CN114920842B - Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof - Google Patents
Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereofDownload PDFInfo
- Publication number
- CN114920842B CN114920842BCN202210501892.8ACN202210501892ACN114920842BCN 114920842 BCN114920842 BCN 114920842BCN 202210501892 ACN202210501892 ACN 202210501892ACN 114920842 BCN114920842 BCN 114920842B
- Authority
- CN
- China
- Prior art keywords
- seq
- ser
- antibody
- thr
- hlpv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
- 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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/10—Antioedematous agents; Diuretics
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
- 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
- G01N33/68—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
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- 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]
- 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
- C07K2317/567—Framework region [FR]
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/16—Ophthalmology
- G01N2800/164—Retinal disorders, e.g. retinopathy
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Hematology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Diabetes (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Obesity (AREA)
- Plant Pathology (AREA)
- Hospice & Palliative Care (AREA)
Abstract
Translated fromChineseDescription
Translated fromChinese技术领域technical field
本发明属于生物技术的单克隆抗体领域，具体涉及特异性结合PV-1蛋白的抗体或其抗原结合片段及其应用。The invention belongs to the field of monoclonal antibodies of biotechnology, and specifically relates to an antibody specifically binding to PV-1 protein or an antigen-binding fragment thereof and application thereof.
背景技术Background technique
生物机体物质交换是由呼吸系统吸进的氧和消化系统吸收的营养物质先进入血液，再通过组织液进入体内细胞；同时，体内细胞新陈代谢产生的废物和二氧化碳，进入组织液，然后再进入血液而被运送到泌尿系统和呼吸系统排出体外的过程。The material exchange of biological organisms is that the oxygen inhaled by the respiratory system and the nutrients absorbed by the digestive system first enter the blood, and then enter the cells in the body through the tissue fluid; at the same time, the waste and carbon dioxide produced by the metabolism of the cells in the body enter the tissue fluid, and then enter the blood. The process of transporting to the urinary system and respiratory system to excrete the body.
血管-组织间的物质交换是生物体物质交换的重要的组成部分，主要通过血管内皮细胞空隙或带有隔膜的微孔(或凹洞)两种途径。质膜膜泡关联蛋白(Plasmalemmavesicle-associated protein，即PLVAP，简称PV-1)分子量约55-60kD，II型跨膜糖蛋白，是目前已知唯一的内皮血管微孔隔膜或质膜凹洞隔膜蛋白。PV-1除在内分泌腺体如脑垂体、肾上腺及肺组织中表达较高之外，其他组织中表达水平都较低或无表达。肿瘤、缺氧、外伤及炎症等异常状态并伴有血管新生的病理变化时，PV-1的表达则有显著上调。The material exchange between blood vessels and tissues is an important part of biological material exchange, mainly through the gap of vascular endothelial cells or micropores (or pits) with septum. Plasmalemmavesicle-associated protein (PLVAP, referred to as PV-1), a type II transmembrane glycoprotein with a molecular weight of about 55-60kD, is currently the only known endothelial vascular microporous diaphragm or plasma membrane cavity diaphragm protein. PV-1 has low or no expression in other tissues except endocrine glands such as pituitary gland, adrenal gland and lung tissue, which have high expression. In abnormal states such as tumor, hypoxia, trauma and inflammation accompanied by pathological changes of angiogenesis, the expression of PV-1 is significantly up-regulated.
生物体内存在诸多血管-组织屏障，在维持生物机体正常生理状态中具有重要意义，如血管-脑组织屏障(blood-brain barrier)和血管-视网膜屏障(blood-retinalbarrier)。血管-组织屏障中内皮血管管壁上无质膜微孔或凹洞，也无PV-1蛋白表达，但病理状态下，如缺血性脑中风、脑部原发性或转移性肿瘤、糖尿病视网膜病变等情况下，血管-组织屏障结构遭到破坏，内皮血管管壁出现有微孔并伴有PV-1抗原表达。There are many blood vessel-tissue barriers in organisms, which are of great significance in maintaining the normal physiological state of organisms, such as blood-brain barrier and blood-retinal barrier. There are no plasma membrane micropores or pits on the endothelial vascular wall in the vascular-tissue barrier, and there is no expression of PV-1 protein, but under pathological conditions, such as ischemic stroke, primary or metastatic brain tumors, and diabetes In conditions such as retinopathy, the vascular-tissue barrier structure is destroyed, and micropores appear in the endothelial vessel wall accompanied by the expression of PV-1 antigen.
综上所述，PV-1蛋白有望成为治疗年龄相关性黄斑变性(Age-related MacularDegeneration，AMD)、糖尿病黄斑水肿(Diabetic Macular Edema，DME)和成年患者视网膜静脉阻塞引起的黄斑水肿等疾病的新靶点。数据库检索可知，市场上治疗此类疾病的靶点有血管内皮生长因子(VEGF)，血管紧张素II(AngII)等，暂无企业布局PV-1靶点相关产品。In summary, PV-1 protein is expected to become a new therapeutic agent for age-related macular degeneration (Age-related Macular Degeneration, AMD), diabetic macular edema (DME) and macular edema caused by retinal vein occlusion in adult patients. target. According to the database search, the targets for the treatment of such diseases on the market include vascular endothelial growth factor (VEGF), angiotensin II (AngII), etc., and there is no company planning PV-1 target related products.
发明内容Contents of the invention
本发明的目的之一在于提供特异性结合PV-1蛋白的抗体或其抗原结合片段。One of the objectives of the present invention is to provide antibodies or antigen-binding fragments thereof that specifically bind to PV-1 protein.
本发明提供了特异性结合PV-1蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区和所述轻链可变区均由决定簇互补区和框架区组成；所述重链可变区和所述轻链可变区的决定簇互补区均由CDR1、CDR2和CDR3组成；The present invention provides an antibody specifically binding to PV-1 protein or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the The light chain variable regions are both composed of determinant complementary regions and framework regions; the heavy chain variable regions and the determinant complementary regions of the light chain variable regions are both composed of CDR1, CDR2 and CDR3;
所述重链可变区的CDR1的氨基酸序列如SEQ ID NO：03、SEQ ID NO：05、SEQ IDNO：06、SEQ ID NO：07、SEQ ID NO：08、SEQ ID NO：09、SEQ ID NO：10、SEQ ID NO：11、SEQ IDNO：12、SEQ ID NO：13或SEQ ID NO：14的第26-35位所示；The amino acid sequence of the CDR1 of the heavy chain variable region is as follows: SEQ ID NO: 03, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 shown in No. 26-35;
所述重链可变区的cDR2的氨基酸序列如SEQ ID NO：03、SEQ ID NO：05、SEQ IDNO：06、SEQ ID NO：07、SEQ ID NO：08、SEQ ID NO：09、SEQ ID NO：10、SEQ ID NO：11、SEQ IDNO：12、SEQ ID NO：13或SEQ ID NO：14的第50-66位所示；The amino acid sequence of cDR2 of the heavy chain variable region is as follows: SEQ ID NO: 03, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 shown in the 50-66 position;
所述重链可变区的CDR3的氨基酸序列如SEQ ID NO：03、SEQ ID NO：05、SEQ IDNO：06、SEQ ID NO：07、SEQ ID NO：08、SEQ ID NO：09、SEQ ID NO：10、SEQ ID NO：11、SEQ IDNO：12、SEQ ID NO：13或SEQ ID NO：14的第97-101位所示；The amino acid sequence of the CDR3 of the heavy chain variable region is as follows: SEQ ID NO: 03, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID No. 97-101 of NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14;
所述轻链可变区的CDR1的氨基酸序列如SEQ ID NO：04、SEQ ID NO：15、SEQ IDNO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ IDNO：22、SEQ ID NO：23、SEQ ID NO：24或SEQ ID NO：25的第24-34位所示；The amino acid sequence of CDR1 of the light chain variable region is as follows: SEQ ID NO: 04, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 shown in the 24th-34th position;
所述轻链可变区的CDR2的氨基酸序列如SEQ ID NO：04、SEQ ID NO：15、SEQ IDNO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ IDNO：22、SEQ ID NO：23、SEQ ID NO：24或SEQ ID NO：25的第50-56位所示；The amino acid sequence of the CDR2 of the light chain variable region is as follows: SEQ ID NO: 04, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 shown in the 50th-56th position;
所述轻链可变区的CDR3的氨基酸序列如SEQ ID NO：04、SEQ ID NO：15、SEQ IDNO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ IDNO：22、SEQ ID NO：23、SEQ ID NO：24或SEQ ID NO：25的第89-97位所示。The amino acid sequence of the CDR3 of the light chain variable region is as follows: SEQ ID NO: 04, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or 89-97 of SEQ ID NO: 25.
上述抗体可为全长抗体，上述抗原结合片段可为Fab片段、Fv片段、Fab′片段、F(ab′)2片段、单链抗体(ScFv)、纳米抗体(单域抗体)、双特异性抗体或最小识别单位(MRU)。The above-mentioned antibodies can be full-length antibodies, and the above-mentioned antigen-binding fragments can be Fab fragments, Fv fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific Antibody or minimal recognition unit (MRU).
可选地，根据上述的抗体或其抗原结合片段，所述重链可变区的氨基酸序列如SEQID NO：03、SEQ ID NO：05、SEQ ID NO：06、SEQ ID NO：07、SEQ ID NO：08、SEQ ID NO：09、SEQID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13或SEQ ID NO：14所示。Optionally, according to the above-mentioned antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is such as SEQ ID NO: 03, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID Shown in NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
可选地，根据上述的抗体或其抗原结合片段，所述轻链可变区的氨基酸序列如SEQID NO：04、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24或SEQ ID NO：25所示。Optionally, according to the above-mentioned antibody or antigen-binding fragment thereof, the amino acid sequence of the light chain variable region is such as SEQ ID NO: 04, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.
可选地，上述抗体或其抗原结合片段为如下任一种：Optionally, the above-mentioned antibody or antigen-binding fragment thereof is any of the following:
名称为HLPV-P01的抗体，所述HLPV-P01重链可变区的氨基酸序列如SEQ ID NO：03所示，轻链可变区的氨基酸序列如SEQ ID NO：04所示；The antibody named HLPV-P01, the amino acid sequence of the HLPV-P01 heavy chain variable region is shown in SEQ ID NO: 03, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 04;
名称为HLPV-P02的抗体，所述HLPV-P02重链可变区的氨基酸序列如SEQ ID NO：05所示，轻链可变区的氨基酸序列如SEQ ID NO：15所示；The antibody named HLPV-P02, the amino acid sequence of the HLPV-P02 heavy chain variable region is shown in SEQ ID NO: 05, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15;
名称为HLPV-P03的抗体，所述抗体HLPV-P03重链可变区的氨基酸序列如SEQ IDNO：06所示，轻链可变区的氨基酸序列如SEQ ID NO：16所示；The antibody named HLPV-P03, the amino acid sequence of the heavy chain variable region of the antibody HLPV-P03 is shown in SEQ ID NO: 06, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16;
名称为HLPV-P04的抗体，所述HLPV-P04重链可变区的氨基酸序列如SEQ ID NO：07所示，轻链可变区的氨基酸序列如SEQ ID NO：17所示；The antibody named HLPV-P04, the amino acid sequence of the HLPV-P04 heavy chain variable region is shown in SEQ ID NO: 07, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17;
名称为HLPV-P05的抗体，所述HLPV-P05重链可变区的氨基酸序列如SEQ ID NO：08所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示；The antibody named HLPV-P05, the amino acid sequence of the HLPV-P05 heavy chain variable region is shown in SEQ ID NO: 08, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18;
名称为HLPV-P06的抗体，所述HLPV-P06重链可变区的氨基酸序列如SEQ ID NO：09所示，轻链可变区的氨基酸序列如SEQ ID NO：16所示；The antibody named HLPV-P06, the amino acid sequence of the HLPV-P06 heavy chain variable region is shown in SEQ ID NO: 09, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16;
名称为HLPV-P07的抗体，所述HLPV-P07重链可变区的氨基酸序列如SEQ ID NO：10所示，轻链可变区的氨基酸序列如SEQ ID NO：19所示；The antibody named HLPV-P07, the amino acid sequence of the HLPV-P07 heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19;
名称为HLPV-P08的抗体，所述HLPV-P08重链可变区的氨基酸序列如SEQ ID NO：11所示，轻链可变区的氨基酸序列如SEQ ID NO：20所示；The antibody named HLPV-P08, the amino acid sequence of the HLPV-P08 heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20;
名称为HLPV-P09的抗体，所述HLPV-P09重链可变区的氨基酸序列如SEQ ID NO：11所示，轻链可变区的氨基酸序列如SEQ ID NO：21所示；The antibody named HLPV-P09, the amino acid sequence of the HLPV-P09 heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21;
名称为HLPV-P10的抗体，所述HLPV-P10重链可变区的氨基酸序列如SEQ ID NO：08所示，轻链可变区的氨基酸序列如SEQ ID NO：22所示；The antibody named HLPV-P10, the amino acid sequence of the HLPV-P10 heavy chain variable region is shown in SEQ ID NO: 08, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22;
名称为HLPV-P11的抗体，所述HLPV-P11重链可变区的氨基酸序列如SEQ ID NO：11所示，轻链可变区的氨基酸序列如SEQ ID NO：23所示；The antibody named HLPV-P11, the amino acid sequence of the HLPV-P11 heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23;
名称为HLPV-P12的抗体，所述HLPV-P12重链可变区的氨基酸序列如SEQ ID NO：12所示，轻链可变区的氨基酸序列如SEQ ID NO：24所示；The antibody named HLPV-P12, the amino acid sequence of the HLPV-P12 heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 24;
名称为HLPV-P13的抗体，所述HLPV-P13重链可变区的氨基酸序列如SEQ ID NO：13所示，轻链可变区的氨基酸序列如SEQ ID NO：25所示；The antibody named HLPV-P13, the amino acid sequence of the HLPV-P13 heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25;
名称为HLPV-P14的抗体，所述HLPV-P14重链可变区的氨基酸序列如SEQ ID NO：14所示，轻链可变区的氨基酸序列如SEQ ID NO：25所示。The antibody named HLPV-P14, the amino acid sequence of the HLPV-P14 heavy chain variable region is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25.
上述抗体亚型可为人源抗体，如IgA，IgD，IgE，IgG1，IgG2，IgG3，IgG4，IgM型抗体等；上述抗体亚型又如为去除ADCC效应的IgG1(N297A)和IgG4(S228P)。The above-mentioned antibody subtypes can be human antibodies, such as IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM antibodies, etc.; the above-mentioned antibody subtypes are IgG1 (N297A) and IgG4 (S228P) that remove the ADCC effect.
可选地，根据上述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包括重链恒定区和轻链恒定区。所述重链恒定区为IgG1的重链恒定区、IgG1变体的重链恒定区、IgG4的重链恒定区或IgG4变体的重链恒定区。所述轻链恒定区为Kappa或其变体的轻链恒定区。所述抗体具体可为重链可变区碳端(C端)融合重链恒定区，轻链可变区碳端(C端)融合轻链恒定区。Optionally, according to the above-mentioned antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region is an IgG1 heavy chain constant region, an IgG1 variant heavy chain constant region, an IgG4 heavy chain constant region or an IgG4 variant heavy chain constant region. The light chain constant region is that of Kappa or a variant thereof. Specifically, the antibody can be a heavy chain variable region carbon-terminal (C-terminal) fused with a heavy chain constant region, and a light-chain variable region carbon-terminal (C-terminal) fused with a light chain constant region.
IgG1的重链恒定区的氨基酸序列如UniProtKB数据库序列号P01857.1(2022.02.23最新更新)所示，IgG1变体的重链恒定区的氨基酸序列为IgG1的重链恒定区引入N297A突变(EU编号)获得。IgG4变体的重链恒定区的氨基酸序列如UniProtKB数据库序列号为P01861.1(2022.02.23最新更新)的IgG4引入S228P(EU编号)氨基酸突变获得。Kappa的轻链恒定区的氨基酸序列如UniProtKB数据库序列号P01834.2(2022.02.23最新更新)所示。The amino acid sequence of the heavy chain constant region of IgG1 is shown in the UniProtKB database sequence number P01857.1 (2022.02.23 latest update), and the amino acid sequence of the heavy chain constant region of the IgG1 variant is that the heavy chain constant region of IgG1 introduces the N297A mutation (EU number) to obtain. The amino acid sequence of the heavy chain constant region of the IgG4 variant was obtained by introducing the S228P (EU numbering) amino acid mutation into IgG4 with the sequence number P01861.1 (the latest update on 2022.02.23) in the UniProtKB database. The amino acid sequence of the light chain constant region of Kappa is shown in the UniProtKB database sequence number P01834.2 (2022.02.23 latest update).
编码重链恒定区的基因核苷酸序列可为SEQ ID No.28第409-1398或SEQ IDNo.29的第409-1389位所示。编码轻链恒定区的基因核苷酸序列可为SEQ ID No.30的第394-714位所示。The nucleotide sequence of the gene encoding the constant region of the heavy chain can be as shown in the 409-1398 of SEQ ID No. 28 or the 409-1389 of SEQ ID No. 29. The nucleotide sequence of the gene encoding the constant region of the light chain can be shown in positions 394-714 of SEQ ID No.30.
本发明还提供了上述抗体或其抗原结合片段的相关生物材料，所述相关生物材料为如下任一种：The present invention also provides related biological materials of the above antibodies or antigen-binding fragments thereof, wherein the related biological materials are any of the following:
B1)编码上述抗体或其抗原结合片段的核酸分子；B1) a nucleic acid molecule encoding the above antibody or an antigen-binding fragment thereof;
B2)编码上述抗体或其抗原结合片段的重链和/或轻链的核酸分子；B2) a nucleic acid molecule encoding the heavy chain and/or light chain of the above antibody or an antigen-binding fragment thereof;
B3)编码上述抗体或其抗原结合片段的重链可变区和/或轻链可变区的核酸分子；B3) a nucleic acid molecule encoding the heavy chain variable region and/or light chain variable region of the above antibody or an antigen-binding fragment thereof;
B4)含有B1)-B3)任一所述核酸分子的表达盒；B4) an expression cassette containing any one of the nucleic acid molecules of B1)-B3);
B5)含有B1)-B3)任一所述核酸分子的重组载体、或含有B4)所述表达盒的重组载体；B5) A recombinant vector containing any of the nucleic acid molecules described in B1)-B3), or a recombinant vector containing the expression cassette described in B4);
B6)含有B1)-B3)任一所述核酸分子的重组微生物、或含有B4)所述表达盒的重组微生物、或含有B5)所述重组载体的重组微生物；B6) A recombinant microorganism containing any of the nucleic acid molecules described in B1)-B3), or a recombinant microorganism containing an expression cassette described in B4), or a recombinant microorganism containing a recombinant vector described in B5);
B7)含有B1)-B3)任一所述核酸分子的细胞系、或含有B4)所述表达盒的细胞系、或含有B5)所述重组载体的细胞系。B7) A cell line containing any of the nucleic acid molecules described in B1)-B3), or a cell line containing the expression cassette described in B4), or a cell line containing the recombinant vector described in B5).
可选地，根据上述的相关生物材料，Optionally, according to the relevant biological material mentioned above,
B2)编码上述抗体或其抗原结合片段的重链的核酸分子为核苷酸序列如SEQ IDNo.28或SEQ ID No.29所示的核酸分子；B2) The nucleic acid molecule encoding the heavy chain of the above-mentioned antibody or antigen-binding fragment thereof is a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID No.28 or SEQ ID No.29;
B2)编码上述抗体或其抗原结合片段的轻链的核酸分子为核苷酸序列如SEQ IDNo.30所示的核酸分子；B2) The nucleic acid molecule encoding the light chain of the above-mentioned antibody or antigen-binding fragment thereof is a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID No.30;
B3)编码上述抗体或其抗原结合片段的重链可变区的核酸分子为核苷酸序列如SEQ ID No.26所示的核酸分子，其中CDR1，CDR2和CDR3分别为对应的76-105位，148-198位，289-303位碱基；B3) The nucleic acid molecule encoding the heavy chain variable region of the above-mentioned antibody or its antigen-binding fragment is a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID No.26, wherein CDR1, CDR2 and CDR3 are corresponding to positions 76-105, respectively , 148-198, 289-303 bases;
B3)编码上述抗体或其抗原结合片段的轻链可变区的核酸分子为核苷酸序列如SEQ ID No.27所示的核酸分子，其中，CDR1，CDR2和CDR3分别为对应的70-102位，148-168位和278-291位碱基。B3) The nucleic acid molecule encoding the light chain variable region of the above-mentioned antibody or its antigen-binding fragment is a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID No.27, wherein CDR1, CDR2 and CDR3 are respectively corresponding to 70-102 148-168 and 278-291 bases.
本文所述载体是本领域技术人员公知的，包括但不限于：质粒、噬菌体(如λ噬菌体或M13丝状噬菌体等)、黏粒(即柯斯质粒)、病毒载体(如杆状病毒载体、逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒或疱疹病毒(如单纯疱疹病毒)等)。在本发明的一个实施例中，载体具体可为pUC57或pCGS3。具体可为下述实施例制备的pCGS3-HLPV-P00至P14重组表达质粒、pCGS3-HLPV-P03(S228P)、pCGS3-HLPV-P05(S228P)、pCGS3-HLPV-P08(S228P)、pCGS3-HLPV-P09(S228P)、pCGS3-HLPV-P10(S228P)、pCGS3-HLPV-P11(S228P)、pCGS3-HLPV-P14(S228P)、pCGS3-HLPV-P03(N297A)、pCGS3-HLPV-P05(N297A)、pCGS3-HLPV-P08(N297A)、pCGS3-HLPV-P09(N297A)、pCGS3-HLPV-P10(N297A)、pCGS3-HLPV-P11(N297A)或pCGS3-HLPV-P14(N297A)。The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, phages (such as lambda phages or M13 filamentous phages, etc.), cosmids (ie Cosmids), viral vectors (such as baculovirus vectors, Retrovirus (including lentivirus), adenovirus, adeno-associated virus or herpes virus (such as herpes simplex virus, etc.). In one embodiment of the present invention, the vector can specifically be pUC57 or pCGS3. Specifically, it can be the recombinant expression plasmids from pCGS3-HLPV-P00 to P14 prepared in the following examples, pCGS3-HLPV-P03(S228P), pCGS3-HLPV-P05(S228P), pCGS3-HLPV-P08(S228P), pCGS3-HLPV -P09(S228P), pCGS3-HLPV-P10(S228P), pCGS3-HLPV-P11(S228P), pCGS3-HLPV-P14(S228P), pCGS3-HLPV-P03(N297A), pCGS3-HLPV-P05(N297A) , pCGS3-HLPV-P08(N297A), pCGS3-HLPV-P09(N297A), pCGS3-HLPV-P10(N297A), pCGS3-HLPV-P11(N297A) or pCGS3-HLPV-P14(N297A).
本文所述微生物可为酵母、细菌或真菌。其中，细菌可来自埃希氏菌属(Escherichia)、欧文氏菌(Erwinia)、根癌农杆菌属(Agrobacterium)、黄杆菌属(Flavobacterium)、产碱菌属(Alcaligenes)、假单胞菌属(Pseudomonas)、芽胞杆菌属(Bacillus)等；酵母可为毕赤酵母(P.pastoris)。The microorganisms described herein may be yeast, bacteria or fungi. Among them, the bacteria can be from the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc.; the yeast can be Pichia pastoris (P. pastoris).
所述细胞系(宿主细胞)是指可用于导入载体的细胞，其包括但不限于：真核细胞(如酵母细胞、曲霉菌)、动物细胞(如哺乳动物细胞、昆虫细胞)或原核细胞。在本发明的一个实施例中，所述细胞系具体可为ExpiCHO-S细胞。The cell line (host cell) refers to a cell that can be used to introduce the vector, including but not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells) or prokaryotic cells. In one embodiment of the present invention, the cell line may specifically be ExpiCHO-S cells.
术语“细胞”和“细胞系”可互换使用，并且所有这类名称都包括其后代。The terms "cell" and "cell line" are used interchangeably and all such designations include progeny.
本发明还提供了上述抗体或其抗原结合片段的制备方法，所述方法具体包括：将上述抗体或其抗原结合片段的编码基因通过含有所述编码基因的重组表达载体导入宿主细胞中，得重组细胞，培养所述重组细胞上清，通过亲和层析纯化获得目的蛋白。The present invention also provides a method for preparing the above-mentioned antibody or its antigen-binding fragment, which specifically includes: introducing the coding gene of the above-mentioned antibody or its antigen-binding fragment into a host cell through a recombinant expression vector containing the coding gene to obtain a recombinant cells, culturing the supernatant of the recombinant cells, and purifying by affinity chromatography to obtain the target protein.
上述方法中，所述宿主细胞可为真核细胞，如CHO细胞，HEK293细胞，酵母细胞或昆虫细胞等。在本发明的一个实施例中，所述动物细胞为CHO细胞。In the above method, the host cells can be eukaryotic cells, such as CHO cells, HEK293 cells, yeast cells or insect cells. In one embodiment of the present invention, the animal cells are CHO cells.
本发明还提供了改善、预防或治疗PV-1过表达相关疾病的药物组合物，所述药物组合物包含上述抗体或其抗原结合片段，以及一种或多种药学上可接受的载体。The present invention also provides a pharmaceutical composition for improving, preventing or treating diseases related to PV-1 overexpression, said pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers.
所述药学上可接受的载体可为稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂但不限于此。The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant, but is not limited thereto.
本发明还提供了用于检测PV-1的试剂或试剂盒，所述试剂或试剂盒含有上述任一所述的抗体或其抗原结合片段。The present invention also provides a reagent or a kit for detecting PV-1, which contains any of the above-mentioned antibodies or antigen-binding fragments thereof.
本发明还提供了缀合物(coniugate)，所述缀合物包含本发明所述的抗体或其抗原结合片段，以及与所述抗体或其抗原结合片段连接的可检测的标记；具体地，所述可检测的标记可选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。The present invention also provides a conjugate (coniugate), said conjugate comprising the antibody or antigen-binding fragment thereof of the present invention, and a detectable label linked to said antibody or antigen-binding fragment thereof; specifically, The detectable label can be selected from enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), Fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.
上述抗体或其抗原结合片段，和/或，上述的生物材料的下述任一种应用也属于本发明的保护范围之内：Any of the following applications of the above-mentioned antibodies or antigen-binding fragments thereof, and/or the above-mentioned biological materials also fall within the protection scope of the present invention:
D1)在制备用于改善、预防或治疗PV-1过表达相关疾病的药物中的应用；D1) Application in the preparation of drugs for improving, preventing or treating diseases related to PV-1 overexpression;
D2)在制备用于诊断或筛查PV-1过表达相关疾病的产品中的应用；D2) Application in the preparation of products for diagnosing or screening PV-1 overexpression-related diseases;
D3)在检测PV-1中的应用；D3) application in detecting PV-1;
D4)在制备用于检测PV-1的产品中的应用；D4) Application in preparing products for detecting PV-1;
D5)在制备用于结合PV-1蛋白的产品中的应用。D5) Use in the preparation of products for binding PV-1 protein.
所述PV-1过表达相关疾病可包括年龄相关性黄斑变性、糖尿病黄斑水肿、成年患者视网膜静脉阻塞引起的黄斑水肿、肝癌、缺血性脑中风和/或脑水肿。The PV-1 overexpression-related diseases may include age-related macular degeneration, diabetic macular edema, macular edema caused by retinal vein occlusion in adult patients, liver cancer, ischemic stroke and/or cerebral edema.
所述用于检测PV-1的产品包括利用酶联免疫吸附法、免疫荧光检测法、放射免疫法、发光免疫测定法、胶体金免疫层析法、凝集法或免疫比浊法等检测抗原抗体结合的产品。The products for detecting PV-1 include detecting antigen and antibody by enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination or immunoturbidimetry. combined products.
进一步地，所述产品可为试剂、试剂盒或芯片。Further, the product can be a reagent, kit or chip.
本发明所述药物、试剂、试剂盒或芯片含有本文任一所述抗体或其抗原结合片段或其组合。所述试剂盒可为化学发光免疫试剂盒、酶联免疫检测试剂盒、胶体金免疫试剂盒、或荧光免疫试剂盒，但不限于此。The medicament, reagent, kit or chip of the present invention contains any of the antibodies or antigen-binding fragments or combinations thereof described herein. The kit can be a chemiluminescent immunoassay kit, an enzyme-linked immunoassay kit, a colloidal gold immunoassay kit, or a fluorescent immunoassay kit, but is not limited thereto.
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。所述抗原结合片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，所述抗原结合片段保留至少10％的母体结合活性。具体地，所述抗原结合片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which typically include at least part of the antigen-binding or variable region (eg, one or more CDRs) of the parental antibody. The antigen-binding fragments retain at least some of the binding specificity of the parent antibody. Typically, the antigen-binding fragment retains at least 10% of the parent-binding activity when the activity is expressed on a molar basis. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parental antibody for the target.
术语“Fab片段”是由重链Fd和完整轻链通过二硫键结合而成的异二聚体，仅含一个抗原结合位点。将重链Fd和完整轻链的编码基因连接，融合细菌蛋白信号肽基因后可在大肠杆菌内分泌表达Fab抗体(Fab片段)，并有完整的立体折叠和链内、链间二硫键。所述重链Fd指Fab中约1/2的H链部分(约含225个氨基酸残基，包括VH、CH1和部分铰链区)。The term "Fab fragment" is a heterodimer composed of the heavy chain Fd and the complete light chain through disulfide bonds, and contains only one antigen-binding site. The heavy chain Fd and the complete light chain coding gene are connected, and the Fab antibody (Fab fragment) can be secreted and expressed in Escherichia coli after the bacterial protein signal peptide gene is fused, and there are complete three-dimensional folding and intra-chain and inter-chain disulfide bonds. The heavy chain Fd refers to about 1/2 of the H chain in the Fab (about 225 amino acid residues, including VH, CH1 and part of the hinge region).
术语“Fv片段”是指可分别构建含有VH和VL基因的载体，共转染细胞，使之分别表达，再组装成功能性Fv抗体；也可在载体中的VH和VL之间设置终止密码，分别表达两个小分子蛋白片段，再通过非共价键结合而形成Fv抗体(Fv片段)。The term "Fv fragment" means that vectors containing VH and VL genes can be constructed separately, co-transfected into cells, expressed separately, and then assembled into functional Fv antibodies; a stop codon can also be set between VH and VL in the vector , respectively express two small molecular protein fragments, and then form Fv antibodies (Fv fragments) through non-covalent bonding.
术语“Fab′片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′)2分子。The term "Fab' fragment" contains one light chain and part of one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby the two heavy chains of the two Fab' fragments Interchain disulfide bonds are formed between them to form F(ab')2 molecules.
术语“F(ab′)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab′)2片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。The term "F(ab')2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
术语“单链抗体(ScFv)”是指用适当的寡核苷酸接头(1inker)连接轻链和重链可变区基因，使之表达单一的多肽链，称为单链抗体(ScFv)。多肽链能自发折叠成天然构象，保持Fv的特异性和亲和力。The term "single-chain antibody (ScFv)" refers to linking the light chain and heavy chain variable region genes with an appropriate oligonucleotide linker (linker) to express a single polypeptide chain, which is called a single-chain antibody (ScFv). The polypeptide chain can spontaneously fold into a natural conformation, maintaining the specificity and affinity of Fv.
术语“纳米抗体(单域抗体)”是指将抗体重链V区通过基因工程方法表达，获得仅含VH片段的抗体。单域抗体与抗原结合的能力及其稳定性，与完全抗体基本一致。The term "nanobody (single-domain antibody)" refers to expressing the V region of the heavy chain of an antibody through genetic engineering to obtain an antibody containing only a VH fragment. The ability and stability of single-domain antibodies to bind to antigens are basically the same as those of complete antibodies.
术语“双特异性抗体”是指将两套轻链、重链基因导入骨髓瘤细胞中，选择合适的抗体恒定区及Ig类型，可获得产量大、均一性和纯度高的双特异性抗体。另外，以化学交联技术或杂交-杂交瘤技术也可获得双特异性抗体。The term "bispecific antibody" refers to the introduction of two sets of light chain and heavy chain genes into myeloma cells, and the selection of appropriate antibody constant regions and Ig types can obtain bispecific antibodies with high yield, high uniformity and purity. In addition, bispecific antibodies can also be obtained by chemical cross-linking technology or hybrid-hybridoma technology.
术语“最小识别单位(MRU)”是指仅含可变区中单一CDR结构，分子质量仅为完整抗体的1％左右，可结合相应抗原。The term "Minimum Recognition Unit (MRU)" refers to a structure containing only a single CDR in the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。本领域技术人员可根据需要选择本领域常规的宿主细胞、表达载体、将表达载体导入宿主细胞的方法以及抗体的分离纯化方法。The antibodies of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. Insert the obtained DNA molecule into the expression vector, then transfect the host cell, culture the transfected host cell under specific conditions, and express the antibody of the present invention. Those skilled in the art can select conventional host cells, expression vectors, methods for introducing expression vectors into host cells, and antibody isolation and purification methods in the art as needed.
本领域技术人员熟知，所述抗原结合片段可以通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies, as is well known to those skilled in the art.
本发明实施例通过两次噬菌体筛选实验，获得了一组特异结合人质膜膜泡关联蛋白PV-1亲和力成熟抗体，亲和力比原始抗体提高1-2个数量级，理论可降低用药剂量，治疗年龄相关性黄斑变性(AMD)和糖尿病性黄斑水肿(DME)等PV-1过表达相关疾病，加速PV-1新靶点的临床药物开发。In the embodiment of the present invention, through two phage screening experiments, a group of affinity-matured antibodies specifically binding to human plasma membrane vesicle-associated protein PV-1 was obtained, and the affinity was 1-2 orders of magnitude higher than that of the original antibody. Accelerate the development of clinical drugs for new PV-1 targets.
附图说明Description of drawings
图1噬菌体展示序列IgG1全抗组装表达质粒构建核酸电泳鉴定图；上图为IgG1全抗组装表达质粒轻链构建核酸电泳鉴定图，采用HindIII和XhoI双酶切，酶切后理论获得载体片段9535bp和轻链723bp，其中泳道A为pCGS3-HLPV-P00L，B为pCGS3-HLPV-P01L，C为pCGS3-HLPV-P02L，D为pCGS3-HLPV-P03/06L和，E为pCGS3-HLPV-P04L，F为pCGS3-HLPV-P05L，G为pCGS3-HLPV-P07L，H为pCGS3-HLPV-P08L，I为pCGS3-HLPV-P09L，J为pCGS3-HLPV-P10L，K为pCGS3-HLPV-P11L，L为pCGS3-HLPV-P12L，M为pCGS3-HLPV-P13/14L；下图为IgG1全抗组装表达质粒重链构建酸电泳鉴定图，采用BstBI和PacI双酶切，酶切后理论获得载体片段10200bp和轻链1409bp，其中泳道编号依次对应抗体标号，即泳道00为pCGS3-HLPV-P00，泳道01为pCGS3-HLPV-P00，泳道02为pCGS3-HLPV-P02...，泳道14为pCGS3-HLPV-P14表达质粒。Figure 1 Phage Display Sequence IgG1 Antibody Assembly Expression Plasmid Construction Nucleic Acid Electrophoresis Identification Diagram; The upper picture is the IgG1 Antibody Assembly Expression Plasmid Light Chain Construction Nucleic Acid Electrophoresis Identification Diagram. HindIII and XhoI double enzyme digestion was used to obtain a vector fragment of 9535bp theoretically. and light chain 723bp, wherein lane A is pCGS3-HLPV-P00L, B is pCGS3-HLPV-P01L, C is pCGS3-HLPV-P02L, D is pCGS3-HLPV-P03/06L and, E is pCGS3-HLPV-P04L, F is pCGS3-HLPV-P05L, G is pCGS3-HLPV-P07L, H is pCGS3-HLPV-P08L, I is pCGS3-HLPV-P09L, J is pCGS3-HLPV-P10L, K is pCGS3-HLPV-P11L, L is pCGS3-HLPV-P12L, M is pCGS3-HLPV-P13/14L; the figure below is the acid electrophoresis identification map of IgG1 full-antibody assembly expression plasmid heavy chain construction, using BstBI and PacI double enzyme digestion, theoretically obtained vector fragment 10200bp and The light chain is 1409bp, and the lane numbers correspond to the antibody labels in turn, that is,
图2噬菌体展示序列IgG1组装全抗蛋白SDS-PAGE蛋白电泳图；野生型IgG1组装全抗蛋白纯化后SDS-PAGE鉴定(非还原为非还原SDS-PAGE，还原为还原SDS-PAGE)，其中泳道编号依次对应抗体标号，即泳道00为HLPV-P00，泳道01为HLPV-P01，泳道02-HLPV-P02...泳道14为HLPV-P14抗体。Fig. 2 SDS-PAGE protein electrophoresis diagram of phage display sequence IgG1 assembled anti-protein; SDS-PAGE identification after purification of wild-type IgG1 assembled anti-protein (non-reduced to non-reduced SDS-PAGE, reduced to reduced SDS-PAGE), in which lane The numbers correspond to the antibody labels in turn, that is,
图3去除ADCC效应全抗组装表达质粒构建核酸电泳鉴定图；构建仅仅改变重链碱基序列，采用BstBI和PacI双酶切，第1至7泳道酶切后理论获得载体片段10200bp和轻链1409bp，第8至14泳道酶切后理论获得载体片段10200bp和轻链1400bp。其中，1为pCGS3-HLPV-P03(N297A)，2为pCGS3-HLPV-P05(N297A)，3为pCGS3-HLPV-P08(N297A)，4为pCGS3-HLPV-P09(N297A)，5为pCGS3-HLPV-P10(N297A)，6为pCGS3-HLPV-P11(N297A)，7为pCGS3-HLPV-P14(N297A)，8为pCGS3-HLPV-P03(S228P)，9为pCGS3-HLPV-P05(S228P)，10为pCGS3-HLPV-P08(S228P)，11为pCGS3-HLPV-P09(S228P)，12为pCGS3-HLPV-P10(S228P)，13为pCGS3-HLPV-P11(S228P)，14为pCGS3-HLPV-P14(S228P)重组表达质粒。Figure 3 Elimination of the ADCC effect of all-antibody assembly expression plasmid construction of nucleic acid electrophoresis identification; the construction only changes the base sequence of the heavy chain, and is digested with BstBI and PacI, and theoretically obtains a vector fragment of 10200bp and a light chain of 1409bp after digesting
图4去除ADCC效应组装全抗纯化SDS-PAGE蛋白电泳图；上图为IgG1(N297)型去除ADCC效应抗体，下图为IgG4(S228P)型去除ADCC效应抗体SDS-PAGE蛋白电泳图，其中AN表示纯化废液非还原SDS-PAGE，BN表示纯化蛋白非还原SDS-PAGE，AR表示纯化废液还原SDS-PAGE，BR表示纯化蛋白还原SDS-PAGE。其中泳道编号依次对应抗体标号，即泳道03为HLPV-P03，泳道05为HLPV-P05，泳道08为HLPV-P08，泳道09为HLPV-P09，泳道10为HLPV-P10，泳道11为HLPV-P11，泳道14为HLPV-P14抗体。Fig. 4 SDS-PAGE protein electrophoresis of purified antibody assembled with ADCC effect removed; the upper picture is the IgG1 (N297) type antibody with ADCC effect removed, and the lower picture is the IgG4 (S228P) type ADCC effect antibody removed SDS-PAGE protein electrophoresis picture, where AN Represents non-reducing SDS-PAGE of purified waste liquid, BN represents non-reducing SDS-PAGE of purified protein, AR represents reduced SDS-PAGE of purified waste liquid, BR represents reduced SDS-PAGE of purified protein. The lane numbers correspond to the antibody labels in turn, that is,
具体实施方式Detailed ways
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
下述实施例中的主要试剂及其厂家信息如下：The main reagents and their manufacturer information in the following examples are as follows:
ExpiCHO-STM Cells：Thermo公司；ExpiCHO-S™ Cells: Thermo Corporation;
ExpiCHOTM Expression Medium：Thermo公司；ExpiCHO™ Expression Medium: Thermo Corporation;
ExpiFectamineTM CHO Transfection Kit：Thermo公司；ExpiFectamineTM CHO Transfection Kit: Thermo Company;
pCGS3表达载体：Merck公司；pCGS3 expression vector: Merck Company;
重组人PLVAP/PV-1蛋白：Abcam公司；Recombinant human PLVAP/PV-1 protein: Abcam Company;
Protein A预装色谱柱：生工生物工程(上海)股份有限公司；Protein A prepacked column: Sangon Bioengineering (Shanghai) Co., Ltd.;
Centrifugal Filters
Centrifugal Filters 0.5ml 10K
PBS pH7.4(1×)：Thermo公司；PBS pH7.4 (1×): Thermo Company;
Sure PAGETM，Bis-Tris，10x8，4-12％，12wells：南京金斯瑞生物科技有限公司。Sure PAGETM , Bis-Tris, 10x8, 4-12%, 12wells: Nanjing GenScript Biotechnology Co., Ltd.
下述实施例中的关键仪器及其厂家信息如下：Key instruments and manufacturer information thereof in the following examples are as follows:
凝胶成像系统：Protein Simple公司；Gel imaging system: Protein Simple;
eStainTML1蛋白染色仪：南京金斯瑞生物科技有限公司；eStainTM L1 protein staining instrument: Nanjing GenScript Biotechnology Co., Ltd.;
Biacore T200分子间相互作用分析系统：GE公司。Biacore T200 intermolecular interaction analysis system: GE Company.
实施例1、亲和力成熟实验
本实施例在原始抗体(该原始抗体命名为HLPV-P00，重链可变区如SEQ ID NO：01所示，轻链可变区如SEQ ID NO：02所示)基础上，通过噬菌体展示进行亲和力成熟筛选。噬菌体展示突变采用四个组合方式，进行单点或双点饱和突变，设计突变文库。理论库容大小的计算方式为CDR1(氨基酸个数)×20(每个位点有20种可能性)×CDR2(氨基酸个数)×20×CDR3(氨基酸个数)×20。以01号库容为例，库容为9×20×11×20×9×20＝3.96×106(表1)。In this example, on the basis of the original antibody (the original antibody is named HLPV-P00, the variable region of the heavy chain is shown in SEQ ID NO: 01, and the variable region of the light chain is shown in SEQ ID NO: 02), through phage display Perform an affinity maturation screen. Phage display mutagenesis uses four combinations to perform single-point or double-point saturation mutation and design a mutation library. The calculation method of the theoretical storage capacity is CDR1 (number of amino acids) × 20 (each site has 20 possibilities) × CDR2 (number of amino acids) × 20 × CDR3 (number of amino acids) × 20. Taking No. 01 storage capacity as an example, the storage capacity is 9×20×11×20×9×20=3.96×106 (Table 1).
表1：抗体亲和力成熟实验库容统计表Table 1: Statistical Table of Storage Capacity of Antibody Affinity Maturation Experiment
第一次噬菌体筛选实验失败，抗体亲和力提高不足一个数量级。第一轮噬菌体筛选实验组装11个全长抗体，其中5个抗体的亲和力提高了2倍以上，亲和力最高的抗体提高4.8倍，亲和力最高的抗体命名为HLPV-P01，重链可变区如SEQ ID NO：03所示，轻链可变区如SEQ ID NO：04所示。The first phage screening experiment failed, and the increase in antibody affinity was less than an order of magnitude. The first round of phage screening experiments assembled 11 full-length antibodies, of which the affinity of 5 antibodies was increased by more than 2 times, and the antibody with the highest affinity was increased by 4.8 times. The antibody with the highest affinity was named HLPV-P01, and the variable region of the heavy chain is shown as SEQ ID NO: 03, and the light chain variable region is shown in SEQ ID NO: 04.
第二次噬菌体筛选实验在第一次实验获得的最佳抗体HLPV-P01基础上，再次通过表1所示设计的抗体文库，进行亲和力成熟筛选，获得亲和力成熟抗体名称依次为HLPV-P02至14，所述编码抗体可变区序列如表2。In the second phage screening experiment, on the basis of the best antibody HLPV-P01 obtained in the first experiment, the antibody library designed as shown in Table 1 was used for affinity maturation screening again, and the names of the affinity matured antibodies were HLPV-P02 to 14 in sequence , the sequence encoding the variable region of the antibody is shown in Table 2.
表2：亲和力成熟筛选抗体序列统计表Table 2: Statistical Table of Antibody Sequences for Affinity Maturation Screening
实施例2、组装全抗分子验证亲和力实验Example 2, assembly of whole antibody molecule verification affinity experiment
1重组表达质粒构建实验1 Recombinant expression plasmid construction experiment
根据实施例1表2亲和力成熟单抗重轻和轻链可变区序列，分别融合IgG1-Fc和κ链恒定区序列(重链可变区碳端融合IgG1-Fc，轻链可变区碳端融合κ恒定区)，密码子优化并委托生工生物合成，重链基因序列连接至pUC57克隆载体的限制性核酸内切酶SmaI单酶切位点之后，轻链基因序列连接至pUC57克隆载体的限制性核酸内切酶SmaI和HindIII的酶切位点之间，对应合成质粒名称如表3。According to Example 1 Table 2, the sequence of heavy light and light chain variable regions of affinity matured monoclonal antibody is respectively fused to IgG1-Fc and κ chain constant region sequences (the carbon end of the heavy chain variable region is fused to IgG1-Fc, and the carbon end of the light chain variable region is fused to Kappa constant region), codon optimization and commissioned by Sangon Biosynthesis, the heavy chain gene sequence was connected to the restriction endonuclease SmaI single enzyme cutting site of the pUC57 cloning vector, and the light chain gene sequence was connected to the pUC57 cloning vector Table 3 shows the names of the corresponding synthetic plasmids between the restriction endonucleases SmaI and HindIII.
表3：全抗表达组合重、轻链质粒统计表Table 3: Statistical table of heavy and light chain plasmids for full antibody expression combination
其中pUC57-00-L，pUC57-01-L，pUC57-02-L，pUC57-03/06-L，pUC57-04-L，pUC57-05-L，pUC57-07-L，pUC57-08-L，pUC57-09-L，pUC57-10-L，pUC57-11-L，pUC57-12-L和pUC57-13/14-L为轻链质粒，其含有完整的轻链基因，该轻链基因是分别将轻链可变区基因(序列如SEQ ID NO：02、SEQ ID NO：04、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQID NO：24和SEQ ID NO：25所示轻链的编码基因)的3’端与人Kappa恒定区(κ恒定区)轻链基因连接得到的融合基因。其中人Kappa恒定区(κ恒定区)轻链基因的核苷酸序列为SEQ IDNo.30的第394-714位所示，人Kappa恒定区(κ恒定区)轻链基因表达氨基酸序列如UniProtKB数据库序列号P01834.2(2022.02.23最新更新)所示的人Kappa恒定区。Where pUC57-00-L, pUC57-01-L, pUC57-02-L, pUC57-03/06-L, pUC57-04-L, pUC57-05-L, pUC57-07-L, pUC57-08-L , pUC57-09-L, pUC57-10-L, pUC57-11-L, pUC57-12-L and pUC57-13/14-L are light chain plasmids, which contain the complete light chain gene, which is The light chain variable region genes (sequences such as SEQ ID NO: 02, SEQ ID NO: 04, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19. The 3' end of the light chain coding gene shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25) and human Kappa constant region (κ constant region) fusion gene obtained by linking light chain genes. Wherein the nucleotide sequence of the light chain gene of the human Kappa constant region (κ constant region) is shown in the 394-714th positions of SEQ ID No.30, and the amino acid sequence of the light chain gene expression of the human Kappa constant region (κ constant region) is as shown in the UniProtKB database The human kappa constant region shown in the sequence number P01834.2 (2022.02.23 latest update).
其中pUC57-00-H，pUC57-01-H，pUC57-02-H，pUC57-03-H，pUC57-04-H，pUC57-05/10-H，pUC57-06-H，pUC57-07-H，pUC57-08/09/11-H，pUC57-12-H，pUC57-13-H和pUC57-14-H为重链质粒，其含有完整的重链基因，该重链基因是分别将重链可变区基因(SEQ ID NO：01、SEQ ID NO：03、SEQ ID NO：05、SEQ ID NO：06、SEQ ID NO：07、SEQ ID NO：08、SEQ IDNO：09、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14的编码基因)的3’端与人IgG1恒定区重链基因(IgG1-Fc)连接得到的融合基因。其中人IgG1恒定区重链基因的核苷酸序列为SEQ ID No.28的第409-1398位所示，人IgG1恒定区重链基因表达氨基酸序列如UniProtKB数据库序列号P01857.1(2022.02.23最新更新)所示的人IgG1恒定区。Where pUC57-00-H, pUC57-01-H, pUC57-02-H, pUC57-03-H, pUC57-04-H, pUC57-05/10-H, pUC57-06-H, pUC57-07-H , pUC57-08/09/11-H, pUC57-12-H, pUC57-13-H and pUC57-14-H are heavy chain plasmids, which contain the complete heavy chain gene, which is respectively the heavy chain Variable region genes (SEQ ID NO: 01, SEQ ID NO: 03, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10. The fusion obtained by linking the 3' end of the coding genes of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 to the human IgG1 constant region heavy chain gene (IgG1-Fc) Gene. The nucleotide sequence of the human IgG1 constant region heavy chain gene is shown in the 409-1398th position of SEQ ID No.28, and the human IgG1 constant region heavy chain gene expression amino acid sequence is as shown in the UniProtKB database sequence number P01857.1 (2022.02.23 The human IgG1 constant region indicated in the latest update).
上述重组表达质粒的构建方案采用为先构建轻链，轻链构建完成后，在此基础上按照表3不同组合方式构建重链，最后形成双表达框重组表达质粒，共计构建13个轻链中间质粒和15个全抗分子，具体步骤如下：The construction scheme of the above-mentioned recombinant expression plasmid adopts to construct the light chain first, after the construction of the light chain is completed, on this basis, construct the heavy chain according to the different combinations in Table 3, and finally form a recombinant expression plasmid with double expression cassettes, and construct 13 intermediate light chains in total. Plasmid and 15 full-antibody molecules, the specific steps are as follows:
第一步构建全抗分子轻链，操作步骤为：采用HindIII(NEB)和XhoI(NEB)双酶切pCGS3(Merck)载体，pUC57-00-L，pUC57-01-L，pUC57-02-L，pUC57-03/06-L，pUC57-04-L，pUC57-05-L，pUC57-07-L，pUC57-08-L，pUC57-09-L，pUC57-10-L，pUC57-11-L，pUC57-12-L和pUC57-13/14-L轻链质粒，获得9535bp载体片段和13条723bp轻链片段。利用T4连接酶(TAKARA)连接，转化，涂板，提取质粒，HindIII和XhoI双酶切鉴定，酶切条带符合预期。测序正确质粒分别命名为pCGS3-HLPV-P00L，pCGS3-HLPV-P01L，pCGS3-HLPV-P02L，pCGS3-HLPV-P03/06L，pCGS3-HLPV-P04L，pCGS3-HLPV-P05L，pCGS3-HLPV-P07L，pCGS3-HLPV-P08L，pCGS3-HLPV-P09L，pCGS3-HLPV-P10L，pCGS3-HLPV-P11L，pCGS3-HLPV-P12L，pCGS3-HLPV-P13/14L中间质粒(图1上)。图中，泳道A-M依次为pCGS3-HLPV-P00L，pCGS3-HLPV-P01L，pCGS3-HLPV-P02L，pCGS3-HLPV-P03/06L，pCGS3-HLPV-P04L，pCGS3-HLPV-P05L，pCGS3-HLPV-P07L，pCGS3-HLPV-P08L，pCGS3-HLPV-P09L，pCGS3-HLPV-P10L，pCGS3-HLPV-P11L，pCGS3-HLPV-P12L，pCGS3-HLPV-P13/14L中间质粒酶切产物电泳结果。The first step is to construct the light chain of the whole antibody molecule. The operation steps are: use HindIII (NEB) and XhoI (NEB) to double digest the pCGS3 (Merck) vector, pUC57-00-L, pUC57-01-L, pUC57-02-L , pUC57-03/06-L, pUC57-04-L, pUC57-05-L, pUC57-07-L, pUC57-08-L, pUC57-09-L, pUC57-10-L, pUC57-11-L , pUC57-12-L and pUC57-13/14-L light chain plasmids, a 9535bp vector fragment and 13 723bp light chain fragments were obtained. T4 ligase (TAKARA) was used to ligate, transform, plate, extract the plasmid, and identify it by double enzyme digestion with HindIII and XhoI, and the digested bands were in line with expectations. The correct plasmids were named pCGS3-HLPV-P00L, pCGS3-HLPV-P01L, pCGS3-HLPV-P02L, pCGS3-HLPV-P03/06L, pCGS3-HLPV-P04L, pCGS3-HLPV-P05L, pCGS3-HLPV-P07L, pCGS3-HLPV-P08L, pCGS3-HLPV-P09L, pCGS3-HLPV-P10L, pCGS3-HLPV-P11L, pCGS3-HLPV-P12L, pCGS3-HLPV-P13/14L intermediate plasmids (Figure 1 top). In the figure, lanes A-M are pCGS3-HLPV-P00L, pCGS3-HLPV-P01L, pCGS3-HLPV-P02L, pCGS3-HLPV-P03/06L, pCGS3-HLPV-P04L, pCGS3-HLPV-P05L, pCGS3-HLPV-P07L , pCGS3-HLPV-P08L, pCGS3-HLPV-P09L, pCGS3-HLPV-P10L, pCGS3-HLPV-P11L, pCGS3-HLPV-P12L, pCGS3-HLPV-P13/14L intermediate plasmid digestion product electrophoresis results.
第二步为构建全抗分子重链以及按照表3不同组合方式构建全抗分子：采用BstBI(NEB)和PacI(NEB)双酶切上述构建完成的13个中间质粒和pUC57-00-H，pUC57-01-H，pUC57-02-H，pUC57-03-H，pUC57-04-H，pUC57-05/10-H，pUC57-06-H，pUC57-07-H，pUC57-08/09/11-H，pUC57-12-H，pUC57-13-H和pUC57-14-H重链质粒，获得13条10200bp载体片段和12条1409bp重链片段，采用T4连接酶(TAKARA)连接，转化，涂板，提取质粒，BstBI和PacI双酶切鉴定，酶切条带符合预期(图1下)。测序正确的质粒分别命名为pCGS3-HLPV-P00至P14。图中泳道01-14分别为pCGS3-HLPV-P00至P14酶切产物电泳结果。The second step is to construct the heavy chain of the whole antibody molecule and construct the whole antibody molecule according to different combinations in Table 3: use BstBI (NEB) and PacI (NEB) to double digest the 13 intermediate plasmids and pUC57-00-H constructed above, pUC57-01-H, pUC57-02-H, pUC57-03-H, pUC57-04-H, pUC57-05/10-H, pUC57-06-H, pUC57-07-H, pUC57-08/09/ 11-H, pUC57-12-H, pUC57-13-H and pUC57-14-H heavy chain plasmids, obtained 13 10200bp vector fragments and 12 1409bp heavy chain fragments, connected with T4 ligase (TAKARA), transformed, Plating, extraction of plasmids, BstBI and PacI double enzyme digestion identification, the digestion bands are in line with expectations (Figure 1 bottom). The plasmids with correct sequencing were named pCGS3-HLPV-P00 to P14, respectively. Lanes 01-14 in the figure are the electrophoresis results of pCGS3-HLPV-P00 to P14 digestion products, respectively.
pCGS3-HLPV-P00至P14重组表达质粒中每一个重组表达质粒均含有1409bp重链基因片段和723bp轻链基因片段，重链基因片段和轻链基因片段核苷酸序列中，只有编码重链可变区和轻链可变区的核苷酸序列不同，其它核苷酸序列均相同。相应的重链可变区和轻链可变区的氨基酸序列如表3所示。其中，本发明亲和力成熟的序列HLPV-P02至P14的重链可变区的编码序列如SEQ ID No.26所示(表4)，轻链可变区的编码序列如SEQ ID No.27所示(表5)，其中，R为A或G，Y为C或T，M为A或C，K为G或T，S为G或C，W为A或T，H为A或T或C，B为G或T或C，V为G或A或C，D为G或A或T，N为A或T或G或C。Each of the recombinant expression plasmids from pCGS3-HLPV-P00 to P14 contains a 1409bp heavy chain gene fragment and a 723bp light chain gene fragment. In the nucleotide sequences of the heavy chain gene fragment and the light chain gene fragment, only the heavy chain can be encoded. The nucleotide sequences of the variable region and the light chain variable region are different, and the other nucleotide sequences are the same. The amino acid sequences of the corresponding heavy chain variable region and light chain variable region are shown in Table 3. Wherein, the coding sequence of the heavy chain variable region of the affinity matured sequence HLPV-P02 to P14 of the present invention is shown in SEQ ID No.26 (Table 4), and the coding sequence of the light chain variable region is shown in SEQ ID No.27 (Table 5), wherein, R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C, W is A or T, H is A or T or C, B is G or T or C, V is G or A or C, D is G or A or T, N is A or T or G or C.
表4 SEQ ID No.26序列简并碱基统计表Table 4 Statistical table of degenerate bases of SEQ ID No.26 sequence
表5 SEQ ID No.27序列简并碱基统计表Table 5 Statistical table of degenerate bases of SEQ ID No.27 sequence
上述重链基因片段如SEQ ID NO：28所示，第1-6位为BstBI酶切位点，第7-15位为Kozak序列，第16-72位为信号肽基因，第73-1398位为重链基因序列，第1399-1401位为终止密码子，第1402-1409位为PacI酶切位点，其中第73-408位核苷酸序列为重链可变区的编码序列，第409-1398位为恒定区序列。The above heavy chain gene fragment is shown in SEQ ID NO: 28, the 1-6 position is the BstBI restriction site, the 7-15 position is the Kozak sequence, the 16-72 position is the signal peptide gene, and the 73-1398 position It is the heavy chain gene sequence, the 1399-1401th position is a stop codon, the 1402-1409th position is a PacI restriction site, and the 73rd-408th nucleotide sequence is the coding sequence of the heavy chain variable region, and the 409th position Position -1398 is the constant region sequence.
上述轻链基因片段如SEQ ID NO：30所示，第1-6位为HindIII酶切位点，第7-15位为Kozak序列，第16-72位为信号肽，第73-714位轻链基因序列，第715-717位为终止密码子，第718-723位为XhoI酶切位点，其中第73-393位核苷酸为轻链可变区的编码序列，第394-714位为恒定区序列。The above light chain gene fragment is shown in SEQ ID NO: 30, the 1-6 position is the HindIII restriction site, the 7-15 position is the Kozak sequence, the 16-72 position is the signal peptide, and the 73-714 position is the light chain sequence. Chain gene sequence, the 715th-717th position is the stop codon, the 718-723rd position is the XhoI restriction site, and the 73rd-393rd nucleotide is the coding sequence of the light chain variable region, and the 394-714th position for the constant region sequence.
2重组表达质粒瞬转表达实验2 Recombinant expression plasmid transient expression experiment
按照每mL细胞培养物转染0.8μg质粒的量及质粒原始浓度，计算转染50mL细胞所需原始质粒的体积。将15个上述重组表达质粒与转染试剂ExpiFectamineTM CHOTransfection Kit(Thermo公司)复合物缓慢滴入含有ExpiCHO-S(Thermo公司)细胞的细胞培养基ExpiCHOTM Expression Medium(Thermo公司)中培养以表达15个抗体分子，边加边摇晃细胞培养物，使DNA与转染试剂复合物分散均匀。按照最大滴度在转染后18至22h添加补料和增强剂，同时将培养条件37℃降至32℃、CO2浓度由8％降至5％；转染后第5天再次添加补料。当活率降至65％-75％时终止培养得到培养物。所得培养物依次命名为CHO-S/pCGS3-HLPV-00至14共计15个培养物。According to the amount of 0.8 μg plasmid transfected per mL of cell culture and the original concentration of the plasmid, calculate the volume of the original plasmid required to transfect 50 mL of cells. The 15 above-mentioned recombinant expression plasmids and the transfection reagent ExpiFectamineTM CHOTransfection Kit (Thermo Company) complex were slowly dropped into the cell culture medium ExpiCHOTM Expression Medium (Thermo Company) containing ExpiCHO-S (Thermo Company) cells and cultivated to express 15 Shake the cell culture while adding antibody molecules, so that the complex of DNA and transfection reagent is evenly dispersed. Add feed and enhancer at 18 to 22 hours after transfection according to the maximum titer, and at the same time reduce the culture conditions from 37°C to 32°C, and reduce theCO2 concentration from 8% to 5%; add feed again on the 5th day after transfection . When the viability drops to 65%-75%, the culture is terminated to obtain a culture. The resulting cultures were sequentially named CHO-S/pCGS3-HLPV-00 to 14, a total of 15 cultures.
3组装全抗蛋白纯化实验3 Assembly whole antibody purification experiment
将上述培养物于6000g离心30min，收集上清，采用超滤管Centrifugal FiltersU1
4全抗分子亲和力检测4 Whole Antibody Molecular Affinity Detection
采用表面等离子共振技术检测亲和力成熟获得的HLPV-P00至P14共计15个抗体与PV-1蛋白结合亲和力。将偶联物重组人PLVAP/PV-1蛋白(Abcam公司)偶联到Series SSensor Chip CM5芯片(GE公司)上，Biacore T200分子间相互作用分析系统(GE公司)上机，将分析物亲和力成熟获得的抗体流过芯片表面(温度25℃，流速度30μl/min，结合时间120s，解离时间180s)，若偶联物与分析物有结合活性，则会引起金膜表面折射率变化，最终导致SPR角变化。通过检测SPR角度变化，Biacore T200 Evaluation Software程序软件输出原始数据，并进行1∶1结合模式进行动力学拟合分析，亲和力成熟抗体与PV-1蛋白的亲和力常数如表6所示。A total of 15 antibodies from HLPV-P00 to P14 obtained by affinity maturation were used to detect the binding affinity of PV-1 protein by surface plasmon resonance technology. The conjugate recombinant human PLVAP/PV-1 protein (Abcam Company) was coupled to the Series SSensor Chip CM5 chip (GE Company), and the Biacore T200 intermolecular interaction analysis system (GE Company) was used to mature the analyte affinity The obtained antibody flows over the surface of the chip (
表6亲和力成熟全抗分子亲和力统计表Table 6 Affinity statistics of affinity matured whole antibody molecules
由表6可知，经过两次噬菌体展示实验和全抗分子亲和力实验验证，共计获得14个亲和力成熟抗体分子，亲和力比原始抗体显著提高，最高抗体HLPV-P11的亲和力提高倍数为40.49倍(表6)。It can be seen from Table 6 that after two phage display experiments and the whole antibody molecular affinity experiment verification, a total of 14 affinity matured antibody molecules were obtained, the affinity was significantly improved compared with the original antibody, and the affinity of the highest antibody HLPV-P11 was increased by 40.49 times (Table 6 ).
实施例3、去除ADCC效应亚型抗体亲和力研究Example 3, Removal of ADCC Effector Subtype Antibody Affinity Research
1重组表达质粒构建实验1 Recombinant expression plasmid construction experiment
靶点PV-1临床应用不需要抗体的ADCC效应，因此任选择七个亲和力成熟抗体，构建弱化ADCC效应的IgG1(N297A)和IgG4(S228P)两个亚型抗体重组表达质粒(表7)，用于验证不同去除ADCC效应亚型抗体对亲和力的影响。The clinical application of the target PV-1 does not require the ADCC effect of the antibody, so seven affinity matured antibodies were randomly selected to construct recombinant expression plasmids for IgG1 (N297A) and IgG4 (S228P) subtype antibodies that weaken the ADCC effect (Table 7). Used to verify the effect of different ADCC effector subtype antibodies on affinity.
表7去除ADCC效应亚型抗体构建所需统计表Table 7 removes the statistical table required for ADCC effector subtype antibody construction
IgG1(N297A)质粒和IgG4(S228P)质粒是将重链基因序列连接至pUC57克隆载体的限制性核酸内切酶SmaI单酶切位点之后获得的。The IgG1 (N297A) plasmid and IgG4 (S228P) plasmid were obtained after linking the heavy chain gene sequence to the restriction endonuclease SmaI single-cutting site of the pUC57 cloning vector.
其中pUC57-03-H(N297A)，pUC57-05/10-H(N297A)，pUC57-08/09/11-H(N297A)，pUC57-14-H(N297A)含有完整的重链基因，该重链基因是分别将重链可变区基因(SEQ IDNO：06、SEQ ID NO：08、SEQ ID NO：11和SEQ ID NO：14的编码基因)的3’端与人IgG1(N297A)基因连接得到的融合基因。其中IgG1(N297A)基因的核苷酸序列为SEQ ID NO：28所示，IgG1(N297A)基因表达的IgG1(N297A)与人IgG1恒定区的不同之处仅在于第297位天冬氨酸突变为丙氨酸。Wherein pUC57-03-H (N297A), pUC57-05/10-H (N297A), pUC57-08/09/11-H (N297A), pUC57-14-H (N297A) contain a complete heavy chain gene, the The heavy chain gene is the 3' end of the heavy chain variable region gene (the coding gene of SEQ ID NO: 06, SEQ ID NO: 08, SEQ ID NO: 11 and SEQ ID NO: 14) and the human IgG1 (N297A) gene The resulting fusion gene was ligated. The nucleotide sequence of the IgG1 (N297A) gene is shown in SEQ ID NO: 28. The IgG1 (N297A) expressed by the IgG1 (N297A) gene differs from the human IgG1 constant region only in the 297th aspartic acid mutation for alanine.
其中pUC57-03-H(S228P)，pUC57-05/10-H(S228P)，pUC57-08/09/11-H(S228P)和pUC57-14-H(S228P)含有完整的重链基因，该重链基因是分别将重链可变区基因(SEQ IDNO：06、SEQ ID NO：08、SEQ ID NO：11和SEQ ID NO：14的编码基因)的3’端与人IgG4(S228P)基因连接得到的融合基因。其中IgG4(S228P)基因的核苷酸序列为SEQ ID No.29的第409-1389位所示，IgG4(S228P)基因表达氨基酸序列如UniProtKB数据库序列号为P01861.1(2022.02.23最新更新)的IgG4并引入S228P(EU编号)氨基酸突变所示的IgG4(S228P)恒定区，即IgG4(S228P)基因表达的IgG4(S228P)与人IgG4恒定区的不同之处仅在于第228位丝氨酸突变为脯氨酸。Wherein pUC57-03-H(S228P), pUC57-05/10-H(S228P), pUC57-08/09/11-H(S228P) and pUC57-14-H(S228P) contain complete heavy chain gene, the The heavy chain gene is the 3' end of the heavy chain variable region gene (the coding gene of SEQ ID NO: 06, SEQ ID NO: 08, SEQ ID NO: 11 and SEQ ID NO: 14) and the human IgG4 (S228P) gene The resulting fusion gene was ligated. The nucleotide sequence of the IgG4 (S228P) gene is shown in the 409-1389th position of SEQ ID No.29, and the amino acid sequence expressed by the IgG4 (S228P) gene is P01861.1 (the latest update on 2022.02.23) as shown in the UniProtKB database IgG4 and introduce the IgG4 (S228P) constant region indicated by the S228P (EU numbering) amino acid mutation. proline.
按照表7构建质粒：采用BstBI(NEB)和PacI(NEB)双酶切上述构建完成的表7中7个中间质粒，pUC57-03-H(N297A)，pUC57-05/10-H(N297A)，pUC57-08/09/11-H(N297A)，pUC57-14-H(N297A)，pUC57-03-H(S228P)，pUC57-05/10-H(S228P)，pUC57-08/09/11-H(S228P)和pUC57-14-H(S228P)质粒，获得10200bp载体片段和1409bp的IgG1(N297A)或1400bp的IgG4(S228P)重链片段，采用T4连接酶(TAKARA)连接，转化，涂版，提取质粒，BstBI和PacI双酶切鉴定，酶切条带符合预期(图3)。质粒命名为pCGS3-HLPV-P03(N297A)，pCGS3-HLPV-P05(N297A)，pCGS3-HLPV-P08(N297A)，pCGS3-HLPV-P09(N297A)，pCGS3-HLPV-P10(N297A)，pCGS3-HLPV-P11(N297A)，pCGS3-HLPV-P14(N297A)，pCGS3-HLPV-P03(S228P)，pCGS3-HLPV-P05(S228P)，pCGS3-HLPV-P08(S228P)，pCGS3-HLPV-P09(S228P)，pCGS3-HLPV-P10(S228P)，pCGS3-HLPV-P11(S228P)，pCGS3-HLPV-P14(S228P)。Construct plasmids according to Table 7: use BstBI (NEB) and PacI (NEB) to double digest the 7 intermediate plasmids in Table 7 constructed above, pUC57-03-H (N297A), pUC57-05/10-H (N297A) , pUC57-08/09/11-H(N297A), pUC57-14-H(N297A), pUC57-03-H(S228P), pUC57-05/10-H(S228P), pUC57-08/09/11 -H (S228P) and pUC57-14-H (S228P) plasmids, obtained 10200bp vector fragment and 1409bp IgG1 (N297A) or 1400bp IgG4 (S228P) heavy chain fragment, connected with T4 ligase (TAKARA), transformed, coated version, extracted the plasmid, identified by double enzyme digestion with BstBI and PacI, and the digested bands were in line with expectations (Figure 3). The plasmids were named pCGS3-HLPV-P03(N297A), pCGS3-HLPV-P05(N297A), pCGS3-HLPV-P08(N297A), pCGS3-HLPV-P09(N297A), pCGS3-HLPV-P10(N297A), pCGS3- HLPV-P11(N297A), pCGS3-HLPV-P14(N297A), pCGS3-HLPV-P03(S228P), pCGS3-HLPV-P05(S228P), pCGS3-HLPV-P08(S228P), pCGS3-HLPV-P09(S228P ), pCGS3-HLPV-P10(S228P), pCGS3-HLPV-P11(S228P), pCGS3-HLPV-P14(S228P).
pCGS3-HLPV-P03(N297A)，pCGS3-HLPV-P05(N297A)，pCGS3-HLPV-P08(N297A)，pCGS3-HLPV-P09(N297A)，pCGS3-HLPV-P10(N297A)，pCGS3-HLPV-P11(N297A)和pCGS3-HLPV-P14(N297A)这7个去除ADCC效应IgG1(N297A)亚型抗体重组表达质粒均含有1409bp的IgG1(N297A)重链片段和723bp轻链基因片段，IgG1(N297A)重链片段和轻链基因片段核苷酸序列中，只有编码重链可变区和轻链可变区的核苷酸序列不同，其它核苷酸序列均相同。IgG1(N297A)重链片段如SEQ ID NO：28所示，第1-6位为BstBI酶切位点，第7-15位为Kozak序列，第16-72位为信号肽基因，第73-1398位为重链基因序列，第1399-1401位为终止密码子，第1402-1409位为PacI酶切位点，其中第73-408位核苷酸序列为重链可变区的编码序列，第409-1398位为恒定区序列。该轻链基因片段如SEQ ID NO：30所示，第1-6位为HindIII酶切位点，第7-15位为Kozak序列，第16-72位为信号肽，第73-714位轻链基因序列，第715-717位为终止密码子，第718-723位为XhoI酶切位点，其中第73-393位核苷酸为轻链可变区的编码序列，394-714为恒定区序列。pCGS3-HLPV-P03(N297A), pCGS3-HLPV-P05(N297A), pCGS3-HLPV-P08(N297A), pCGS3-HLPV-P09(N297A), pCGS3-HLPV-P10(N297A), pCGS3-HLPV-P11 (N297A) and pCGS3-HLPV-P14(N297A), the seven recombinant expression plasmids for IgG1(N297A) subtype antibody without ADCC effect, all contain 1409bp IgG1(N297A) heavy chain fragment and 723bp light chain gene fragment, IgG1(N297A) Among the nucleotide sequences of the heavy chain fragment and the light chain gene fragment, only the nucleotide sequences encoding the variable region of the heavy chain and the variable region of the light chain are different, and the other nucleotide sequences are the same. The IgG1 (N297A) heavy chain fragment is shown in SEQ ID NO: 28, the 1-6 position is the BstBI restriction site, the 7-15 position is the Kozak sequence, the 16-72 position is the signal peptide gene, and the 73- The 1398th position is the heavy chain gene sequence, the 1399-1401th position is the stop codon, the 1402-1409th position is the PacI restriction site, and the 73rd-408th nucleotide sequence is the coding sequence of the heavy chain variable region, Positions 409-1398 are constant region sequences. The light chain gene fragment is shown in SEQ ID NO: 30, the 1-6 position is the HindIII restriction site, the 7-15 position is the Kozak sequence, the 16-72 position is the signal peptide, and the 73-714 position is the light chain gene fragment. Chain gene sequence, the 715-717th position is a stop codon, the 718-723rd position is an XhoI restriction site, and the 73rd-393rd nucleotide is the coding sequence of the light chain variable region, and the 394-714 is a constant zone sequence.
pCGS3-HLPV-P03(S228P)，pCGS3-HLPV-P05(S228P)，pCGS3-HLPV-P08(S228P)，pCGS3-HLPV-P09(S228P)，pCGS3-HLPV-P10(S228P)，pCGS3-HLPV-P11(S228P)和pCGS3-HLPV-P14(S228P)这7个去除ADCC效应IgG4(SP28P)亚型抗体重组表达质粒均含有1400bp的IgG4(S228P)重链片段和723bp轻链基因片段，IgG4(S228P)重链片段和723bp轻链基因片段核苷酸序列中，只有编码重链可变区和轻链可变区的核苷酸序列不同，其它核苷酸序列均相同。IgG4(S228P)重链片段如SEQ ID NO：29所示，第1-6位为BstBI酶切位点，第7-15位为Kozak序列，第16-72位为信号肽基因，第73-1389位为重链基因序列，第1390-1392位为终止密码子，第1393-1400位为PacI酶切位点，其中第73-408位核苷酸序列为重链可变区的编码序列，第409-1389位为恒定区序列。该轻链基因片段如SEQ ID NO：30所示，第1-6位为HindIII酶切位点，第7-15位为Kozak序列，第16-72位为信号肽，第73-714位轻链基因序列，第715-717位为终止密码子，第718-723位为XhoI酶切位点，其中第73-393位核苷酸为轻链可变区的编码序列，394-714为恒定区序列。pCGS3-HLPV-P03(S228P), pCGS3-HLPV-P05(S228P), pCGS3-HLPV-P08(S228P), pCGS3-HLPV-P09(S228P), pCGS3-HLPV-P10(S228P), pCGS3-HLPV-P11 (S228P) and pCGS3-HLPV-P14(S228P), the seven recombinant expression plasmids for removing the ADCC effect IgG4 (SP28P) subtype antibody, all contain a 1400bp IgG4 (S228P) heavy chain fragment and a 723bp light chain gene fragment, IgG4 (S228P) Among the nucleotide sequences of the heavy chain fragment and the 723bp light chain gene fragment, only the nucleotide sequences encoding the variable region of the heavy chain and the variable region of the light chain are different, and the other nucleotide sequences are the same. The IgG4 (S228P) heavy chain fragment is shown in SEQ ID NO: 29, the 1-6 position is the BstBI restriction site, the 7-15 position is the Kozak sequence, the 16-72 position is the signal peptide gene, and the 73- The 1389th position is the heavy chain gene sequence, the 1390-1392th position is the stop codon, the 1393-1400th position is the PacI restriction site, and the 73rd-408th nucleotide sequence is the coding sequence of the heavy chain variable region, Positions 409-1389 are constant region sequences. The light chain gene fragment is shown in SEQ ID NO: 30, the 1-6 position is the HindIII restriction site, the 7-15 position is the Kozak sequence, the 16-72 position is the signal peptide, and the 73-714 position is the light chain gene fragment. Chain gene sequence, the 715-717th position is a stop codon, the 718-723rd position is an XhoI restriction site, and the 73rd-393rd nucleotide is the coding sequence of the light chain variable region, and the 394-714 is a constant zone sequence.
2去除ADCC效应亚型抗体亲和力检测2 Removal of ADCC effector subtype antibody affinity detection
按照实施例2中重组表达质粒瞬转表达方法对去除ADCC效应IgG1(N297A)和IgG4(SP28P)亚型抗体重组表达质粒进行表达获得培养物，按照实施例2中抗体蛋白纯化对获得的培养物进行纯化并对纯化后培养物进行SDS-PAGE鉴定纯化单克隆抗体，检测结果显示单克隆抗体条带符合预期(图4)，表明前述培养物分别含有抗体亚型为IgG1(N297A)的抗体HLPV-P03、HLPV-P05、HLPV-P08、HLPV-P09、HLPV-P10、HLPV-P11和HLPV-P14以及抗体亚型为IgG4(S228P)的抗体HLPV-P03、HLPV-P05、HLPV-P08、HLPV-P09、HLPV-P10、HLPV-P11和HLPV-P14。According to the recombinant expression plasmid transient expression method in Example 2, the recombinant expression plasmids for removing the ADCC effect IgG1 (N297A) and IgG4 (SP28P) subtype antibodies were expressed to obtain cultures, and the obtained cultures were purified according to Example 2 for antibody protein purification Purify and identify the purified monoclonal antibody by SDS-PAGE on the purified culture, and the test results show that the monoclonal antibody band is in line with expectations (Figure 4), indicating that the aforementioned cultures contain the antibody subtype of IgG1 (N297A) antibody HLPV -P03, HLPV-P05, HLPV-P08, HLPV-P09, HLPV-P10, HLPV-P11 and HLPV-P14 and antibodies of the antibody subtype IgG4 (S228P) HLPV-P03, HLPV-P05, HLPV-P08, HLPV - P09, HLPV-P10, HLPV-P11 and HLPV-P14.
按照实施例2中亲和力检测方法对前述制备的抗体以及实施例2制备的HLPV-P00进行检测，检测去除ADCC效应亚型抗体与PV-1蛋白的亲和力常数，实验结果如表8所示。The antibody prepared above and the HLPV-P00 prepared in Example 2 were detected according to the affinity detection method in Example 2, and the affinity constant between the ADCC effector subtype antibody and PV-1 protein was detected. The experimental results are shown in Table 8.
表8去除ADCC效应亚型抗体亲和力统计表Table 8 Remove ADCC Effect Subtype Antibody Affinity Statistical Table
表8可知，IgG1(N297A)和IgG4(S228P)两种去除ADCC效应的抗体亲和力均比原始抗体(HLPV-P00)提高1-2个数量级，亲和力成熟实验符合预期，可用于下一步药物开发实验。It can be seen from Table 8 that the affinity of IgG1 (N297A) and IgG4 (S228P) antibodies that remove the ADCC effect is 1-2 orders of magnitude higher than that of the original antibody (HLPV-P00). .
综上所述，本发明通过两次噬菌体筛选实验，获得亲和力提高1-2个数量级的14个亲和力成熟抗体序列HLPV-P00至14。更进一步的，PV-1靶点抗体不需抗体依赖的细胞介导的细胞毒性效应(ADCC)，本发明采用IgG1(N297A)和IgG4(S228P)两种去除ADCC效应的抗体亚型验证，亲和力依然提高1-2个数量级。本发明所述亲和力成熟抗体预期降低给药剂量，用于治疗年龄相关性黄斑变性(AMD)、糖尿病黄斑水肿(DME)和成年患者视网膜静脉阻塞引起的黄斑、肝癌等疾病。In summary, the present invention obtains 14 affinity matured antibody sequences HLPV-P00 to 14 whose affinity is increased by 1-2 orders of magnitude through two phage screening experiments. Furthermore, the PV-1 target antibody does not require antibody-dependent cell-mediated cytotoxicity (ADCC). The present invention uses IgG1 (N297A) and IgG4 (S228P) two antibody subtypes to remove the ADCC effect. The affinity Still improved by 1-2 orders of magnitude. The affinity matured antibody described in the present invention is expected to reduce the dosage, and is used for treating diseases such as age-related macular degeneration (AMD), diabetic macular edema (DME), macula and liver cancer caused by retinal vein occlusion in adult patients.
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.
序列表sequence listing
<110> 华兰基因工程有限公司<110> Hualan Gene Engineering Co., Ltd.
<120> 特异性结合PV-1蛋白的抗体或其抗原结合片段及其应用<120> Antibody or antigen-binding fragment thereof specifically binding to PV-1 protein and application thereof
<130> 221127<130> 221127
<160> 30<160> 30
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 1<400> 1
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile His Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile His Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 2<210> 2
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 2<400> 2
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Thr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Thr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 3<210> 3
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 3<400> 3
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 4<210> 4
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 4<400> 4
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 5<210> 5
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 5<400> 5
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Thr Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Thr Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 6<210> 6
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 6<400> 6
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Val Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Val Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 7<210> 7
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 7<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Thr Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Thr Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 8<210> 8
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 8<400> 8
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 9<210> 9
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 9<400> 9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Ser Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Ser Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 10<210> 10
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 10<400> 10
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Glu Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Gln Gly Asp Val Arg Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Gln Gly Asp Val Arg Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 11<210> 11
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 11<400> 11
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Gln Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Thr Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Thr Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 12<210> 12
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 12<400> 12
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Ser Gln Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Ser Gln Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 13<210> 13
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 13<400> 13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asp TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Val Phe Thr Asp Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Ser Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Ser Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 14<210> 14
<211> 112<211> 112
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 14<400> 14
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30 20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp IleTyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 35 40 45
Gly Glu Ile Asn Pro Thr Ser Gly Asp Val Asn Phe Asn Glu Met PheGly Glu Ile Asn Pro Thr Ser Gly Asp Val Asn Phe Asn Glu Met Phe
50 55 60 50 55 60
Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala TyrLys Ser Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerThr Ser Ile Phe Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 100 105 110
<210> 15<210> 15
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 15<400> 15
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ser Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ser Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 16<210> 16
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 16<400> 16
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Phe Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Phe Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 17<210> 17
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 17<400> 17
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 18<210> 18
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 18<400> 18
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asn Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asn Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 19<210> 19
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 19<400> 19
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Tyr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Tyr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 20<210> 20
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 20<400> 20
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Ser His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Ser His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 21<210> 21
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 21<400> 21
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Gly Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Gly Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Tyr Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Tyr Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 22<210> 22
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 22<400> 22
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Ser Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Ser Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 23<210> 23
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 23<400> 23
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile LysSer Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 24<210> 24
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 24<400> 24
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Thr Asp Arg Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Thr Asp Arg Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 25<210> 25
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 25<400> 25
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr GlnAsp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu IleLeu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45 35 40 45
Tyr Tyr Ala Gly Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Ala Gly Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro PheGlu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysThr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 100 105
<210> 26<210> 26
<211> 336<211> 336
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 26<400> 26
caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60
tcctgcaagg cttctggcta crybtttvvs rmbtactaca tgtactgggt ccgacaggct 120tcctgcaagg cttctggcta crybtttvvs rmbtactaca tgtactgggt ccgacaggct 120
cctggacagg gactcgagtg gatcggcgag rtyaatcctw cwhmkggcga cgtgmrcttc 180cctggacagg gactcgagtg gatcggcgag rtyaatcctw cwhmkggcga cgtgmrcttc 180
aacgagatgt tcaagtcccg cgtgaccatg accgtggaca cctctacctc taccgcctac 240aacgagatgt tcaagtcccg cgtgaccatg accgtggaca cctctacctc taccgcctac 240
atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcac cwcyatcttt 300atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcac cwcyatcttt 300
tattggggcc agggcacact ggtcaccgtg tcctct 336tattggggcc agggcacact ggtcaccgtg tcctct 336
<210> 27<210> 27
<211> 321<211> 321
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 27<400> 27
gacatcgtga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60gacatcgtga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60
atcacatgca aggcsagcca ggacatcrrg tatcagytgw cbtggtatca gcagaagcct 120atcacatgca aggcsagcca ggacatcrrg tatcagytgw cbtggtatca gcagaagcct 120
ggcaaggccc ctaagacact gatctactac kcyrsykhtc kggccgatgg cgtgccctct 180ggcaaggccc ctaagacact gatctactac kcyrsykhtc kggccgatgg cgtgccctct 180
agattttccg gctctggctc tggccaagag tttaccctga caatctccag cctgcagcct 240agattttccg gctctggctc tggccaagag tttaccctga caatctccag cctgcagcct 240
gaggacttcg ccacctacta ctgcgttymg cackmyraya gacccttcwc ytttggccag 300gaggacttcg ccacctacta ctgcgttymg cackmyraya gacccttcwc ytttggccag 300
ggcaccaagc tggaaatcaa g 321ggcaccaagc tggaaatcaa g 321
<210> 28<210> 28
<211> 1409<211> 1409
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 28<400> 28
ttcgaagccg ccaccatggg atggtcttgt atcatcctgt ttctggtggc taccgccaca 60ttcgaagccg ccaccatggg atggtcttgt atcatcctgt ttctggtggc taccgccaca 60
ggcgtgcata gccaggttca gctggttcag tctggcgccg aagtgaagaa acctggcgcc 120ggcgtgcata gccaggtca gctggttcag tctggcgccg aagtgaagaa acctggcgcc 120
tctgtgaagg tgtcctgcaa ggcttctggc tacrybtttv vsrmbtacta catgtactgg 180tctgtgaagg tgtcctgcaa ggcttctggc tacrybtttv vsrmbtacta catgtactgg 180
gtccgacagg ctcctggaca gggactcgag tggatcggcg agrtyaatcc twcwhmkggc 240gtccgacagg ctcctggaca gggactcgag tggatcggcg agrtyaatcc twcwhmkggc 240
gacgtgmrct tcaacgagat gttcaagtcc cgcgtgacca tgaccgtgga cacctctacc 300gacgtgmrct tcaacgagat gttcaagtcc cgcgtgacca tgaccgtgga cacctctacc 300
tctaccgcct acatggaact gtccagcctg agatctgagg acaccgccgt gtactactgc 360tctaccgcct acatggaact gtccagcctg agatctgagg acaccgccgt gtactactgc 360
accwcyatct tttattgggg ccagggcaca ctggtcaccg tgtcctctgc ttccaccaag 420accwcyatct tttattgggg ccagggcaca ctggtcaccg tgtcctctgc ttccaccaag 420
ggcccctccg tgttccccct ggctccctct tccaagagca ccagcggcgg caccgctgct 480ggcccctccg tgttccccct ggctccctct tccaagagca ccagcggcgg caccgctgct 480
ctgggatgtc tggtgaagga ctacttccct gagcctgtga ccgtgtcctg gaattccggc 540ctgggatgtc tggtgaagga ctacttccct gagcctgtga ccgtgtcctg gaattccggc 540
gccctgacct ccggcgtgca cacattccct gctgtgctgc agtcctccgg cctgtatagc 600gccctgacct ccggcgtgca cacattccct gctgtgctgc agtcctccgg cctgtatagc 600
ctgtcctccg tggtgacagt gcctagctcc agcctgggca cccagaccta tatctgcaac 660ctgtcctccg tggtgacagt gcctagctcc agcctgggca cccagaccta tatctgcaac 660
gtgaaccaca agcctagcaa taccaaggtg gacaagaagg tggagcctaa gagctgcgac 720gtgaaccaca agcctagcaa taccaaggtg gacaagaagg tggagcctaa gagctgcgac 720
aagacccaca cctgtcctcc atgtcctgct ccagaactgc tcggcggacc ttccgtgttc 780aagaccccaca cctgtcctcc atgtcctgct ccagaactgc tcggcggacc ttccgtgttc 780
ctgtttcctc caaagcctaa ggacaccctg atgatcagca gaacccctga agtgacctgc 840ctgtttcctc caaagcctaa ggacaccctg atgatcagca gaacccctga agtgacctgc 840
gtggtggtgg atgtgtccca cgaggatccc gaagtgaagt tcaattggta cgtggacggc 900gtggtggtgg atgtgtccca cgaggatccc gaagtgaagt tcaattggta cgtggacggc 900
gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacrmcag cacctacaga 960gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacrmcag cacctacaga 960
gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 1020gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 1020
aaggtgtcca acaaggccct gcctgctcct atcgagaaaa ccatcagcaa ggccaagggc 1080aaggtgtcca acaaggccct gcctgctcct atcgagaaaa ccatcagcaa ggccaagggc 1080
cagcctaggg aaccccaggt ttacacactg cctccaagca gggacgagct gaccaagaat 1140cagcctaggg aaccccaggt ttacacactg cctccaagca gggacgagct gaccaagaat 1140
caggtgtccc tgacctgcct ggtcaagggc ttctaccctt ccgatatcgc cgtggaatgg 1200caggtgtccc tgacctgcct ggtcaagggc ttctaccctt ccgatatcgc cgtggaatgg 1200
gagagcaatg gccagcctga gaacaactac aagacaaccc ctcctgtgct ggacagcgac 1260gagagcaatg gccagcctga gaacaactac aagacaaccc ctcctgtgct ggacagcgac 1260
ggctcattct tcctgtacag caagctgaca gtggacaaga gcagatggca gcagggcaac 1320ggctcattct tcctgtacag caagctgaca gtggacaaga gcagatggca gcagggcaac 1320
gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1380gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1380
agcctgtctc ctggcaaata attaattaa 1409agcctgtctc ctggcaaata attaattaa 1409
<210> 29<210> 29
<211> 1400<211> 1400
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)
<400> 29<400> 29
ttcgaagccg ccaccatggg atggtcttgt atcatcctgt ttctggtggc taccgccaca 60ttcgaagccg ccaccatggg atggtcttgt atcatcctgt ttctggtggc taccgccaca 60
ggcgtgcata gccaggttca gctggttcag tctggcgccg aagtgaagaa acctggcgcc 120ggcgtgcata gccaggtca gctggttcag tctggcgccg aagtgaagaa acctggcgcc 120
tctgtgaagg tgtcctgcaa ggcttctggc tacrybtttv vsrmbtacta catgtactgg 180tctgtgaagg tgtcctgcaa ggcttctggc tacrybtttv vsrmbtacta catgtactgg 180
gtccgacagg ctcctggaca gggactcgag tggatcggcg agrtyaatcc twcwhmkggc 240gtccgacagg ctcctggaca gggactcgag tggatcggcg agrtyaatcc twcwhmkggc 240
gacgtgmrct tcaacgagat gttcaagtcc cgcgtgacca tgaccgtgga cacctctacc 300gacgtgmrct tcaacgagat gttcaagtcc cgcgtgacca tgaccgtgga cacctctacc 300
tctaccgcct acatggaact gtccagcctg agatctgagg acaccgccgt gtactactgc 360tctaccgcct acatggaact gtccagcctg agatctgagg acaccgccgt gtactactgc 360
accwcyatct tttattgggg ccagggcaca ctggtcaccg tgtcctctgc ctccaccaag 420accwcyatct tttattgggg ccagggcaca ctggtcaccg tgtcctctgc ctccaccaag 420
ggcccaagcg tgtttccact ggctccctgc tctcggtcca ccagcgagtc tacagccgct 480ggcccaagcg tgtttccact ggctccctgc tctcggtcca ccagcgagtc tacagccgct 480
ctgggctgtc tggtgaagga ttatttccct gagccagtga cagtgtcttg gaactccggc 540ctgggctgtc tggtgaagga ttatttccct gagccagtga cagtgtcttg gaactccggc 540
gccctgacct ctggagtgca cacatttcct gctgtgctgc agagctctgg cctgtactcc 600gccctgacct ctggagtgca cacatttcct gctgtgctgc agagctctgg cctgtactcc 600
ctgtccagcg tggtgaccgt gccatcttcc agcctgggca caaagaccta tacatgcaac 660ctgtccagcg tggtgaccgt gccatcttcc agcctgggca caaagaccta tacatgcaac 660
gtggaccata agccctccaa tacaaaggtg gataagagag tggagagcaa gtacggcccc 720gtggaccata agccctccaa tacaaaggtg gataagagag tggagagcaa gtacggcccc 720
ccttgcccac cctgtccagc tcctgagttc ttaggaggac catccgtgtt cctgtttcct 780ccttgcccac cctgtccagc tcctgagttc ttaggaggac catccgtgtt cctgtttcct 780
ccaaagccta aggacaccct gatgatcagc cgcaccccag aggtgacatg cgtggtggtg 840ccaaagccta aggacaccct gatgatcagc cgcaccccag aggtgacatg cgtggtggtg 840
gacgtgtctc aggaggatcc tgaggtgcag ttcaactggt atgtggatgg cgtggaggtg 900gacgtgtctc aggaggatcc tgaggtgcag ttcaactggt atgtggatgg cgtggaggtg 900
cacaatgcta agaccaagcc cagggaggag cagtttaata gcacctaccg ggtggtgtct 960cacaatgcta agaccaagcc cagggaggag cagtttaata gcacctaccg ggtggtgtct 960
gtgctgacag tgctgcatca ggactggctg aacggcaagg agtataagtg caaggtgtcc 1020gtgctgacag tgctgcatca ggactggctg aacggcaagg agtataagtg caaggtgtcc 1020
aataagggcc tgccttcttc catcgagaag accatcagca aggccaaggg ccagcctagg 1080aataagggcc tgccttcttc catcgagaag accatcagca aggccaaggg ccagcctagg 1080
gagccacagg tgtacacact gcccccttct caggaggaga tgaccaagaa ccaggtgtcc 1140gagccacagg tgtacacact gcccccttct caggaggaga tgaccaagaa ccaggtgtcc 1140
ctgacatgtc tggtgaaggg cttctatcct tctgacatcg ctgtggagtg ggagtccaat 1200ctgacatgtc tggtgaaggg cttctatcct tctgacatcg ctgtggagtg ggagtccaat 1200
ggccagccag agaacaatta caagaccaca ccacccgtgc tggacagcga tggctctttc 1260ggccagccag agaacaatta caagaccaca ccaccccgtgc tggacagcga tggctctttc 1260
tttctgtatt ccagactgac cgtggataag agccgctggc aggagggcaa cgtgttttcc 1320tttctgtatt ccagactgac cgtggataag agccgctggc aggagggcaa cgtgttttcc 1320
tgcagcgtga tgcatgaggc cctgcacaat cattacacac agaagtctct gtccctgagc 1380tgcagcgtga tgcatgaggc cctgcacaat cattacacac agaagtctct gtccctgagc 1380
ctgggcaagt aattaattaa 1400ctgggcaagt aattaattaa 1400
<210> 30<210> 30
<211> 723<211> 723
<212> DNA<212>DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)
<400> 30<400> 30
aagcttgccg ccaccatggg ctggtcttgt attatcctgt ttctggtggc tacagctaca 60aagcttgccg ccaccatggg ctggtcttgt attatcctgt ttctggtggc tacagctaca 60
ggcgtgcact ctgacatcgt gatgacccag tctccatcct ctctgtccgc ctctgtgggc 120ggcgtgcact ctgacatcgt gatgacccag tctccatcct ctctgtccgc ctctgtgggc 120
gacagagtga ccatcacatg caaggcsagc caggacatcr rgtatcagyt gwcbtggtat 180gacagagtga ccatcacatg caaggcsagc caggacatcr rgtatcagyt gwcbtggtat 180
cagcagaagc ctggcaaggc ccctaagaca ctgatctact ackcyrsykh tckggccgat 240cagcagaagc ctggcaaggc ccctaagaca ctgatctact ackcyrsykh tckggccgat 240
ggcgtgccct ctagattttc cggctctggc tctggccaag agtttaccct gacaatctcc 300ggcgtgccct ctagattttc cggctctggc tctggccaag agtttaccct gacaatctcc 300
agcctgcagc ctgaggactt cgccacctac tactgcgtty mgcackmyra yagacccttc 360agcctgcagc ctgaggactt cgccacctac tactgcgtty mgcackmyra yagacccttc 360
wcytttggcc agggcaccaa gctggaaatc aagaggaccg tggctgcccc cagcgtgttc 420wcytttggcc agggcaccaa gctggaaatc aagaggaccg tggctgcccc cagcgtgttc 420
atcttccctc ctagcgacga gcagctgaag agcggcaccg ctagcgtggt gtgtctgctg 480atcttccctc ctagcgacga gcagctgaag agcggcaccg ctagcgtggt gtgtctgctg 480
aataacttct atcccaggga ggccaaggtg cagtggaagg tggataacgc cctgcagagc 540aataacttct atcccaggga ggccaaggtg cagtggaagg tggataacgc cctgcagagc 540
ggcaactccc aggagtccgt gaccgagcag gactccaagg acagcaccta ctccctgagc 600ggcaactccc aggagtccgt gaccgagcag gactccaagg acagcaccta ctccctgagc 600
tccaccctga ccctgtccaa ggctgattat gagaagcaca aggtgtatgc ttgcgaggtg 660tccaccctga ccctgtccaa ggctgattat gagaagcaca aggtgtatgc ttgcgaggtg 660
acacaccagg gcctgtccag ccctgtgacc aagagcttca accggggcga gtgctaactc 720acacaccagg gcctgtccag ccctgtgacc aagagcttca accggggcga gtgctaactc 720
gag 723gag 723
Claims (8)
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310427097.3ACN116239694B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof |
| CN202210501892.8ACN114920842B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427094.XACN116514979B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments HLPV-P09 thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427100.1ACN116239695B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof HLPV-P11 that specifically bind to PV-1 protein and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210501892.8ACN114920842B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof |
Related Child Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310427094.XADivisionCN116514979B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments HLPV-P09 thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427100.1ADivisionCN116239695B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof HLPV-P11 that specifically bind to PV-1 protein and uses thereof |
| CN202310427097.3ADivisionCN116239694B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114920842A CN114920842A (en) | 2022-08-19 |
| CN114920842Btrue CN114920842B (en) | 2023-07-04 |
Family
ID=82809213
Family Applications (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210501892.8AActiveCN114920842B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427097.3AActiveCN116239694B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427094.XAActiveCN116514979B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments HLPV-P09 thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427100.1AActiveCN116239695B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof HLPV-P11 that specifically bind to PV-1 protein and uses thereof |
Family Applications After (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310427097.3AActiveCN116239694B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427094.XAActiveCN116514979B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments HLPV-P09 thereof that specifically bind to PV-1 protein and uses thereof |
| CN202310427100.1AActiveCN116239695B (en) | 2022-05-09 | 2022-05-09 | Antibodies or antigen binding fragments thereof HLPV-P11 that specifically bind to PV-1 protein and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (4) | CN114920842B (en) |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100092470A1 (en)* | 2008-09-22 | 2010-04-15 | Icb International, Inc. | Antibodies, analogs and uses thereof |
| CN102153647B (en)* | 2011-01-27 | 2012-07-04 | 中国人民解放军军事医学科学院生物工程研究所 | Anti-EGFR (epidemic growth factor receptor) humanized antibody L3-H3 and coding gene and application thereof |
| US9493552B2 (en)* | 2013-11-15 | 2016-11-15 | China Synthetic Rubber Corporation | Therapeutic biologic for treatment of hepatocellular carcinoma |
| EP3666901A1 (en)* | 2015-10-30 | 2020-06-17 | F. Hoffmann-La Roche AG | Methods of measuring factor d activity and potency of factor d inhibitors |
| TW201730211A (en)* | 2015-10-30 | 2017-09-01 | 建南德克公司 | Anti-Factor D antibodies and conjugates |
| CN105924524A (en)* | 2016-04-23 | 2016-09-07 | 同济大学苏州研究院 | Anti-c-Met chimeric antibody and application thereof |
| CN108997497B (en)* | 2018-03-30 | 2022-02-25 | 华兰基因工程有限公司 | Monoclonal antibody specifically binding to human plasma membrane vesicle associated protein PV-1 and preparation method and application thereof |
| EP4039707A4 (en)* | 2019-09-30 | 2022-11-16 | Harbour Biomed (Shanghai) Co., Ltd | CD3-TARGETING ANTIBODY, BISPECIFIC ANTIBODY AND USE THEREOF |
- 2022
- 2022-05-09CNCN202210501892.8Apatent/CN114920842B/enactiveActive
- 2022-05-09CNCN202310427097.3Apatent/CN116239694B/enactiveActive
- 2022-05-09CNCN202310427094.XApatent/CN116514979B/enactiveActive
- 2022-05-09CNCN202310427100.1Apatent/CN116239695B/enactiveActive
Also Published As
| Publication number | Publication date |
|---|---|
| CN116239695B (en) | 2024-03-22 |
| CN116239694B (en) | 2023-12-15 |
| CN114920842A (en) | 2022-08-19 |
| CN116514979B (en) | 2024-05-07 |
| CN116239694A (en) | 2023-06-09 |
| CN116239695A (en) | 2023-06-09 |
| CN116514979A (en) | 2023-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109937212B (en) | B7-H3 antibody, its antigen-binding fragment and its medical use | |
| JP6127043B2 (en) | AGR2 blocking antibody and use thereof | |
| CN111744013A (en) | Methods and pharmaceutical combinations for treating diseases using anti-TIGIT antibodies in combination with PD-1 inhibitors | |
| US20230138315A1 (en) | Anti-angptl3 antibody and use thereof | |
| WO2023208182A1 (en) | Anti-ccr8 antibody and use thereof | |
| KR20250027562A (en) | Antigen binding molecule specifically binding to DLL3 and CD3 and medicinal uses thereof | |
| CN115975034A (en) | TRBC2 antibody and its application | |
| CN114375308B (en) | Monoclonal antibodies against TFPI | |
| CN111378045B (en) | Bivalent bispecific antibody and preparation method thereof, encoding gene, host cell, and composition | |
| CN114920842B (en) | Antibodies or antigen binding fragments thereof that specifically bind to PV-1 protein and uses thereof | |
| CN118576543A (en) | A bispecific antibody preparation binding to human CD3/IL13Rα and its application | |
| WO2022156739A1 (en) | Antigen binding molecule specifically binding to bcma and cd3, and medical use thereof | |
| CN115925934A (en) | Humanized monoclonal antibody with improved stability and application thereof | |
| WO2021130717A1 (en) | Method for purifying biologically active peptide by using protein a affinity chromatography | |
| CN117567625B (en) | Claudin 18.2-resistant monoclonal antibody and application thereof | |
| EP4400512A1 (en) | Anti-ang2 antibody, preparation method therefor, and application thereof | |
| CN113980135B (en) | A kind of antidrug antibody combined with coronavirus bispecific antibody and preparation method and application thereof | |
| US20240417468A1 (en) | Trispecific Engineered Antibodies | |
| EP4585618A1 (en) | Anti-ilt4 antibody and pharmaceutical use thereof | |
| EP4435006A1 (en) | Bispecific antibody and use thereof | |
| CN117915950A (en) | Multispecific antibody and application thereof | |
| CN115850499A (en) | Antigen binding molecule specifically binding to ANGPTL3 and PCSK9 and medical application thereof | |
| CN117903299A (en) | Nano antibody capable of specifically recognizing AAV-DJ and application thereof | |
| CN120230205A (en) | Multispecific antibodies comprising a CCR8 antigen binding domain | |
| CN117304320A (en) | Nanobodies targeting mouse CLEC12B protein and their applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right | Effective date of registration:20230608 Address after:9/F, Building B, Innovation Incubation Base, South Zhongyuan Technopole, East Sanquan Road, Longhu Middle Ring West Road, Zhengzhou Area, Zhengzhou Pilot Free Trade Zone, 450000 Henan Province Applicant after:Hualan Gene Engineering (Henan) Co.,Ltd. Address before:453000 No.1-1 Huanghe Road, Pingyuan demonstration area, Xinxiang City, Henan Province Applicant before:HUALAN GENETIC ENGINEERING Co.,Ltd. | |
| GR01 | Patent grant | ||
| GR01 | Patent grant |