CN114920842A - Antibody or antigen binding fragment thereof specifically binding to PV-1 protein and application thereof - Google Patents

Antibody or antigen binding fragment thereof specifically binding to PV-1 protein and application thereof Download PDF

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CN114920842A
CN114920842A CN202210501892.8A CN202210501892A CN114920842A CN 114920842 A CN114920842 A CN 114920842A CN 202210501892 A CN202210501892 A CN 202210501892A CN 114920842 A CN114920842 A CN 114920842A
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amino acid
acid sequence
variable region
chain variable
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CN114920842B (en
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张静静
王斌
安文琪
邢体坤
宋路萍
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Hualan Gene Engineering Henan Co ltd
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Hualan Genetic Engineering Co ltd
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Priority to CN202310427097.3A priority Critical patent/CN116239694B/en
Priority to CN202310427100.1A priority patent/CN116239695B/en
Priority to CN202210501892.8A priority patent/CN114920842B/en
Priority to CN202310427094.XA priority patent/CN116514979B/en
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Abstract

The invention discloses an antibody or an antigen-binding fragment thereof specifically binding to PV-1 protein and application thereof. The antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the determinant complementary regions of the heavy chain variable region and the light chain variable region each consisting of CDR1, CDR2 and CDR 3; the amino acid sequence of CDR1 of the heavy chain variable region is set forth in SEQ ID NO: 03, etc. at positions 26-35; the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 03, etc. at positions 50-66; the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 03, etc. from position 97-101; the amino acid sequence of CDR1 of the light chain variable region is set forth in SEQ ID NO: 04, etc. from position 24 to 34; the amino acid sequence of CDR2 of the light chain variable region is as shown in SEQ ID NO: 04, etc. from position 50 to 56; the amino acid sequence of CDR3 of the light chain variable region is set forth in SEQ ID NO: 04, et al, 89-97. The affinity of the antibody or the antigen binding fragment thereof is improved by 1-2 orders of magnitude compared with the original antibody, the dosage can be theoretically reduced, and the clinical drug development of a new PV-1 target is accelerated.

Description

Antibody or antigen binding fragment thereof specifically binding to PV-1 protein and application thereof
Technical Field
The invention belongs to the field of monoclonal antibodies of biotechnology, and particularly relates to an antibody or an antigen binding fragment thereof specifically binding to PV-1 protein and application thereof.
Background
The biological organism substance exchange is that oxygen sucked by a respiratory system and nutrient substances absorbed by a digestive system firstly enter blood and then enter cells in vivo through tissue fluid; meanwhile, the waste and carbon dioxide generated by the metabolism of cells in the body enter tissue fluid and then enter blood to be conveyed to the urinary system and the respiratory system to be discharged out of the body.
The material exchange between blood vessel and tissue is an important component of the material exchange of biological body, and mainly passes through two routes of vascular endothelial cell gaps or micropores (or pits) with membranes. Plasma membrane vesicle associated protein (PLVAP, abbreviated as PV-1) has a molecular weight of about 55-60kD, and is the only endothelial vascular micropore membrane or plasma membrane caveolar membrane protein known at present. PV-1 is expressed at a low or no level in other tissues than in endocrine glands such as pituitary, adrenal gland and lung. When abnormal states such as tumor, anoxia, trauma and inflammation are accompanied by pathological changes of angiogenesis, the expression of PV-1 is obviously up-regulated.
The existence of many vascular-tissue barriers in a living body is of great importance in maintaining the normal physiological state of the living body, such as the vascular-brain barrier (blood-brain barrier) and the vascular-retinal barrier (blood-retinal barrier). There are no plasma membrane micropores or caves on the endothelial vessel wall in the vessel-tissue barrier, and no PV-1 protein expression, but in pathological conditions, such as ischemic cerebral apoplexy, primary or metastatic brain tumor, diabetic retinopathy, etc., the vessel-tissue barrier structure is destroyed, and micropores are formed on the endothelial vessel wall and PV-1 antigen expression is accompanied.
In summary, the PV-1 protein is expected to be a new target for treating Age-related Macular Degeneration (AMD), Diabetic Macular Edema (DME), and Macular Edema caused by retinal vein occlusion in adult patients. Database retrieval shows that the targets for treating the diseases on the market are Vascular Endothelial Growth Factor (VEGF), angiotensin II (AngII) and the like, and no products related to the PV-1 target are distributed in enterprises temporarily.
Disclosure of Invention
It is an object of the present invention to provide antibodies or antigen-binding fragments thereof that specifically bind to PV-1 protein.
The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to a PV-1 protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of the heavy chain variable region and the light chain variable region each consist of a CDR1, a CDR2, and a CDR 3;
the amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 03. the amino acid sequence of SEQ ID NO: 05. SEQ ID NO: 06. SEQ ID NO: 07. SEQ ID NO: 08. the amino acid sequence of SEQ ID NO: 09. SEQ ID NO: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO: 13 or SEQ ID NO: positions 26-35 of 14;
the amino acid sequence of cDR2 of the heavy chain variable region is shown as SEQ ID NO: 03. the amino acid sequence of 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 amino acid sequence of SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 or SEQ ID NO: positions 50-66 of 14;
the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 03. the amino acid sequence of SEQ ID NO: 05. the amino acid sequence of SEQ ID NO: 06. SEQ ID NO: 07. SEQ ID NO: 08. SEQ ID NO: 09. SEQ ID NO: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: positions 97-101 of 14;
the amino acid sequence of the CDR1 of the light chain variable region is as shown in SEQ ID NO: 04. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. the amino acid sequence of SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 20. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25 at positions 24-34;
the amino acid sequence of CDR2 of the light chain variable region is shown as SEQ ID NO: 04. SEQ ID NO: 15. the amino acid sequence of SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, positions 50-56;
the amino acid sequence of the CDR3 of the light chain variable region is as shown in SEQ ID NO: 04. SEQ ID NO: 15. SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO: 22. the amino acid sequence of SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25 at positions 89-97.
The antibody can be a full-length antibody, and the antigen-binding fragment can be a Fab fragment, a Fv fragment, a Fab 'fragment, a F (ab') 2 fragment, a single chain antibody (ScFv), a nanobody (single domain antibody), a bispecific antibody, or a Minimal Recognition Unit (MRU).
Alternatively, according to the antibody or antigen-binding fragment thereof described above, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 03. the amino acid sequence of SEQ ID NO: 05. the amino acid sequence of SEQ ID NO: 06. SEQ ID NO: 07. the amino acid sequence of SEQ ID NO: 08. the amino acid sequence of SEQ ID NO: 09. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 or SEQ ID NO: as shown at 14.
Alternatively, according to the antibody or antigen-binding fragment thereof described above, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 04. SEQ ID NO: 15. SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: shown at 25.
Alternatively, the antibody or antigen-binding fragment thereof is any one of the following:
an antibody designated HLPV-P01, the HLPV-P01 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 03, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 04 is shown;
an antibody designated HLPV-P02, the HLPV-P02 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 05, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 15 is shown in the figure;
an antibody designated HLPV-P03, the antibody HLPV-P03 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 06, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 16 is shown;
an antibody designated HLPV-P04, the HLPV-P04 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 07, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 17 is shown;
an antibody designated HLPV-P05, the HLPV-P05 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 08, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 18 is shown in the figure;
an antibody designated HLPV-P06, the HLPV-P06 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 09, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16 is shown;
an antibody designated HLPV-P07, the HLPV-P07 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 19 is shown in the figure;
an antibody designated HLPV-P08, the HLPV-P08 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 20 is shown;
an antibody designated HLPV-P09, the HLPV-P09 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 11, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 21 is shown;
an antibody designated HLPV-P10, the HLPV-P10 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 08, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 22;
an antibody designated HLPV-P11, the HLPV-P11 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 11, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 23 is shown;
an antibody designated HLPV-P12, the HLPV-P12 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 24, respectively;
an antibody designated HLPV-P13, the HLPV-P13 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 25 is shown;
an antibody designated HLPV-P14, the HLPV-P14 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 25.
The antibody subtype may be human antibody such as IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM type antibody, etc.; the antibody subtypes are IgG1(N297A) and IgG4(S228P) which remove ADCC effect.
Optionally, the antibody or antigen-binding fragment thereof according to the above further comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region is a heavy chain constant region of an IgG1, an IgG1 variant, an IgG4 or an IgG4 variant. The light chain constant region is that of Kappa or a variant thereof. The antibody can be specifically a heavy chain constant region fused at the carbon end (C end) of a heavy chain variable region, and a light chain constant region fused at the carbon end (C end) of a light chain variable region.
The amino acid sequence of the heavy chain constant region of IgG1 was obtained by introducing a N297A mutation (EU numbering) into the heavy chain constant region of IgG1 as shown in UniProtKB database sequence No. P01857.1(2022.02.23 latest update). 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, as in the UniProtKB database with sequence No. P01861.1(2022.02.23 latest update) IgG 4. The amino acid sequence of the Kappa light chain constant region is shown in the UniProtKB database sequence No. P01834.2(2022.02.23 latest updates).
The nucleotide sequence of the gene encoding the heavy chain constant region can be shown as positions 409-1398 of SEQ ID No.28 or 1389 of SEQ ID No. 29. The nucleotide sequence of the gene encoding the light chain constant region may be represented by positions 394-714 of SEQ ID No. 30.
The invention also provides a related biological material of the antibody or the antigen binding fragment thereof, wherein the related biological material is any one of the following materials:
B1) nucleic acid molecules encoding the above antibodies or antigen-binding fragments thereof;
B2) nucleic acid molecules encoding the heavy and/or light chains of the above antibodies or antigen-binding fragments thereof;
B3) a nucleic acid molecule encoding the heavy chain variable region and/or the light chain variable region of the above antibody or an antigen-binding fragment thereof;
B4) an expression cassette comprising any one of the nucleic acid molecules of B1) -B3);
B5) a recombinant vector containing any one of the nucleic acid molecules B1) -B3), or a recombinant vector containing the expression cassette of B4);
B6) a recombinant microorganism comprising any one of the nucleic acid molecules B1) -B3), or a recombinant microorganism comprising B4) the expression cassette, or a recombinant microorganism comprising B5) the recombinant vector;
B7) a cell line comprising any one of the nucleic acid molecules B1) -B3), or a cell line comprising the expression cassette B4), or a cell line comprising the recombinant vector B5).
Alternatively, the biological material may be, according to the related biological material described above,
B2) the nucleic acid molecule of the heavy chain of the coded antibody or the antigen binding fragment thereof is the nucleic acid molecule with the nucleotide sequence shown as SEQ ID No.28 or SEQ ID No. 29;
B2) the nucleic acid molecule of the light chain of the antibody or the antigen binding fragment thereof is a nucleic acid molecule with the nucleotide sequence shown as SEQ ID No. 30;
B3) the nucleic acid molecule encoding the heavy chain variable region of the antibody or the antigen binding fragment thereof is the nucleic acid molecule with the nucleotide sequence shown in SEQ ID No.26, wherein the CDR1, CDR2 and CDR3 are the corresponding 76-105, 148-198 and 289-303 bases respectively;
B3) the nucleic acid molecule encoding the light chain variable region of the antibody or the antigen binding fragment thereof is the nucleic acid molecule with the nucleotide sequence shown in SEQ ID No.27, wherein the CDR1, CDR2 and CDR3 are the corresponding bases 70-102, 148-168 and 278-291.
Vectors described herein are well known to those skilled in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), viral vectors (e.g., baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpes viruses (e.g., herpes simplex virus), etc.). In one embodiment of the invention, the vector may specifically be pUC57 or pCGS 3. Specifically, the recombinant expression plasmids include pCGS3-HLPV-P00 to P14 recombinant expression plasmids, pCGS3-HLPV-P03(S228P), pCGS3-HLPV-P05(S228P), pCGS 3-HLPV-P3 (S228 3), pCGS 3-HLPV-P3 (N297 3), pCGS 3-HLPV-P3 (N36297), pCGS 3-HLPV-3 (N36297), pCGS3-HLPV 36297 (N3-3) and pCGS3-HLPV 3 (N3-36297), pCGS3-HLPV 3-3) or pCGS3-HLPV 3.
The microorganism described herein may be a yeast, a bacterium or a fungus. Wherein the bacteria can be derived from Escherichia (Escherichia), Erwinia (Erwinia), Agrobacterium tumefaciens (Agrobacterium), Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc.; the yeast can be Pichia pastoris (P.
The cell line (host cell) refers to a cell that can be used for introducing a vector, and includes, but is not limited to: eukaryotic cells (e.g., yeast cells, aspergillus), animal cells (e.g., mammalian cells, insect cells), or prokaryotic cells. In one embodiment of the invention, the cell line may specifically be an ExpicHO-S cell.
The terms "cell" and "cell line" are used interchangeably, and all such designations include progeny thereof.
The invention also provides a preparation method of the antibody or the antigen binding fragment thereof, which specifically comprises the following steps: introducing the coding gene of the antibody or the antigen binding fragment thereof into host cells through a recombinant expression vector containing the coding gene to obtain recombinant cells, culturing the supernatant of the recombinant cells, and purifying through affinity chromatography to obtain the target protein.
In the above method, the host cell may be a eukaryotic cell, such as a CHO cell, a HEK293 cell, a yeast cell, an insect cell, or the like. In one embodiment of the invention, the animal cell is a CHO cell.
The invention also provides a pharmaceutical composition for improving, preventing or treating diseases related to PV-1 overexpression, wherein the pharmaceutical composition comprises the antibody or the antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.
The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, or a lubricant, but is not limited thereto.
The invention also provides a reagent or a kit for detecting PV-1, wherein the reagent or the kit contains any one of the antibodies or the antigen-binding fragments thereof.
The invention also provides a conjugate (conjugate) comprising an antibody or antigen-binding fragment thereof as described herein, and a detectable label linked to the antibody or antigen-binding fragment thereof; in particular, the detectable label may be selected from enzymes (e.g. horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g. acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g. fluorescein or fluorescent proteins), radionuclides or biotin.
The following uses of the above antibody or antigen binding fragment thereof, and/or the above biomaterial are also within the scope of the present invention:
D1) the use in the manufacture of a medicament for ameliorating, preventing or treating a disease associated with PV-1 overexpression;
D2) the application in preparing products for diagnosing or screening diseases related to PV-1 overexpression;
D3) the application in detecting PV-1;
D4) use in the preparation of a product for detecting PV-1;
D5) use in the manufacture of a product for binding to a PV-1 protein.
The PV-1 overexpression-related diseases can comprise age-related macular degeneration, diabetic macular edema, macular edema caused by retinal vein occlusion of an adult patient, liver cancer, ischemic cerebral stroke and/or cerebral edema.
The products for detecting PV-1 comprise products for detecting antigen-antibody combination by utilizing an enzyme-linked immunosorbent assay, an immunofluorescence detection method, a radioimmunoassay, a luminescence immunoassay method, a colloidal gold immunochromatography, an agglutination method, an immunoturbidimetry and the like.
Further, the product may be a reagent, a kit or a chip.
The medicament, reagent, kit or chip of the invention contains any one of the antibodies or antigen binding fragments thereof or combination thereof. The kit can be a chemiluminescence immunoassay kit, an enzyme-linked immunoassay kit, a colloidal gold immunoassay kit or a fluorescence immunoassay kit, but is not limited thereto.
The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, the antigen-binding fragment retains at least 10% of maternal binding activity when expressed on a molar basis. In particular, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target.
The term "Fab fragment" is a heterodimer of a heavy chain Fd and an intact light chain, which contains only one antigen binding site, bound by a disulfide bond. The encoding genes of the heavy chain Fd and the complete light chain are connected, and the Fab antibody (Fab fragment) can be expressed in the escherichia coli incretion after the fusion of the bacterial protein signal peptide gene, and the Fab antibody has complete three-dimensional folding and intra-chain and inter-chain disulfide bonds. The heavy chain Fd refers to the H chain portion of about 1/2 (about 225 amino acid residues including VH, CH1 and part of the hinge region) in a Fab.
The term "Fv fragment" refers to a vector comprising the VH and VL genes, respectively, which can be co-transfected into a cell, expressed separately, and assembled into a functional Fv antibody; a termination code can also be arranged between VH and VL in the vector, two small molecular protein fragments are respectively expressed and combined through a non-covalent bond to form an Fv antibody (Fv fragment).
The term "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.
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, a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
The term "single chain antibody (ScFv)" refers to the expression of a single polypeptide chain by linking the light and heavy chain variable region genes using an appropriate oligonucleotide linker (1inker), and is called a single chain antibody (ScFv). The polypeptide chain is capable of spontaneously folding into its native conformation, retaining the specificity and affinity of the Fv.
The term "nanobody (single domain antibody)" refers to an antibody obtained by genetically engineering the heavy chain V region of an antibody to contain only VH fragments. The ability of single domain antibodies to bind antigen and their stability are essentially identical to those of full antibodies.
The term "bispecific antibody" refers to a bispecific antibody obtained by introducing two sets of light chain and heavy chain genes into myeloma cells, and selecting appropriate antibody constant regions and Ig classes, with high yield, uniformity and purity. In addition, bispecific antibodies can also be obtained by chemical cross-linking techniques or hybrid-hybridoma techniques.
The term "Minimal Recognition Unit (MRU)" means that it contains only a single CDR structure in the variable region, has a molecular mass of only about 1% of that of the whole antibody, and binds to the corresponding antigen.
The antibodies of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, followed by transfection of host cells, culture of the transfected host cells under specific conditions, and expression of the antibody of the present invention. The host cell, the expression vector, a method for introducing the expression vector into the host cell, and a method for isolating and purifying the antibody, which are conventional in the art, can be selected as necessary by those skilled in the art.
It is well known to those skilled in the art that such antigen-binding fragments can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
According to the embodiment of the invention, a group of affinity mature antibodies specifically binding to human plasma membrane vesicle associated protein PV-1 is obtained through two phage screening experiments, the affinity is improved by 1-2 orders of magnitude compared with the original antibody, the dosage can be theoretically reduced, the age-related macular degeneration (AMD), the Diabetic Macular Edema (DME) and other PV-1 overexpression related diseases are treated, and the clinical drug development of a new PV-1 target is accelerated.
Drawings
FIG. 1 shows the construction of a nucleic acid electrophoresis identification chart of the full anti-assembly expression plasmid with the phage display sequence IgG 1; the upper diagram is an IgG1 full-antibody assembly expression plasmid light chain construction nucleic acid electrophoresis identification diagram, a vector fragment 9535bp and a light chain 723bp are obtained by double enzyme digestion of HindIII and XhoI, wherein a lane A is pCGS3-HLPV-P00L, B is pCGS3-HLPV-P01L, C is pCGS3-HLPV-P02L, D is pCGS3-HLPV-P03/06L, E is pCGS3-HLPV-P04L, F is pCGS3-HLPV-P05L, G is pCGS3-HLPV-P07L, H is pCGS3-HLPV-P L, I is pC 3-HLPV-P3609, J is pCGS L-HLPV-P10L, K is pCGS L-HLPV-P11L, GS 72-HLPV 72 is pCGS L-P L, HLPV 72-P L is HLPV 72, and HLPV-P14/L is pCGS L/L; the lower diagram is an IgG1 full-anti-assembly expression plasmid heavy chain construction acid electrophoresis identification diagram, a vector fragment 10200bp and a light chain 1409bp are obtained theoretically after enzyme digestion by BstBI and PacI, wherein lane numbers correspond to antibody labels in sequence, namely a lane 00 is pCGS3-HLPV-P00, a lane 01 is pCGS3-HLPV-P00, a lane 02 is pCGS3-HLPV-P02, and a lane 14 is pCGS3-HLPV-P14 expression plasmid.
FIG. 2 shows the SDS-PAGE protein electrophoretogram of the assembled full-antibody protein of the phage display sequence IgG 1; wild-type IgG1 was assembled into a total anti-protein and identified by SDS-PAGE after purification (non-reduced to non-reduced SDS-PAGE, reduced to reduced SDS-PAGE), wherein lane numbering corresponds to antibody designation in the order, i.e., lane 00 is HLPV-P00, lane 01 is HLPV-P01, lane 02-HLPV-P02.. lane 14 is HLPV-P14 antibody.
FIG. 3 shows the nucleic acid electrophoresis identification chart of the construction of the ADCC effect-removed full anti-assembly expression plasmid; construction only changes heavy chain base sequence, BstBI and PacI are adopted for double enzyme digestion, a vector fragment 10200bp and a light chain 1409bp are obtained theoretically after enzyme digestion in lanes 1 to 7, and a vector fragment 10200bp and a light chain 1400bp are obtained theoretically after enzyme digestion in lanes 8 to 14. Wherein, 1 is pCGS3-HLPV-P03(N297A), 2 is pCGS3-HLPV-P05(N297A), 3 is pCGS3-HLPV-P08(N297 695 297A), 4 is pCGS3-HLPV-P09(N297A), 5 is pCGS3-HLPV-P10(N297A), 6 is pCGS 3-HLPV-P3 (N297 3), 7 is pCGS 3-HLPV-P3 (N297 3), 8 is pCGS 3-HLPV-P3 (S228 3), 9 is pCGS 3-HLPV-P3 (S228 3), 10 is pCGS 3-HLPV-P3 (S36228), 11 is pCGS 3-HLPV-P3 (S228), 12 is pCGS3-HLPV 228-HLPV-P3), 10 is pCGS 3-HLPV-36228 (S3), and expression is pCGS 3-HLPV-3 (S3) or pCGS3-HLPV 3-3 plasmid (S3) or pCGS3-HLPV 3-36228.
FIG. 4 shows an assembled SDS-PAGE protein electrophoretogram of total antibody purified by eliminating ADCC effect; the upper graph is AN IgG1(N297) type ADCC effector antibody-removed SDS-PAGE protein electrophoresis graph, and the lower graph is AN IgG4(S228P) type ADCC effector antibody-removed SDS-PAGE protein electrophoresis graph, wherein AN represents purified waste liquid non-reduced SDS-PAGE, BN represents purified protein non-reduced SDS-PAGE, AR represents purified waste liquid reduced SDS-PAGE, and BR represents purified protein reduced SDS-PAGE. Wherein the lane numbering corresponds to the antibody designation in the order of lane 03 being HLPV-P03, lane 05 being HLPV-P05, lane 08 being HLPV-P08, lane 09 being HLPV-P09, lane 10 being HLPV-P10, lane 11 being HLPV-P11, lane 14 being HLPV-P14 antibody.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The main reagents and their manufacturer information in the following examples are as follows:
ExpiCHO-S TM cells: thermo corporation;
ExpiCHO TM expression Medium: thermo corporation;
ExpiFectamine TM CHO transduction Kit: thermo corporation;
pCGS3 expression vector: merck, Inc.;
recombinant human PLVAP/PV-1 protein: abcam Corp;
protein a prepackaged chromatography column: bio-engineering (shanghai) corporation;
Centrifugal Filters
Figure BDA0003633779020000091
-10K: millipore Corp;
Centrifugal Filters 0.5ml 10K
Figure BDA0003633779020000092
-10K: millipore corporation;
PBS ph7.4(1 ×): thermo corporation;
Sure PAGE TM Bis-Tris, 10 × 8, 4-12%, 12 wells: nanjing Kinsrui Biotech Ltd.
The key instruments and their manufacturer information in the following examples are as follows:
gel imaging system: protein Simple corporation;
eStain TM l1 protein staining instrument: nanjing Kinsrui Biotechnology Ltd;
biacore T200 intermolecular interaction analysis system: GE company.
Example 1 affinity maturation assay
This example was based on the original antibody (designated HLPV-P00, with the heavy chain variable region shown in SEQ ID NO: 01 and the light chain variable region shown in SEQ ID NO: 02) screened for affinity maturation by phage display. The phage display mutation adopts four combination modes to carry out single-point or double-point saturation mutation and design a mutation library. The theoretical library capacity size was calculated as CDR1 (number of amino acids) × 20 (20 possibilities per site) × CDR2 (number of amino acids) × 020 × CDR3 (number of amino acids) × 20. Taking the storage capacity No. 01 as an example, the storage capacity is 9 × 20 × 11 × 20 × 9 × 20 ═ 3.96 × 10 6 (Table 1).
Table 1: antibody affinity maturation experiment library volume statistical table
Library number Combination mode Mode of mutation Theoretical storage capacity
01 LCDR1+LCDR3+HCDR3 Single point combinatorial mutagenesis 3.96×10 06
02 LCDR2+HCDR1+HCDR2 Single point combinatorial mutagenesis 5.60×10 06
03 LCDR3 Double point mutation 3.20×10 06
04 LCDR3+HCDR3 Double point combined mutation 5.12×10 06
The first phage screening experiment failed, and the antibody affinity was improved by less than one order of magnitude. The first round of phage screening experiment assembled 11 full-length antibodies, wherein 5 antibodies improved affinity more than 2 times, the highest affinity antibody improved 4.8 times, the highest affinity antibody was named as HLPV-P01, and the heavy chain variable region is shown as SEQ ID NO: 03, and the light chain variable region is shown as SEQ ID NO: 04, respectively.
And in the second phage screening experiment, affinity maturation screening is carried out on the basis of the optimal antibody HLPV-P01 obtained in the first experiment through the designed antibody library shown in the table 1 again to obtain affinity maturation antibody names HLPV-P02-14 in sequence, wherein the sequences of the variable regions of the encoding antibodies are shown in the table 2.
Table 2: statistical table of affinity maturation screening antibody sequences
Antibodies Heavy chain variable region Light chain variable region Remarks to note
HLPV-P00 SEQ ID NO：01 SEQ ID NO：02 Original antibody
HLPV-P01 SEQ ID NO：03 SEQ ID NO：04 Intermediate antibodies
HLPV-P02 SEQ ID NO：05 SEQ ID NO：15
HLPV-P03 SEQ ID NO：06 SEQ ID NO：16
HLPV-P04 SEQ ID NO：07 SEQ ID NO：17
HLPV-P05 SEQ ID NO：08 SEQ ID NO：18
HLPV-P06 SEQ ID NO：09 SEQ ID NO：16
HLPV-P07 SEQ ID NO：10 SEQ ID NO：19
HLPV-P08 SEQ ID NO：11 SEQ ID NO：20
HLPV-P09 SEQ ID NO：11 SEQ ID NO：21
HLPV-P10 SEQ ID NO：08 SEQ ID NO：22
HLPV-P11 SEQ ID NO：11 SEQ ID NO：23
HLPV-P12 SEQ ID NO：12 SEQ ID NO：24
HLPV-P13 SEQ ID NO：13 SEQ ID NO：25
HLPV-P14 SEQ ID NO：14 SEQ ID NO：25
Example 2 Assembly of full-antibody molecular validation affinity experiments
1 recombinant expression plasmid construction experiment
Affinity matured monoclonal antibody heavy and light chain variable region sequences were fused to IgG1-Fc and kappa chain constant region sequences (heavy chain variable region carbon end fused to IgG1-Fc, light chain variable region carbon end fused to kappa constant region), respectively, codon optimized and committed for synthetic biosynthesis according to example 1 table 2, heavy chain gene sequences were ligated after restriction endonuclease SmaI single cleavage site of the pUC57 cloning vector, light chain gene sequences were ligated between restriction endonuclease SmaI and HindIII cleavage site of the pUC57 cloning vector, corresponding to synthetic plasmid names as in table 3.
Table 3: total antibody expression combined heavy and light chain plasmid statistical table
Antibodies Heavy chain variable region Heavy chain plasmid Light chain variable region Light chain plasmids
HLPV-P00 SEQ ID NO：01 pUC57-00-H SEQ ID NO：02 pUC57-00-L
HLPV-P01 SEQ ID NO：03 pUC57-01-H SEQ ID NO：04 pUC57-01-L
HLPV-P02 SEQ ID NO：05 pUC57-02-H SEQ ID NO：15 pUC57-02-L
HLPV-P03 SEQ ID NO：06 pUC57-03-H SEQ ID NO：16 pUC57-03/06-L
HLPV-P04 SEQ ID NO：07 pUC57-04-H SEQ ID NO：17 pUC57-04-L
HLPV-P05 SEQ ID NO：08 pUC57-05/10-H SEQ ID NO：18 pUC57-05-L
HLPV-P06 SEQ ID NO：09 pUC57-06-H SEQ ID NO：16 pUC57-03/06-L
HLPV-P07 SEQ ID NO：10 pUC57-07-H SEQ ID NO：19 pUC57-07-L
HLPV-P08 SEQ ID NO：11 pUC57-08/09/11-H SEQ ID NO：20 pUC57-08-L
HLPV-P09 SEQ ID NO：11 pUC57-08/09/11-H SEQ ID NO：21 pUC57-09-L
HLPV-P10 SEQ ID NO：08 pUC57-05/10-H SEQ ID NO：22 pUC57-10-L
HLPV-P11 SEQ ID NO：11 pUC57-08/09/11-H SEQ ID NO：23 pUC57-11-L
HLPV-P12 SEQ ID NO：12 pUC57-12-H SEQ ID NO：24 pUC57-12-L
HLPV-P13 SEQ ID NO：13 pUC57-13-H SEQ ID NO：25 pUC57-13/14-L
HLPV-P14 SEQ ID NO：14 pUC57-14-H SEQ ID NO：25 pUC57-13/14-L
Wherein 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 complete light chain genes respectively encoding light chain variable region genes (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, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 20, SEQ ID NO: 24 and SEQ ID NO: 25) and a human Kappa constant region (Kappa constant region) light chain gene. Wherein the nucleotide sequence of the human Kappa constant region (Kappa constant region) light chain gene is shown as position 394-714 of SEQ ID No.30, and the human Kappa constant region (Kappa constant region) light chain gene expresses the human Kappa constant region having an amino acid sequence shown as UniProtKB database sequence No. P01834.2(2022.02.23 latest update).
Wherein 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 comprising an intact heavy chain gene obtained by separately ligating the 3' end of a heavy chain variable region gene (encoding genes of 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, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14) with a human 1 constant region (IgG 1-IgG-H) The resulting fusion gene was ligated. Wherein the nucleotide sequence of the heavy chain gene of the constant region of the human IgG1 is shown as 409-1398 of SEQ ID No.28, and the amino acid sequence of the heavy chain gene of the constant region of the human IgG1 is shown as the constant region of the human IgG1 shown by the UniProtKB database sequence number P01857.1(2022.02.23 is updated newly).
The construction scheme of the recombinant expression plasmid comprises the steps of firstly constructing a light chain, constructing a heavy chain on the basis of the light chain construction according to different combination modes in a table 3 after the light chain construction is finished, finally forming the double-expression-frame recombinant expression plasmid, and constructing 13 light chain intermediate plasmids and 15 full-antibody molecules in total, wherein the specific steps are as follows:
the first step is to construct a full antibody light chain, and the operation steps are as follows: a9535 bp vector fragment and a 13-723 bp light chain fragment were obtained by double-digesting 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 with HindIII (NEB) and XhoI (NEB). T4 ligase (TAKARA) is used for ligation, transformation, plate coating, plasmid extraction, HindIII and XhoI double enzyme digestion identification, and the enzyme digestion band is in accordance with the expectation. The correctly sequenced plasmids were designated 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 (FIG. 1). In the figure, lanes A-M are the electrophoresis results of the intermediate plasmid cut products of 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, pCGS 3-HLPV-GS P11L, pCGS3-HLPV-P12L, pCGS3-HLPV-P13/14L in sequence.
The second step is to construct the heavy chain of the whole antibody molecule and construct the whole antibody molecule according to different combination modes in table 3: the 13 intermediate plasmids thus constructed and 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 heavy chain plasmids were double-digested with BstBI (NEB) and PacI (NEB) to obtain 13 10200bp vector fragments and 12 1409bp heavy chain fragments, which were ligated with T4 ligase (TAKARA), transformed, plated, extracted plasmids BstBI, BstBI and PacI, and the digested bands were identified as expected (FIG. 1). The plasmids with correct sequencing were designated pCGS3-HLPV-P00 to P14, respectively. Lanes 01-14 in the figure are the electrophoresis results of the cleavage products of pCGS 3-HLPV-P00-P14, respectively.
Each recombinant expression plasmid of pCGS 3-HLPV-P00-P14 contains 1409bp heavy chain gene fragment and 723bp light chain gene fragment, and in the nucleotide sequences of the heavy chain gene fragment and the light chain gene fragment, only the nucleotide sequences for encoding the heavy chain variable region and the light chain variable region are different, and 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 sequences HLPV-P02 to P14 of the invention is shown as SEQ ID No.26 (Table 4), and the coding sequence of the light chain variable region is shown as 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, and N is A or T or G or C.
TABLE 4 statistical table of degenerate bases of SEQ ID No.26 sequence
Figure BDA0003633779020000131
TABLE 5 statistical table of degenerate bases of the sequence SEQ ID No.27
Figure BDA0003633779020000132
Figure BDA0003633779020000141
The heavy chain gene fragment is shown as SEQ ID NO: 28, BstBI enzyme cutting sites are positioned at 1-6, a Kozak sequence is positioned at 7-15, a signal peptide gene is positioned at 16-72, a heavy chain gene sequence is positioned at 73-1398, a stop codon is positioned at 1399-1401, a PacI enzyme cutting site is positioned at 1402-1409, wherein the nucleotide sequence is positioned at 73-408 and is a coding sequence of a heavy chain variable region, and the constant region sequence is positioned at 409-1398.
The light chain gene segment is shown as SEQ ID NO: 30, HindIII restriction site at the 1-6 position, a Kozak sequence at the 7-15 position, a signal peptide at the 16-72 position, a light chain gene sequence at the 73-714 position, a stop codon at the 715-717 position, an XhoI restriction site at the 718-723 position, wherein the nucleotide at the 73-393 position is a coding sequence of a light chain variable region, and a constant region sequence at the 394-714 position.
2 transient expression experiment of recombinant expression plasmid
The volume of original plasmid required to transfect 50mL of cells was calculated according to the amount of plasmid transfected at 0.8. mu.g per mL of cell culture and the original concentration of plasmid. 15 recombinant expression plasmids and transfection reagent Expifeactine TM CH OTransfection Kit (Thermo Co.) Compound was slowly dropped into cell culture medium ExpCHO containing cells of ExpCHO-S (Thermo Co.) to thereby obtain a cell culture TM Expression Medium (Thermo corporation) was cultured to express 15 antibody molecules, and the cell culture was shaken with the Medium being added to disperse the DNA and transfection reagent complexes uniformly. Feeding and enhancing agents were added at 18 to 22h after transfection at maximum titer while lowering the culture conditions from 37 ℃ to 32 ℃ with CO 2 The concentration is reduced from 8% to 5%; feeding was again added on day 5 post transfection. When the survival rate is reduced to 65% -75%, the culture is stopped to obtain the culture. The resulting cultures were designated CHO-S/pCGS3-HLPV-00 to 14 in sequence for a total of 15 cultures.
3 assembled Total antibody protein purification experiment
The above culture was centrifuged at 6000g for 30min, and the supernatant was collected and centrifuged using an ultrafiltration tube, Centrifugal Filters U1
Figure BDA0003633779020000142
And (3) carrying out ultrafiltration concentration at-10K (Millipore company), uniformly mixing the supernatant and binding/washing buffer (kit component of the biological Prot A prepacked chromatographic column) according to the volume ratio of 1: 1, standing for 20min, and fully incubating to obtain a uniform mixing solution of the ultrafiltration concentrated supernatant and the binding/washing buffer. Binding/washing buffer solution of five column volumes equilibrates the column (kit component for raw biological Protein a pre-packed chromatography column), and the ultrafiltrated concentrated supernatant and the binding/washing buffer mix are added to the column and run through the pre-packed column by gravity flow. The column was washed with 10-15 column volumes of binding/washing buffer and the flow-through was collected. Repeating this step using a new collection tube until the absorbance of the flow-through solution at 280nm approaches the baseline; eluting the recombinant protein on the column with 5-10 column volumes of elution buffer, Centrifugal Filters 0.5ml 10K
Figure BDA0003633779020000151
Ultrafiltration concentration at-10K (Millipore) was replaced with PBS (pH7.4) buffer. The purified monoclonal antibody was identified using reduced SD S-PAGE and the detection showed that the monoclonal antibody bands were as expected (FIG. 2). Total anti-sample HLPV-P00-P14 antibodies were from CHO-S/HLPV-00-14 cultures, respectively.
4 full antibody molecular affinity assay
The surface plasmon resonance technology is adopted to detect the binding affinity of 15 antibodies in total obtained from HLPV-P00 to P14 and PV-1 protein. Conjugate recombinant human PLVAP/PV-1 protein (Abcam company) is coupled to a Series S Sensor Chip CM5 Chip (GE company), a Biacore T200 intermolecular interaction analysis system (GE company) is arranged on the machine, an antibody obtained by the affinity maturation of an analyte flows through the surface of the Chip (the temperature is 25 ℃, the flow rate is 30 mu l/min, the combination time is 120S, and the dissociation time is 180S), if the conjugate and the analyte have the combination activity, the refractive index change of the surface of a gold film is caused, and finally the SPR angle is changed. By detecting SPR angle changes, the Biacore T200 Evaluation Software program Software outputs raw data and performs kinetic fitting analysis in a 1: 1 binding mode, and affinity constants of the affinity matured antibody and the PV-1 protein are shown in Table 6.
TABLE 6 statistical table of affinity maturation holo-antibodies
Antibodies Antibody types Affinity constant Multiple of Remarks to note
HLPV-P00 IgG1-Fc 3.002×10 -8 1.00 Original antibody
HLPV-P01 IgG1-Fc 4.107×10 -9 7.31 Intermediate antibodies
HLPV-P02 IgG1-Fc 2.477×10 -9 6.29
HLPV-P03 IgG1-Fc 2.239×10 -9 13.41
HLPV-P04 IgG1-Fc 2.141×10 -9 14.02
HLPV-P05 IgG1-Fc 1.163×10 -9 25.81
HLPV-P06 IgG1-Fc 3.353×10 -9 8.95
HLPV-P07 IgG1-Fc 2.636×10 -9 11.39
HLPV-P08 IgG1-Fc 7.823×10 -10 38.37
HLPV-P09 IgG1-Fc 8.149×10 -10 36.84
HLPV-P10 IgG1-Fc 1.284×10 -9 23.38
HLPV-P11 IgG1-Fc 7.414×10 -10 40.49
HLPV-P12 IgG1-Fc 7.317×10 -9 4.10
HLPV-P13 IgG1-Fc 3.230×10 -9 9.29
HLPV-P14 IgG1-Fc 3.499×10 -9 8.58
As can be seen from table 6, after verification of phage display experiments and all-anti molecule affinity experiments twice, 14 affinity mature antibody molecules are obtained in total, the affinity is significantly improved compared with the original antibody, and the affinity of the highest antibody HLPV-P11 is improved by 40.49 times (table 6).
Example 3 affinity study of antibody to remove ADCC Effect subtypes
1 recombinant expression plasmid construction experiment
The clinical application of the target PV-1 does not need the ADCC effect of the antibody, so seven affinity mature antibodies are optionally selected, and IgG1(N297A) and IgG4(S228P) subtype antibody recombinant expression plasmids (Table 7) for weakening the ADCC effect are constructed and used for verifying the influence of different antibodies for removing the ADCC effect subtype on the affinity.
TABLE 7 statistical tables required for ADCC Effector subtype antibody construction
Figure BDA0003633779020000161
The IgG1(N297A) and IgG4(S228P) plasmids were obtained after ligating the heavy chain gene sequence into the restriction endonuclease SmaI single cleavage site of the pUC57 cloning vector.
Wherein pUC57-03-H (N297A), pUC57-05/10-H (N297A), pUC57-08/09/11-H (N297A), pUC57-14-H (N297A) contain the whole heavy chain gene which is a fusion gene obtained by respectively linking the 3' end of the heavy chain variable region gene (the encoding genes of SEQ ID NO: 06, SEQ ID NO: 08, SEQ ID NO: 11 and SEQ ID NO: 14) with the human IgG1(N297A) gene. Wherein the nucleotide sequence of the IgG1(N297A) gene is SEQ ID NO: 28, IgG1(N297A) expressed by IgG1(N297A) gene differed from the human IgG1 constant region only in that the 297 th aspartic acid was mutated to alanine.
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 genes which are fusion genes obtained by respectively connecting the 3' end of the heavy chain variable region gene (encoding genes of SEQ ID NO: 06, SEQ ID NO: 08, SEQ ID NO: 11 and SEQ ID NO: 14) with the human IgG4(S228P) gene. Wherein the nucleotide sequence of the IgG4(S228P) gene is shown as 409-1389 th site of SEQ ID No.29, the IgG4(S228P) gene expresses IgG4 with an amino acid sequence shown as uniProtKB database serial number P01861.1(2022.02.23 updated recently), and an IgG4(S228P) constant region shown as S228P (EU numbering) amino acid mutation is introduced, namely the IgG4(S228P) expressed by the IgG4(S228P) gene is different from the human IgG4 constant region only in that serine at the 228 position is mutated into proline.
Plasmids were constructed as per table 7: the 7 intermediate plasmids in Table 7, constructed as described 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) were double-digested with BstBI (NEB) and PacI (NEB) to obtain the 10200bp vector fragment and the 1409bp IgG1(N297A) or 1400bp IgG4(S228P) heavy chain fragment, which was ligated with T4 ligase (TAKARA), transformed, smeared, extracted, and the plasmids, BstBI and PacI (identified as expected double restriction map 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), pCGS 3-HLPV-P3 (N297 3), pCGS 3-HLPV-P3 (N297 863672), pCGS 3-HLPV-P3 (S228 3), pCGS 3-HLPV-P3 (S36228), pCGS 3-HLPV-P3 (S3), pCGS 3-HLPV-P3 (S228), pCGS 3-HLPV-P36228 (S3), pCGS 3-HLPV-P3 (S3) and pCGS3-HLPV-P09 (S297 HLPV-3), pCGS3 (S297 HLPV-3) and S3 (S3) and pCGS3 (S297 HLPV-3).
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), wherein among the 7 ADCC-removed IgG1(N297A) subtype antibody recombinant expression plasmids, the nucleotide sequences encoding only the heavy chain variable region and the light chain variable region are different, and the other nucleotide sequences are the same, wherein the nucleotide sequences excluding the ADCC effect IgG1(N297A) subtype antibody recombinant expression plasmids comprise 1409bp IgG1(N297A) heavy chain fragment and 723bp light chain gene fragment, and the IgG1(N297A) heavy chain fragment. The heavy chain fragment of IgG1(N297A) is as set forth in SEQ ID NO: 28, BstBI enzyme cutting sites are positioned at the 1 st to 6 th sites, a Kozak sequence is positioned at the 7 th to 15 th sites, a signal peptide gene is positioned at the 16 th to 72 th sites, a heavy chain gene sequence is positioned at the 73 th to 1398 th sites, a stop codon is positioned at the 1399 st minus 1401 th sites, a PacI enzyme cutting site is positioned at the 1402 nd sites and 1409 th sites, wherein the nucleotide sequence is positioned at the 73 th to 408 th sites and is a coding sequence of a heavy chain variable region, and the nucleotide sequence is positioned at the 409 st minus 1398 th sites and is a constant region sequence. The light chain gene segment is shown as SEQ ID NO: 30, HindIII restriction site at the 1-6 position, a Kozak sequence at the 7-15 position, a signal peptide at the 16-72 position, a light chain gene sequence at the 73-714 position, a stop codon at the 715-717 position, an XhoI restriction site at the 718-723 position, wherein the nucleotide at the 73-393 position is a coding sequence of a light chain variable region, and the nucleotide at 394-714 is a constant region 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) and pCGS3-HLPV-P14(S228P), wherein the 7 ADCC-removed IgG4(SP28P) subtype antibody recombinant expression plasmids all contain 1400bp IgG4(S228P) heavy chain fragment and 723bp light chain gene fragment, IgG4(S228P) heavy chain fragment and 723bp light chain gene fragment nucleotide sequences, only the nucleotide sequences of the coding heavy chain variable region and the light chain variable region are different, and other nucleotide sequences are the same. The heavy chain fragment of IgG4(S228P) is as shown in SEQ ID NO: 29, BstBI enzyme cutting sites are positioned at the 1 st to 6 th sites, a Kozak sequence is positioned at the 7 th to 15 th sites, a signal peptide gene is positioned at the 16 th to 72 th sites, a heavy chain gene sequence is positioned at the 73 th to 1389 th sites, a stop codon is positioned at the 1390 th and 1392 th sites, a PacI enzyme cutting site is positioned at the 1393 th and 1381400 th sites, wherein the nucleotide sequence at the 73 th to 408 th sites is a coding sequence of a heavy chain variable region, and a constant region sequence is positioned at the 409 th and 1389 th sites. The light chain gene segment is shown as SEQ ID NO: 30, HindIII restriction sites at the 1 st to 6 th positions, a Kozak sequence at the 7 th to 15 th positions, a signal peptide at the 16 th to 72 th positions, a light chain gene sequence at the 73 th to 714 th positions, a stop codon at the 715 th position, a XhoI restriction site at the 718 th position, 723 th position, wherein the nucleotides at the 73 th to 393 th positions are coding sequences of a light chain variable region, and 394 th position 714 is a constant region sequence.
2 affinity detection of antibody for removing ADCC effect subtype
The culture obtained by expressing the recombinant expression plasmid from which the ADCC effect was removed IgG1(N297A) and IgG4(SP28P) subtype antibody was obtained according to the transient expression method of the recombinant expression plasmid in example 2, the obtained culture was purified according to the purification of the antibody protein in example 2 and the purified monoclonal antibody was identified by SDS-PAGE of the purified culture, and the detection results showed that the monoclonal antibody bands were as expected (FIG. 4), indicating that the aforementioned cultures contained the antibodies HLPV-P03, HLPV-P05, HLPV-P08, HLPV-P09, HLPV-P10, HLPV-P11 and HLPV-P14 having antibody subtypes IgG1(N297 228) and HLPV-P03, HLPV-P05, HLPV-P08, HLPV-P09, HLPV-P5, HLPV-P11 and HLPV-57324, respectively, having antibody subtypes IgG 1(S P).
The antibody prepared above and the HLPV-P00 prepared in example 2 were tested according to the affinity test method in example 2 to determine the affinity constant between the antibody with the ADCC-removed effector subtype and the PV-1 protein, and the results are shown in Table 8.
TABLE 8 statistical table of affinity of ADCC effector subtype removed antibodies
Figure BDA0003633779020000181
Figure BDA0003633779020000191
Table 8 shows that the affinity of the IgG1(N297A) and IgG4(S228P) for removing ADCC effect is 1-2 orders of magnitude higher than that of the original antibody (HLPV-P00), and the affinity maturation experiment is expected and can be used for the next drug development experiment.
In conclusion, 14 affinity mature antibody sequences HLPV-P00-14 with 1-2 orders of magnitude improved affinity are obtained through two phage screening experiments. Furthermore, the PV-1 target antibody does not need antibody-dependent cell-mediated cytotoxicity (ADCC), and the affinity is still improved by 1-2 orders of magnitude by adopting two antibody subtypes of IgG1(N297A) and IgG4(S228P) for removing the ADCC effect. The affinity matured antibody of the invention is expected to be administered at a reduced dose for the treatment of age-related macular degeneration (AMD), Diabetic Macular Edema (DME), and macular, liver cancer, etc. caused by retinal vein occlusion in adult patients.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Sequence listing
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85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 20
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Ser His Asp Asp Arg Pro Phe
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 21
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Gly Tyr Gln
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Tyr Asp Arg Pro Phe
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 22
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Ser Asp Arg Pro Phe
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 23
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 24
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Thr Asp Arg Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 25
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Tyr Gln
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Tyr Ala Gly Asp Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gln Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asp Asp Arg Pro Phe
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 26
<211> 336
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60
tcctgcaagg cttctggcta crybtttvvs rmbtactaca tgtactgggt ccgacaggct 120
cctggacagg gactcgagtg gatcggcgag rtyaatcctw cwhmkggcga cgtgmrcttc 180
aacgagatgt tcaagtcccg cgtgaccatg accgtggaca cctctacctc taccgcctac 240
atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcac cwcyatcttt 300
tattggggcc agggcacact ggtcaccgtg tcctct 336
<210> 27
<211> 321
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gacatcgtga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60
atcacatgca aggcsagcca ggacatcrrg tatcagytgw cbtggtatca gcagaagcct 120
ggcaaggccc ctaagacact gatctactac kcyrsykhtc kggccgatgg cgtgccctct 180
agattttccg gctctggctc tggccaagag tttaccctga caatctccag cctgcagcct 240
gaggacttcg ccacctacta ctgcgttymg cackmyraya gacccttcwc ytttggccag 300
ggcaccaagc tggaaatcaa g 321
<210> 28
<211> 1409
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ttcgaagccg ccaccatggg atggtcttgt atcatcctgt ttctggtggc taccgccaca 60
ggcgtgcata gccaggttca gctggttcag tctggcgccg aagtgaagaa acctggcgcc 120
tctgtgaagg tgtcctgcaa ggcttctggc tacrybtttv vsrmbtacta catgtactgg 180
gtccgacagg ctcctggaca gggactcgag tggatcggcg agrtyaatcc twcwhmkggc 240
gacgtgmrct tcaacgagat gttcaagtcc cgcgtgacca tgaccgtgga cacctctacc 300
tctaccgcct acatggaact gtccagcctg agatctgagg acaccgccgt gtactactgc 360
accwcyatct tttattgggg ccagggcaca ctggtcaccg tgtcctctgc ttccaccaag 420
ggcccctccg tgttccccct ggctccctct tccaagagca ccagcggcgg caccgctgct 480
ctgggatgtc tggtgaagga ctacttccct gagcctgtga ccgtgtcctg gaattccggc 540
gccctgacct ccggcgtgca cacattccct gctgtgctgc agtcctccgg cctgtatagc 600
ctgtcctccg tggtgacagt gcctagctcc agcctgggca cccagaccta tatctgcaac 660
gtgaaccaca agcctagcaa taccaaggtg gacaagaagg tggagcctaa gagctgcgac 720
aagacccaca cctgtcctcc atgtcctgct ccagaactgc tcggcggacc ttccgtgttc 780
ctgtttcctc caaagcctaa ggacaccctg atgatcagca gaacccctga agtgacctgc 840
gtggtggtgg atgtgtccca cgaggatccc gaagtgaagt tcaattggta cgtggacggc 900
gtggaagtgc acaacgccaa gaccaagcct agagaggaac agtacrmcag cacctacaga 960
gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 1020
aaggtgtcca acaaggccct gcctgctcct atcgagaaaa ccatcagcaa ggccaagggc 1080
cagcctaggg aaccccaggt ttacacactg cctccaagca gggacgagct gaccaagaat 1140
caggtgtccc tgacctgcct ggtcaagggc ttctaccctt ccgatatcgc cgtggaatgg 1200
gagagcaatg gccagcctga gaacaactac aagacaaccc ctcctgtgct ggacagcgac 1260
ggctcattct tcctgtacag caagctgaca gtggacaaga gcagatggca gcagggcaac 1320
gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1380
agcctgtctc ctggcaaata attaattaa 1409
<210> 29
<211> 1400
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ttcgaagccg ccaccatggg atggtcttgt atcatcctgt ttctggtggc taccgccaca 60
ggcgtgcata gccaggttca gctggttcag tctggcgccg aagtgaagaa acctggcgcc 120
tctgtgaagg tgtcctgcaa ggcttctggc tacrybtttv vsrmbtacta catgtactgg 180
gtccgacagg ctcctggaca gggactcgag tggatcggcg agrtyaatcc twcwhmkggc 240
gacgtgmrct tcaacgagat gttcaagtcc cgcgtgacca tgaccgtgga cacctctacc 300
tctaccgcct acatggaact gtccagcctg agatctgagg acaccgccgt gtactactgc 360
accwcyatct tttattgggg ccagggcaca ctggtcaccg tgtcctctgc ctccaccaag 420
ggcccaagcg tgtttccact ggctccctgc tctcggtcca ccagcgagtc tacagccgct 480
ctgggctgtc tggtgaagga ttatttccct gagccagtga cagtgtcttg gaactccggc 540
gccctgacct ctggagtgca cacatttcct gctgtgctgc agagctctgg cctgtactcc 600
ctgtccagcg tggtgaccgt gccatcttcc agcctgggca caaagaccta tacatgcaac 660
gtggaccata agccctccaa tacaaaggtg gataagagag tggagagcaa gtacggcccc 720
ccttgcccac cctgtccagc tcctgagttc ttaggaggac catccgtgtt cctgtttcct 780
ccaaagccta aggacaccct gatgatcagc cgcaccccag aggtgacatg cgtggtggtg 840
gacgtgtctc aggaggatcc tgaggtgcag ttcaactggt atgtggatgg cgtggaggtg 900
cacaatgcta agaccaagcc cagggaggag cagtttaata gcacctaccg ggtggtgtct 960
gtgctgacag tgctgcatca ggactggctg aacggcaagg agtataagtg caaggtgtcc 1020
aataagggcc tgccttcttc catcgagaag accatcagca aggccaaggg ccagcctagg 1080
gagccacagg tgtacacact gcccccttct caggaggaga tgaccaagaa ccaggtgtcc 1140
ctgacatgtc tggtgaaggg cttctatcct tctgacatcg ctgtggagtg ggagtccaat 1200
ggccagccag agaacaatta caagaccaca ccacccgtgc tggacagcga tggctctttc 1260
tttctgtatt ccagactgac cgtggataag agccgctggc aggagggcaa cgtgttttcc 1320
tgcagcgtga tgcatgaggc cctgcacaat cattacacac agaagtctct gtccctgagc 1380
ctgggcaagt aattaattaa 1400
<210> 30
<211> 723
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aagcttgccg ccaccatggg ctggtcttgt attatcctgt ttctggtggc tacagctaca 60
ggcgtgcact ctgacatcgt gatgacccag tctccatcct ctctgtccgc ctctgtgggc 120
gacagagtga ccatcacatg caaggcsagc caggacatcr rgtatcagyt gwcbtggtat 180
cagcagaagc ctggcaaggc ccctaagaca ctgatctact ackcyrsykh tckggccgat 240
ggcgtgccct ctagattttc cggctctggc tctggccaag agtttaccct gacaatctcc 300
agcctgcagc ctgaggactt cgccacctac tactgcgtty mgcackmyra yagacccttc 360
wcytttggcc agggcaccaa gctggaaatc aagaggaccg tggctgcccc cagcgtgttc 420
atcttccctc ctagcgacga gcagctgaag agcggcaccg ctagcgtggt gtgtctgctg 480
aataacttct atcccaggga ggccaaggtg cagtggaagg tggataacgc cctgcagagc 540
ggcaactccc aggagtccgt gaccgagcag gactccaagg acagcaccta ctccctgagc 600
tccaccctga ccctgtccaa ggctgattat gagaagcaca aggtgtatgc ttgcgaggtg 660
acacaccagg gcctgtccag ccctgtgacc aagagcttca accggggcga gtgctaactc 720
gag 723

Claims (10)

1. An antibody or antigen-binding fragment thereof that specifically binds to a PV-1 protein, characterized in that: the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, both of which are composed of a determinant complementary region and a framework region; the determinant complementary regions of the heavy chain variable region and the light chain variable region each consist of a CDR1, a CDR2, and a CDR 3;
the amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 03. the amino acid sequence of SEQ ID NO: 05. SEQ ID NO: 06. the amino acid sequence of SEQ ID NO: 07. the amino acid sequence of SEQ ID NO: 08. SEQ ID NO: 09. SEQ ID NO: 10. SEQ ID NO: 11. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: positions 26-35 of 14;
the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 03. SEQ ID NO: 05. the amino acid sequence of SEQ ID NO: 06. the amino acid sequence of SEQ ID NO: 07. SEQ ID NO: 08. SEQ ID NO: 09. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 11. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: positions 50-66 of 14;
the amino acid sequence of CDR3 of the heavy chain variable region is shown as SEQ ID NO: 03. SEQ ID NO: 05. the amino acid sequence of SEQ ID NO: 06. the amino acid sequence of SEQ ID NO: 07. SEQ ID NO: 08. SEQ ID NO: 09. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: positions 97-101 of 14;
the amino acid sequence of the CDR1 of the light chain variable region is as shown in 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. the amino acid sequence of SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25 at positions 24-34;
the amino acid sequence of the CDR2 of the light chain variable region is as shown in 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. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25 at positions 50-56;
the amino acid sequence of CDR3 of the light chain variable region is shown as SEQ ID NO: 04. the amino acid sequence of SEQ ID NO: 15. the amino acid sequence of SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. the amino acid sequence of SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: 25 at positions 89-97.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 03. SEQ ID NO: 05. SEQ ID NO: 06. the amino acid sequence of SEQ ID NO: 07. the amino acid sequence of SEQ ID NO: 08. the amino acid sequence of SEQ ID NO: 09. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 or SEQ ID NO: as shown at 14.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein: the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 04. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 20. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24 or SEQ ID NO: shown at 25.
4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein: the antibody or antigen-binding fragment thereof is any one of the following:
an antibody designated HLPV-P01, the HLPV-P01 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 03, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 04 is shown in the specification;
an antibody designated HLPV-P02, the HLPV-P02 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 05, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 15 is shown in the figure;
an antibody designated HLPV-P03, the antibody HLPV-P03 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 06, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 16 is shown in the figure;
an antibody designated HLPV-P04, the HLPV-P04 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 07, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 17 is shown;
an antibody designated HLPV-P05, the HLPV-P05 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 08, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 18 is shown in the figure;
an antibody designated HLPV-P06, the HLPV-P06 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 09, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16 is shown in the figure;
an antibody designated HLPV-P07, the HLPV-P07 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 19, respectively;
an antibody designated HLPV-P08, the HLPV-P08 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 11, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 20 is shown in the figure;
an antibody designated HLPV-P09, the HLPV-P09 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 11, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 21 is shown in the figure;
an antibody designated HLPV-P10, the HLPV-P10 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 08, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 22;
an antibody designated HLPV-P11, the HLPV-P11 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 23 is shown;
an antibody designated HLPV-P12, the HLPV-P12 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 24;
an antibody designated HLPV-P13, the HLPV-P13 heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 13, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 25 is shown;
an antibody designated HLPV-P14, the HLPV-P14 heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 25, respectively.
5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein: 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 a heavy chain constant region of an IgG1, an IgG1 variant, an IgG4 or an IgG4 variant;
the light chain constant region is that of Kappa or a variant thereof.
6. Biomaterial associated with an antibody or antigen-binding fragment thereof according to claims 1-5, characterized in that: the related biological material is any one of the following materials:
B1) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5;
B2) a nucleic acid molecule encoding the heavy and/or light chain of the antibody or antigen-binding fragment thereof of any one of claims 1-5;
B3) a nucleic acid molecule encoding the heavy chain variable region and/or the light chain variable region of the antibody or antigen-binding fragment thereof of any one of claims 1-5;
B4) an expression cassette comprising any one of the nucleic acid molecules described in B1) -B3);
B5) a recombinant vector containing the nucleic acid molecule of any one of B1) -B3) or a recombinant vector containing the expression cassette of B4);
B6) a recombinant microorganism comprising any one of the nucleic acid molecules B1) -B3), or a recombinant microorganism comprising B4) the expression cassette, or a recombinant microorganism comprising B5) the recombinant vector;
B7) a cell line containing the nucleic acid molecule of any one of B1) -B3), or a cell line containing the expression cassette of B4), or a cell line containing the recombinant vector of B5).
7. The related biological material according to claim 6, wherein:
B2) the nucleic acid molecule encoding the heavy chain of the antibody or antigen-binding fragment thereof of any one of claims 1-5 is a nucleic acid molecule having the nucleotide sequence shown in SEQ ID No.28 or SEQ ID No. 29;
B2) the nucleic acid molecule encoding the light chain of the antibody or antigen-binding fragment thereof of any one of claims 1-5 is a nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 30;
B3) the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1-5 is a nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 26;
B3) the nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of any one of claims 1-5 is a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID No. 27.
8. A pharmaceutical composition for ameliorating, preventing or treating a disease associated with PV-1 overexpression, wherein the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and one or more pharmaceutically acceptable carriers.
9. A reagent or kit for detecting PV-1, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.
10. Use of any one of the antibodies or antigen-binding fragments thereof of claims 1-5, and/or the biological material of claim 6 or 7 for any one of the following:
D1) the use in the manufacture of a medicament for ameliorating, preventing or treating a disease associated with PV-1 overexpression;
D2) the application in preparing products for diagnosing or screening diseases related to PV-1 overexpression;
D3) the application in detecting PV-1;
D4) use in the preparation of a product for detecting PV-1;
D5) use in the manufacture of a product for binding a PV-1 protein.
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