality control of the light chain library, and FIG. 4B shows the results of the sequencing quality control of the heavy chain library. As can be seen from fig. 4A and 4B, the library sequences cover the germline of most of the heavy and light chain variable regions of the human antibodies, and library diversity meets the expected requirements.
1.5 expression of Dsg3 protein
The artificially synthesized gene of the Dsg3 domain is recombined into an expression vector plasmid pFastBac1 to obtain a Dsg3-pFastBac1 expression vector. The Dsg3-pFastBac1 expression vector is transfected into DH10Bac competence to be cultured, and the Dsg3 antigen protein is obtained by purification. Artificially synthesizing a Dsg3 gene sequence, and recombining the Dsg3 gene into an expression vector plasmid pFastBac1 to obtain a Dsg3-pFastBac1 expression vector; the amino acid sequence of the cloning site BamHI/XhoI, dsg3 is shown in SEQ ID NO: 69:
EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEKDAVCSSSPSVVVSARTLNNRYTGPYTFALEDQPVKLPAVWSITTLNATSALLRAQEQIPPGVYHISLVLTDSQNNRCEMPRSLTLEVCQCDNRGICGTSYPTTSPGTRYGRPHSGR。
transfecting a Dsg3-pFastBac1 expression vector into DH10Bac competence for culture, collecting precipitate, and carrying out GST tag affinity chromatography to obtain Dsg3 protein; the purified Dsg3 also needs SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis) to verify the purity, and the purified Dsg3 SDS-PAGE electrophoresis chart is shown in FIG. 5, wherein the protein size is 64.95kDa, and the purity is more than 90%.
EXAMPLE 2 screening for monoclonal antibodies that specifically bind to Dsg3
The phage antibody libraries are enriched for the desired specific antibody clones using a suitable panning strategy. The main reagents used in this example are shown in Table 8.
TABLE 8
| Reagent(s) | Numbering device | Manufacturer (S) |
| 96-well plate (96-well plate) | 42592 | Costar |
| Tween 20 (Tween 20) | P2287 | Sigma |
| Tris (Tris-hydroxymethyl aminomethane) | RES3098T-B7 | Sigma |
| Glycine (Glycine) | G8200 | Solarbio |
| PEG (polyethylene glycol) | 181986 | Sigma |
| PBS (phosphate buffered saline) | C10010500BT | Life |
| Casein Na salt (Casein sodium) | S12003-100g | shyuanye |
| Skim milk (Skim milk) | 6342932 | BD |
| HRP-labeled M13 antibodies | MVV05401H | AntibodySystem |
1. First round panning
1.1 biopanning
1.1.1 coating: the tubes were coated and incubated overnight at 4 ℃. Antigen group: 1mL Dsg3 transfection solution (50. Mu.g/mL), control: 500. Mu.L of transfection solution (0. Mu.g/mL).
1.1.2 washing: the centrifuge tube was discarded and washed three times with 5mL of 0.05% PBST.
1.1.3 blocking: 5mL of 5% skim milk or 1% casein (PBST solubilized) was added to the tube and incubated at 37℃for 2h.
1.1.4 washing: the centrifuge tube was discarded and washed once with 5mL of 0.05% PBST.
1.1.5 incubation: phage library was diluted with 1% skim milk or 0.2% casein (PBST lysis), 1mL was added to the centrifuge tube and incubated for 2h at 32 ℃.
1.1.6 washing: the centrifuge tube was discarded, washed three times with 5mL of 0.05% PBST, and twice with PBS.
1.1.7 elution: phage bound to Dsg3 were eluted with 1mL glycine-HCl (pH 2.2) and neutralized to pH 7.0 with Tris-HCl.
1.2 determination of titer of diluted phage
1.2.1 coating of centrifuge tubes with 1mL Dsg3 transfection solution (50. Mu.g/mL) and incubation overnight at 4 ℃.
1.2.2 discard the tube and wash three times with 5mL of 0.05% PBST.
1.2.3 5mL of 5% skim milk (PBST solubilized) was added to the tube and incubated at 37℃for 2h.
1.2.4 discard the tube and wash once with 5mL of 0.05% PBST.
1.2.5 phage of 1.1.7 were transferred to centrifuge tubes and incubated for 2h at 32 ℃.
1.2.6 culturing E.coli TG1 to OD 600 ＝0.4-0.6。
1.2.7 mix 10. Mu.L diluted post-elution phage with 180. Mu.L E.coli TG1.
1.2.8 The mixture was incubated at 37℃for 30min, then poured into 2 XYT-A (Amp 100. Mu.g/mL) medium and the medium was back-incubated overnight at 37 ℃.
1.3 phage library amplification
1.3.1 adding 10. Mu.L E.coli TG1 to 800. Mu.L 2YT culture solution, and mixing and culturing at 37deg.C to OD 600 ＝0.4-0.6。
1.3.2 transfer TG1 cultured to logarithmic phase into 10mL of 2YT-G culture solution (final concentration of 2% glucose), culture to OD at 37℃on a shaker 600 ＝0.4-0.6。
1.3.3 adding the eluted product, incubating at 37℃for 30min, and shaking at 37℃for 30min.
1.3.4 30mL of 2YT-AG broth (final concentration 0.1% Amp,2% glucose) was added and shake cultured at 37℃for 1h.
1.3.5M 13KO7 (M13 KO7: TG 1=20:1) was added, incubated at 37℃for 30min, and shake incubated at 37℃for 30min.
1.3.6 bacterial solution was centrifuged at 5000rpm for 5min, resuspended in 40mL 2YT-AK (final concentration Amp 100. Mu.g/mL, kan 100. Mu.g/mL) and incubated overnight at 30℃in a shaker.
1.3.7 Centrifuging at 8000rpm for 10min, and taking out supernatant. 1/5 (v/v) PEG/NaCl was added to the supernatant, mixed and incubated on ice for 2h.
1.3.8 resuspended in 1mL PBS, centrifuged at 12000rpm for 5min, and the supernatant transferred to a new 1.5mL centrifuge tube.
1.4 phage library titer determination after amplification, procedure 1.2 was followed.
2. Second to third round panning
2.1 biopanning
Step 1 was repeated twice in cycles, and phage library was eluted after the previous round of amplification for each input. Biopanning results are shown in table 9.
TABLE 9
As can be seen from Table 9, the phage/input phage ratio obtained for the antigen group increased from round to round through three rounds of panning, indicating that there was some enrichment of phage.
3. Polyclonal phage ELISA
3.1 coating: the ELISA plate was coated and incubated overnight at 4 ℃. Antigen group: 100. Mu.L/Dsg 3 protein per well (4. Mu.g/mL), control: 100. Mu.L/protein dilution per well (0. Mu.g/mL).
3.2 washing: the liquid in the ELISA plate was discarded and each well was washed three times with 300. Mu.L of 0.05% PBST.
3.3 closing: mu.L of 5% skimmed milk (PBS dissolved) was added to each well, and the wells were blocked at 37℃for 2 hours.
3.4 phage incubation: 100. Mu.L of diluted amplified phage was added to each well and incubated at 32℃for 2h as shown in Table 10.
3.5 washing: the same as in step 3.2.
3.6 secondary antibody incubation: mu.L of anti-M13-HRP anti-ibody (HRP-labeled M13 antibody) (1:6000) diluted with blocking solution was added to each well and incubated at 32℃for 1h.
3.7 washing: the same as in step 3.2.
3.8 color development: mu.L TMB was added to each well, incubated at room temperature, and then the reaction was stopped by adding 100. Mu.L 2M HCl to each well.
3.9 reading the plate: values were read using a microplate reader at 450-630 nm. The results of the polyclonal phage ELISA are shown in Table 10.
Table 10
As can be seen from Table 10, as the number of rounds increased, the ELISA results of the antigen groups increased round by round, indicating that positive phages were enriched round by round, with the second round of antigen groups being widely separated from the control group, so that the elution products of the second group were selected for ELISA screening of monoclonal phages.
4. Monoclonal phage ELISA (second round of elution product was selected for monoclonal based on the polyclonal results)
4.1 96 clones were selected from the petri dishes for detection of elution titer; these clones were cultured at 37℃and 250rpm until OD 600nm ＝0.4-0.6。
4.2M13KO7 infection cultures (moi=20:1), incubation for 30min at 37 ℃, shaking culture for 30min at 37 ℃; the bacterial solution was centrifuged and the pellet was resuspended in an equal volume of 2 XYT-AK (final concentration Amp 100. Mu.g/mL, kan 100. Mu.g/mL) and incubated overnight at 30 ℃.
4.3 the cultures were centrifuged and the supernatant was used for ELISA.
4.4 coating: coating an ELISA plate, and incubating at 4 ℃ overnight; antigen group: 100. Mu.L/Dsg 3 protein per well (4. Mu.g/mL), control: 100. Mu.L/protein dilution per well (0. Mu.g/mL).
4.5 washing: the liquid in the ELISA plate was discarded and each well was washed three times with 300. Mu.L of 0.05% PBST.
4.6 closing: mu.L of 5% skimmed milk (PBS dissolved) was added to each well, and the wells were blocked at 37℃for 2 hours.
4.7 phage incubation: 100. Mu.L of phage supernatant was added to each well and incubated at 32℃for 2h.
4.8 washing: the same as in step 4.5.
4.9 secondary antibody incubation: mu.L of anti-M13-HRP anti-ibody (HRP-labeled M13 antibody) (1:6000) diluted with blocking solution was added to each well and incubated at 32℃for 1h.
4.10 washing: the same as in step 4.5.
4.11 color development: mu.L TMB was added to each well, incubated at room temperature, and then the reaction was stopped by adding 100. Mu.L 2M HCl to each well.
4.12 reading the plate: values were read using a microplate reader at 450-630nm and highly specific clones were sequenced. The results of the antigen group monoclonal phage ELISA are shown in Table 11.
TABLE 11
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The results of the control monoclonal phage ELISA are shown in table 12.
Table 12
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| 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 |
As is clear from the results in tables 11 and 12, the difference between the results of the antigen groups of the G1, F2, E6, C9, F9 clones and the control group was greater than 0.1, and thus 5 clones were once identified as positive clones, and the secondary verification was performed.
5. Positive clone validation ELISA
5.1 50. Mu.L of positive clones were incubated in 2mL of 2YT-AG medium (final concentration 0.1% Amp,2% glucose) to OD 600 ＝0.4-0.6。
5.2M13KO7 infection cultures (MOI=20:1), incubation for 30min at 37℃and shaking culture for 30min at 37 ℃. The bacterial solution was centrifuged and the pellet was resuspended in an equal volume of 2 XYT-AK (final concentration Amp 100. Mu.g/mL, kan 100. Mu.g/mL) and incubated overnight at 30 ℃.
5.3 the cultures were centrifuged and the supernatant was used in ELISA.
5.4 coating: the ELISA plate was coated and incubated overnight at 4 ℃. Antigen group: 100. Mu.L/Dsg 3 protein per well (4. Mu.g/mL), control: 100. Mu.L/protein dilution per well (0. Mu.g/mL).
5.5 washing: the liquid in the ELISA plate was discarded and each well was washed three times with 300. Mu.L of 0.05% PBST.
5.6 closing: mu.L of 5% skimmed milk (PBS dissolved) was added to each well, and the wells were blocked at 37℃for 2 hours.
5.7 phage incubation: 100. Mu.L of phage supernatant was added to each well and incubated at 32℃for 2h.
5.8 washing: the same as in step 4.5.
5.9 secondary antibody incubation: mu.L of anti-M13-HRP anti-ibody (HRP-labeled M13 antibody) (1:6000) diluted with blocking solution was added to each well and incubated at 32℃for 1h.
5.10 washing: the same as in step 4.5.
5.11 color development: mu.L TMB was added to each well, incubated at room temperature, and then the reaction was stopped by adding 100. Mu.L 2M HCl to each well.
5.12 reading the plate: values were read using a microplate reader at 450-630nm and highly specific clones were sequenced. The results of the positive monoclonal phage ELISA are shown in table 13.
TABLE 13
As can be seen from the results of Table 13, the secondary validation results of these 5 positive clones were positive. The positive clones were sent to sequencing, and the sequences of the resulting high specificity antibodies were obtained.
Example 3 affinity assay for highly specific antibodies
In this example, the antibodies obtained by screening in example 2 were subjected to affinity assay, and the OD values at different dilution concentrations were detected by enzyme-linked immunosorbent assay. The ELISA experimental steps of the ELISA reaction are as follows:
1, coating: 100. Mu.L/well of Dsg3 protein (4. Mu.g/mL) coated ELISA plates were incubated overnight at 4 ℃.
2, washing: the liquid in the ELISA plate was discarded and each well was washed three times with 300. Mu.L of 0.05% PBST.
And 3, closing: mu.L of 5% skimmed milk (PBS dissolved) was added to each well, and the wells were blocked at 37℃for 2 hours.
4 positive antibody incubation: the 76F-DSG3-IC-R2P1-G1, 76F-DSG3-IC-R2P1-F2, 76F-DSG3-IC-R2P1-E6, 76F-DSG3-IC-R2P1-C9, 76F-DSG3-IC-R2P1-H9 antibodies were each subjected to gradient dilution (4000 ng/mL, 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.6ng/mL, 7.8ng/mL, 3.9ng/mL, 1.95ng/mL, 0.98ng/mL, 0.49ng/mL and 0.25ng/mL, 100. Mu.L of diluted antibody solution was added to each well, and incubated at 37℃for 1H.
5, washing: the same as in step 4.5.
And 6, secondary antibody incubation: the Goat Anti-Human IgG (H+L) Anti-Human IgG secondary antibody (Jackson, code: 109-035-088) was diluted 10000-fold with blocking solution, 100. Mu.L of the diluted secondary antibody was added to each well, and incubated at 37℃for 30min.
7, washing: the same as in step 4.5.
8 developing: 100. Mu.L TMB was added to each well, incubated at 37℃for 10min, and then 50. Mu.L 2M HCl was added to each well to terminate the reaction.
9, reading a plate: values were read using a microplate reader at 450-630 nm.
The results of the ELISA reaction are shown in FIGS. 6A-6E. FIG. 6A shows the results of ELISA detection of the 76F-DSG3-IC-R2P1-G1 antibody and Dsg3, FIG. 6B shows the results of ELISA detection of the 76F-DSG3-IC-R2P1-F2 antibody and Dsg3, FIG. 6C shows the results of ELISA detection of the 76F-DSG3-IC-R2P1-E6 antibody and Dsg3, FIG. 6D shows the results of ELISA detection of the 76F-DSG3-IC-R2P1-C9 antibody and Dsg3, and FIG. 6E shows the results of ELISA detection of the 76F-DSG3-IC-R2P1-H9 antibody and Dsg 3. From 6A-6E, the screened 5 humanized anti-Dsg 3 monoclonal antibodies and Dsg3 proteins have strong specific binding capacity, and the antibodies can be used as reference standards for qualitatively detecting PV positivity and can also be used for quantitatively detecting the level of the anti-Dsg 3 autoantibody in a PV patient.
In conclusion, the invention constructs phage humanized antibody library by PBMC of Dsg3 antibody positive patient, and screens monoclonal antibody specifically binding to Dsg3 antigen by specific binding to Dsg 3. The monoclonal antibody has high activity, good stability and stronger specificity, can be used as a reference standard for qualitatively detecting the positive of the PV, and can also quantitatively detect the level of the anti-Dsg 3 autoantibody in the PV patient, thereby monitoring the activity of the disease condition and being applicable to the clinical diagnosis of the PV patient. The research of the invention provides theoretical and experimental basis for specific treatment of PV, and simultaneously provides a new research direction for treating autoimmune diseases with autoantibodies as main pathogenic mechanisms.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (11)
1. A monoclonal antibody combined with Dsg3 is characterized in that the heavy chain CDR3 of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 23;
the light chain CDR3 of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 26;
the heavy chain CDR1 of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 21;
the heavy chain CDR2 of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 22;
the light chain CDR1 of the monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 24;
the amino acid sequence of the light chain CDR2 of the monoclonal antibody is WAS.
2. The monoclonal antibody of claim 1 which binds Dsg3, wherein the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID No. 27.
3. The monoclonal antibody of claim 1 which binds Dsg3, wherein the light chain variable region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID No. 28.
4. The monoclonal antibody of claim 1 which binds Dsg3, wherein the heavy chain of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID No. 29.
5. The monoclonal antibody of claim 1 which binds Dsg3, wherein the light chain of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID No. 30.
6. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-5 that binds Dsg 3.
7. An expression vector comprising the nucleic acid molecule of claim 6.
8. A host cell comprising at least one copy of the expression vector of claim 7.
9. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 1-5 that binds Dsg3, and a pharmaceutically acceptable carrier and/or diluent.
10. A kit for detecting Dsg3 protein in a sample, comprising the monoclonal antibody of any one of claims 1-5 that binds Dsg 3.
11. Use of any one or a combination of at least two of the monoclonal antibody binding Dsg3 of any one of claims 1-5, the host cell of claim 8 or the pharmaceutical composition of claim 9 in the preparation of a kit for diagnosing pemphigus.
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| CN116496395A (en) | 2023-07-28 |
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