CN116375859A - anti-vWF/PF 4 protein monoclonal antibody and application thereof - Google Patents

anti-vWF/PF 4 protein monoclonal antibody and application thereof Download PDF

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CN116375859A
CN116375859A CN202310655089.4A CN202310655089A CN116375859A CN 116375859 A CN116375859 A CN 116375859A CN 202310655089 A CN202310655089 A CN 202310655089A CN 116375859 A CN116375859 A CN 116375859A
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monoclonal antibody
binding fragment
host cell
antigen
vwf
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崔一民
刘志艳
向倩
莫文益
高磊
李龙图
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Peking University First Hospital
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Abstract

The invention discloses a monoclonal antibody of anti-vWF/PF 4 protein and application thereof, and relates to a monoclonal antibody of anti-vWF/PF 4 protein or an antigen binding fragment thereof, wherein the monoclonal antibody or the antigen binding fragment thereof comprises a sequence shown in SEQ ID NO:1, a heavy chain variable region CDR1, SEQ ID NO:2, a heavy chain variable region CDR2, SEQ ID NO:3, a heavy chain variable region CDR3 as set forth in SEQ ID NO:4, the light chain variable region CDR1, SEQ ID NO:5, the light chain variable region CDR2, SEQ ID NO:6, and a light chain variable region CDR3. The invention also provides nucleotide molecules, vectors, host cells, methods and uses encoding the monoclonal antibodies or antigen binding fragments thereof. The invention also provides products, compositions and derivatives comprising the monoclonal antibodies of the invention or antigen binding fragments thereof.

Description

anti-vWF/PF 4 protein monoclonal antibody and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a monoclonal antibody for resisting vWF/PF4 protein and application thereof.
Background
Classical heparin-induced thrombocytopenia (Heparin induced thrombocytopenia, HIT) was first discovered in 1973 and characterized by the formation of PF 4-heparin complex, heparin-dependent antibodies were generated. Clinically, thrombocytopenia and thrombosis occur, with morbidity of about 0.1% -5.0% and multiple life threatening venous and arterial thrombosis that is difficult to reverse when 20% -64% of patients are diagnosed, amputation rates and mortality rates can reach 5-10% and 10-20%. PF4 is a high cationic tetrameric protein that, upon binding to a polyanion to form an immune complex, can be targeted by immunoglobulin G (IgG) of platelet-activated anti-PF 4 antibodies, inducing immune thrombocytopenia and thrombotic events. PF4 exists in vivo in monomeric, dimeric and tetrameric forms.
Von willebrand factor (Von Willebrand factor, VWF) is a large adhesive multimeric protein synthesized primarily by vascular endothelial cells and bone marrow megakaryocytes and involved in hemostasis. VWF plays a critical role in maintaining the balance of coagulation and hemorrhage, and VWF deficiency or defect can lead to von willebrand disease and cause bleeding, whereas an increase in VWF may create an environment that promotes thrombosis.
vWF/PF4 was proposed as a novel biomarker for HIT, spontaneous HIT and VITT, and its elevated expression level induces antibody formation and platelet consumption, a key cause of immune thrombosis. However, there is currently no method for antibody detection against vWF/PF4 complex, and development of new antibody complexes is needed to meet clinical detection needs.
Disclosure of Invention
In order to overcome the shortcomings of the prior art, the present invention aims to provide a monoclonal antibody or antigen binding fragment thereof against vWF/PF4 protein.
The invention provides a monoclonal antibody or antigen binding fragment thereof of anti-vWF/PF 4 protein, which comprises the amino acid sequence of SEQ ID NO:1, a heavy chain variable region CDR1, SEQ ID NO:2, a heavy chain variable region CDR2, SEQ ID NO:3, a heavy chain variable region CDR3 as set forth in SEQ ID NO:4, the light chain variable region CDR1, SEQ ID NO:5, the light chain variable region CDR2, SEQ ID NO:6, and a light chain variable region CDR3.
In certain specific embodiments, the antibodies include multispecific antibodies, naked antibodies, antibody conjugates, and antibody fragments so long as they exhibit the biological activity of interest. In a particular embodiment, the antibodies comprise intact (i.e., complete) immunoglobulin molecules or antigen-binding fragments of antibodies comprising paratopes, including Fab, fab ', F (ab') 2, and Fv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. The single chain Fv or "sFv" antibody fragment comprises antibody heavy and light chain variable regions, which are on a single polypeptide chain.
Further, the monoclonal antibody or antigen binding fragment thereof further comprises heavy chain variable region framework regions FR1, FR2, FR3, FR4, and light chain variable region framework regions FR1, FR2, FR3, FR4.
Further, the monoclonal antibody or antigen binding fragment thereof further comprises a heavy chain constant region and a light chain constant region.
In some embodiments, the monoclonal antibodies or antigen binding fragments thereof provided herein comprise all or a portion of a heavy chain variable domain and/or all or a portion of a light chain variable domain. In one embodiment, the monoclonal antibody or antigen binding fragment thereof provided herein is a single domain antibody consisting of all or a portion of the heavy chain variable domain provided herein.
In certain embodiments, the monoclonal antibodies and functional fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, which optionally further comprises a heavy chain and/or a light chain constant region. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and/or CH2, CH3 region (or optionally a CH2, CH3, CH4 region). The constant regions of the monoclonal antibodies or antigen binding fragments thereof provided herein may be identical to or differ from the wild-type constant region sequence by one or more mutations.
In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof provided herein comprise one or more amino acid residue substitutions in one or more of the CDR sequences and/or one or more of the FR sequences. In certain embodiments, the affinity variants comprise no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in total in the CDR sequence and/or FR sequence.
The invention also provides a nucleotide molecule encoding the monoclonal antibody or antigen binding fragment thereof described above.
Further, the antigen binding fragment of the monoclonal antibody includes a nucleic acid functional fragment or an amino acid functional fragment.
In certain specific embodiments, the monoclonal antibody or antigen-binding fragment thereof can be conjugated to a functional fragment thereof (e.g., a nucleic acid functional fragment) and a detectable label, wherein the antigen-binding fragment can function to bind to an antigen gene to silence it, or to cleave an antigen gene, or the like.
The invention also provides a vector comprising the nucleotide molecule as described above.
Further, the vector includes a lentiviral vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, an artificial chromosome, a phage, SV40, CMV, TMV, a transposon, an IS element, a phagemid, a cosmid, a linear or circular DNA.
In certain specific embodiments, the vectors are capable of directing the expression of those genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or "recombinant vectors". The recombinant vector may comprise a nucleic acid of the present application in a form suitable for expressing the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements, which may be selected according to the nucleic acid molecule to be expressed or the requirements of the host cell to which it is linked.
In certain embodiments, the vector may be a recombinant expression vector or a cloning vector comprising a nucleic acid sequence provided herein encoding an anti-vWF/PF 4 protein antibody, at least one promoter operably linked to the nucleic acid sequence, and/or at least one selectable marker. In a specific embodiment, the vector includes, but is not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papovavirus (e.g., SV 40), lambda phage and M13 phage, plasmids such as pcdna3.3, pMD 18T, pOptivec, pCMV, pEGFP, pIRES, pdd Hyg GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, psg5L, pBABE, pWPXL, pBI, p TVL, pPro18, pTD, pRS10, pLexA, pact2.2, pCMV script.rtm., pCDM8, pcdna1.1/amp, pcdna3.1, pRc/RSV, PCR 2.1, pEF 1, pFB, pSG5, pvt 1, pCDEF3, pSVSPORT, pEF Bos, and the like.
The invention also provides an engineered host cell or population of host cells comprising the same, comprising a monoclonal antibody or antigen binding fragment thereof, a nucleotide molecule, or a vector as described above.
Further, the host cell includes a eukaryotic cell or a prokaryotic cell.
Further, the eukaryotic cells include animal cells, plant cells, fungal cells having a nucleus.
Further, the prokaryotic cells include bacteria, chlamydia, mycoplasma, cyanobacteria, rickettsia, actinomycetes, which are not nuclear membrane-bound nuclei.
Further, the host cell includes a hybridoma cell.
In certain specific embodiments, the vector DNA may be introduced into prokaryotic and eukaryotic cells by conventional transformation or transfection techniques. In a further embodiment, the host cell is an antigen presenting cell, especially a dendritic cell, a monocyte, and a macrophage. The nucleic acid may be present in the host cell in a single copy or in more than two copies, and in one embodiment, the nucleic acid is expressed in the host cell.
The present invention also provides an antibody derivative of a monoclonal antibody against vWF/PF4 protein or an antigen-binding fragment thereof, comprising a complex of the monoclonal antibody or antigen-binding fragment thereof as described above, coupled directly or indirectly to a detectable label or drug.
Further, the detectable label includes an enzyme label such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, lysozyme or malate dehydrogenase, a fluorescent dye such as fluorescein, rhodamine and its derivatives, cyanine fluorescent dyes, coumarin and its derivatives, acridine, phenanthridine, chalcone and coumarin, polycyclic aromatic hydrocarbons, thiazines/oxazines, dansylphthalamine, porphyrins, lanthanoids, up-conversion luminescent materials or quantum dot fluorescent materials, a biotin label such as succinimidyl ester, colloidal gold label, SPA label or ferritin label.
The present invention also provides a product for detecting vWF/PF4 protein, which product comprises the monoclonal antibody or antigen-binding fragment thereof, the nucleotide molecule, the vector, the engineered host cell or host cell population comprising the same, or the antibody derivative.
Further, the product comprises a kit, test paper, a nucleic acid membrane strip and a chip.
The invention also provides a product for detecting heparin-induced thrombocytopenia, the product comprising a monoclonal antibody or antigen binding fragment thereof as described above, a nucleotide molecule as described above, a vector as described above, an engineered host cell or population of host cells comprising the same as described above, or an antibody derivative as described above.
The invention also provides a composition comprising a monoclonal antibody or antigen binding fragment thereof as described above, a nucleotide molecule as described above, a vector as described above, an engineered host cell as described above, or a population of host cells comprising the same.
Further, the composition comprises pharmaceutically acceptable excipients.
Further, the auxiliary materials comprise auxiliary materials required by intravenous drip, subcutaneous injection, intravenous push injection, intravitreal injection and intramuscular injection.
Further, the auxiliary materials comprise normal saline, D5W, ringer's lactic acid, hyaluronidase, a solution tension regulator, a solution osmotic pressure regulator, a freeze-drying protective agent of freeze-drying powder, viscosity-reducing auxiliary materials, buffer solution or surfactant.
The present invention also provides a method for producing the monoclonal antibody or antigen-binding fragment thereof described above, comprising the steps of: transforming the nucleotide molecule as described above or the vector as described above into a host cell; culturing the transformed host cell under suitable conditions; the monoclonal antibody or the antigen binding fragment thereof is obtained by separation and purification in a host cell culture solution.
Further, the method of transformation into a host cell includes calcium phosphate or calcium chloride co-precipitation, DEAE dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, electroporation, or particle bombardment.
The invention also provides a method for inhibiting vWF/PF4 protein in vitro, which comprises the following steps: introducing the monoclonal antibody or antigen-binding fragment thereof, the nucleotide molecule thereof, or the vector thereof into a cell of an organism, and inhibiting vWF/PF4 protein activity by expressing the monoclonal antibody or antigen-binding fragment thereof.
The present invention also provides a method for detecting vWF/PF4 protein or a nucleic acid molecule encoding the same in a sample to be tested, for non-diagnostic and non-therapeutic purposes, comprising the steps of: contacting a test sample with the monoclonal antibody or antigen binding fragment thereof described above, or contacting a test sample with the antibody derivative described above; detecting the formation of complexes of vWF/PF4 protein or nucleic acid molecules encoding the same with the monoclonal antibodies or antigen-binding fragments thereof, or with the antibody derivatives, as described above.
The invention also provides the use of a monoclonal antibody or antigen binding fragment thereof as described hereinbefore, a nucleotide molecule as described hereinbefore, a vector as described hereinbefore, an engineered host cell or population of host cells comprising the same as described hereinbefore, or an antibody derivative as described hereinbefore for the detection of vWF/PF4 protein or a nucleic acid fragment encoding the same.
The invention also provides the use of a monoclonal antibody or antigen binding fragment thereof as described hereinbefore, a nucleotide molecule as described hereinbefore, a vector as described hereinbefore, or an engineered host cell or population of host cells comprising the same as described hereinbefore in the preparation of a product for detecting vWF/PF4 protein.
The invention also provides the use of a monoclonal antibody as described hereinbefore or an antigen binding fragment thereof, a nucleotide molecule as described hereinbefore, a vector as described hereinbefore, or an engineered host cell as described hereinbefore or a population of host cells comprising the same in the manufacture of a product for the detection of heparin-induced thrombocytopenia.
The invention also provides the use of a monoclonal antibody or antigen binding fragment thereof as described hereinbefore, a nucleotide molecule as described hereinbefore, a vector as described hereinbefore, or an engineered host cell or host cell population comprising the same as described hereinbefore in the manufacture of a medicament for the prevention or treatment of heparin-induced thrombocytopenia.
The invention also provides the use of a monoclonal antibody or antigen binding fragment thereof as described hereinbefore, a nucleotide molecule as described hereinbefore, a vector as described hereinbefore, or an engineered host cell or population of host cells comprising the same as described hereinbefore in the manufacture of a medicament for modulating vWF/PF4 protein activity or expression level.
Detailed Description
Before describing the present methods, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Definition of the definition
The term "antibody" or "antibody molecule" as used herein refers to an immunoglobulin molecule (including IgG, igE, igA, igM, igD) and/or an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that comprises an antibody binding site or paratope and is capable of binding an antigen. An "antigen binding fragment", "antibody binding site" or "antigen binding site" is a structural portion of an antibody molecule comprising heavy and light chain variable regions and hypervariable regions (CDRs) that specifically binds an antigen. As is known in the art, specific properties of antibodies relate to immunoglobulin isotypes. In representative embodiments, the antibody or antigen binding fragment is an IgG1 isotype molecule. Antibodies or fragments can also be from any source of species including avian (e.g., chicken, turkey, duck, geese, quail, etc.) and mammalian (e.g., human, non-human primate, mouse, rat, rabbit, cow, goat, sheep, horse, pig, dog, cat, etc.) species.
The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies contained in the population are identical, except for the possibility of naturally occurring mutations that occur in small amounts. mabs are highly specific and directed against a single epitope (i.e., epitope) on an antigen. This property is in contrast to polyclonal antibody preparations, which generally include antibodies directed against different epitopes.
The terms "protein" and "peptide" as used herein refer to a polymer of amino acid residues (amino acid sequence) and do not imply a specific length of the molecule. The term also refers to or includes modification (e.g., post-translational) of any polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. The term "level" or "quantitative" when used in the context of quantifying, or determining the protein level of a protein or peptide in a sample, may refer to absolute or relative quantification. Absolute quantification can be achieved by adding one or more control analytes at known concentrations and comparing the levels of the detected target protein or peptide to the known control analytes (e.g., by generating a standard curve).
The term "vector" as used herein refers to a nucleic acid molecule capable of transporting other nucleic acids to which such nucleic acid molecule binds. One type of vector is a "plasmid", i.e., a circular double-stranded DNA loop into which additional DNA fragments can be ligated. Another type of vector is a viral vector, and it is possible for additional DNA segments to be ligated into the genome of such a virus. Some vectors are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors, when introduced into a host cell, advantageously integrate within the host cell's genome and thus replicate with the host genome. And certain vectors are capable of controlling the expression of genes operably linked to such vectors. These vectors are referred to in this context as "expression vectors". Expression vectors commonly used in DNA recombination techniques take the form of plasmids. In this specification, the "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention will also cover other forms of expression vectors such as viral vectors that have similar functions.
The terms "transformation" and "transfection", conjugation and transduction as used in this context are intended to encompass a variety of methods known in the art for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemically-mediated transfer, electroporation or particle bombardment.
The term "host cell" as used herein relates to any cell that can be transformed or transfected with an exogenous nucleic acid. The term "host cell" according to the invention comprises prokaryotic (e.g.E.coli) or eukaryotic cells (e.g.dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast cells and insect cells). Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, apes, are particularly preferred. The cells may be derived from a wide variety of tissue cells and comprise primary cells and cell lines. Specific samples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells.
The term "expression" as used in the present invention refers to the expression in the most general sense and encompasses the production of RNA or RNA and proteins. It also comprises partial expression of the nucleic acid.
The term "detectable label" as used herein refers to any moiety that generates a detectable signal through a change in an optical, electrical or other physical indicator of the state of a molecule coupled to the moiety. Such physical indicators include spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical and chemical means such as, but not limited to, fluorescence, chemiluminescence and the like.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.
EXAMPLE 1 preparation of a hybridoma of a vWF/PF4 Complex monoclonal antibody
1. Preparation of vWF/PF4 Complex
The naturally extracted VWF protein and recombinant PF4 protein were dialyzed against PBS buffer, respectively. After the completion, the VWF protein and the recombinant PF4 protein were diluted to 0.5mg/mL with PBS, and mixed at a molar ratio of 1:1, and incubated at 4℃for 2 hours. Purifying by molecular sieve high-efficiency chromatography to obtain the VWF/PF4 compound.
2. Immunized mice
After mixing 0.5mg/mL of VWF/PF4 complex (antigen) with Freund's complete adjuvant at 1:1 and emulsifying 500. Mu.L, 6-8 week old female Balb/c mice were subcutaneously multi-injected, each with 100. Mu.g of VWF/PF4 complex. After three weeks, 0.5mg/mL antigen was mixed with Freund's incomplete adjuvant at 1:1 and emulsified in 500. Mu.L for subcutaneous multipoint injection, with 50. Mu.g of each mouse antigen vaccinated at the boost. Three weeks, six weeks, and nine weeks apart were followed by a second immunization following the procedure of boosting previously described, for a total of 4 boosting.
3. Immune serum potency assay
Tail of mice 10 days after fourth booster immunizationVein blood collection, immune serum titers were determined by indirect ELISA. The indirect ELISA method was as follows: the VWF/PF4 complex was diluted to 50. Mu.g/mL with 50mM PBS and added to polystyrene 96-well plates at 100. Mu.L/well and coated overnight at 4 ℃. After the coating solution was discarded, 200. Mu.L of blocking solution (PBS containing 1% fetal bovine serum) was added to a polystyrene 96-well plate per well, and blocked at 37℃for 2 hours. The plates are washed three times by PBST, dried by an air drying oven at 37 ℃ for 4 hours, sealed by an aluminum plastic sealing bag and preserved at 4 ℃ for standby. Murine immune serum was subjected to 10 with a dilution (PBS containing 1% fetal bovine serum) 2 ～10 6 After dilution by a factor of 100. Mu.L/well in 96-well plates followed by incubation at 37℃for 1h, followed by three washes with PBST, goat anti-mouse IgG was labeled with horseradish peroxidase by 1:50000 dilution by a factor of 100. Mu.L/well, followed by incubation at 37℃for 30min, followed by three washes with PBST. TMB was then added to each well to develop 100. Mu.L/well, and the wells were protected from light at room temperature for 10min, after which 1M H was added in an amount of 50. Mu.L/well 2 SO 4 The reaction was terminated. The absorbance at 450nm was measured, the serum of mice before immunization was used as a negative control, and the positive judgment value was obtained by the ratio of the measured value to the control value of 2.1 or more, and the results are shown in Table 1.
Figure SMS_1
4. Preparation of hybridomas
Mice with serum titers greater than 1:105 were taken, 3 days prior to fusion, and after complex proteins were mixed with an equal volume of PBS, BALB/c was intraperitoneally injected in an amount of 50 μg/500 μl each for booster immunization.
The preparation flow of the feeder layer cells comprises the following steps: balb/c (about 4 weeks) mice were euthanized by orbital venous exsanguination, 75% ethanol soaked for 3 min with ventral side facing up; the chest was opened to isolate thymus, ground using a cell sieve, and cells were resuspended using a pre-warmed basal medium. Preparation of celiac macrophages: on the day of fusion, 1 healthy mouse (one from 1 spleen) was selected, and the eyeballs were collected for exsanguination until no blood drop was produced. The cervical vertebra dislocation is fatal, 75% alcohol is soaked for 5 minutes for sterilization, the cervical vertebra dislocation is moved into an ultra-clean workbench, and the abdomen is fixed on an anatomic plate upwards. The skin of the mouse abdomen was lifted with forceps, a small opening was cut with scissors (note that the peritoneum was not damaged, so as not to flow out of the peritoneal fluid), the peritoneum was fully exposed by blunt dissection, and the skin was sterilized by wiping with alcohol. 5-10 mL of basic culture solution is sucked by a disposable sterile syringe and injected into the abdominal cavity of the mouse, the fixed syringe is kept still by the right hand, and the abdomen of the mouse is gently rubbed by the left hand by clamping the alcohol cotton ball with forceps for 1-2 minutes, so that macrophages are promoted to swim out. The culture medium in the abdominal cavity was aspirated by syringe and transferred to a 15mL centrifuge tube. After the peritoneal macrophages and the thymus cells were mixed uniformly, the mixture was centrifuged at 1200r/min for 10 minutes, and the supernatant was discarded. The medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, sigma)) was resuspended in pre-warmed 20% FBS 1 XHAT medium, and kept at 37 ℃.
Cell fusion the preparation procedure of splenocytes at morning: mice 3-4 days after having been boosted were taken, the eyeballs were removed to collect blood, and the isolated serum was collected as a positive control serum at the time of anti-detection. Meanwhile, the mice are killed by cervical dislocation, soaked in 75% alcohol for 5 minutes for sterilization, and immediately placed in an ultra clean bench. Fixing a mouse on a dissecting platen, aseptically opening the abdomen, then lifting the skin on the right side, allowing the spleen to be seen, changing the eye scissors, cutting the peritoneum by using an aseptic operation scissors, taking out the spleen by using forceps, flushing the spleen with normal saline, cutting the spleen into pieces by using scissors, placing the pieces in a disposable cell sieve, lightly squeezing the spleen by using a syringe inner core, repeatedly flushing the cell sieve with normal saline until only connective tissue remains in the cell sieve, and then filtering the cell suspension by using the disposable cell sieve again. The spleen cell suspension was harvested, centrifuged at 1200r/min for 10min and washed 1 time (as much as possible to remove the red blood cell mass) with 30 40ml of heavy suspension centrifugation. The spleen cells are resuspended in basic culture medium containing 10% FBS, and placed in T75 cell bottle at 37deg.C and 5% CO 2 Culturing in an incubator for 2-3 hours to adhere macrophages in the cell suspension.
Preparation procedure of confluent cells at the afternoon, myeloma cells: 3 bottles of T75 myeloma cells were discarded, and the supernatant was blown down with 50mL of pre-warmed physiological saline, centrifuged together with spleen cells, and centrifuged at 1200r/min for 10 minutes. Spleen cells were thoroughly mixed with 3 bottles of T75 myeloma cells, the supernatant was discarded, resuspended in 40ml of pre-warmed saline, and centrifuged at 1200r/min for 10 min.
Cell fusion process: the supernatant was discarded, the supernatant was discarded as much as possible, and the residual liquid was pipetted to remove the erythrocytes as much as possible without affecting the concentration of the cell fusion agent. The bottom of the centrifugal tube is lightly flicked by a finger to mix evenly, so that the precipitated cells are loose and uniform into paste. Fusion at room temperature: the cell fusion agent and the culture medium containing the feeder layer are placed into an incubator for heat preservation when preparing spleen cells. The packed 1mL of the cell fusion agent solution (added as close to the wall of the centrifuge tube at the cell site as possible in one revolution) was aspirated with a Pasteur pipette. Gently mix for 60s-90 s. After timing, 30mL of basal medium preheated to 37 ℃ is added at a time to dilute the cell fusion agent to lose the fusion promoting effect, and the cell fusion agent is kept stand at 37 ℃ for 5min. Centrifuge at 800r/min for 6 min, discard supernatant. Adding preheated
The precipitated cells were gently aspirated in 20% FBS 1×hat medium, suspended and mixed (thoroughly mixed to reduce cell clumps) with gentle action. This experiment was followed by preparation of 10 96-well cell culture plates, 200. Mu.L per well, requiring 200mL of 20% FBS 1 XHAT medium.
37℃、5% CO 2 Incubator culture, observation under a 7d microscope after cell fusion, counting culture wells in which obvious cell clones appear in a 96-well cell culture plate, and calculating the fusion rate, fusion rate= (number of fused cells/total number of cells) ×100%.
5. Screening of hybridoma cells secreting anti-VWF/PF 4 protein monoclonal antibodies
The indirect ELISA method screens cell culture supernatant, selects positive clone hybridoma cells with higher titer for subcloning, and uses a limiting dilution method to clone continuously for 2-3 times until the cell positive rate reaches 100%. The culture supernatants of the obtained 10 hybridoma cell lines with higher cost are detected by an indirect ELISA method, and meanwhile, the culture supernatants are subjected to dilution detection by using 0.02MPBS, and the detection results are shown in Table 2.
Figure SMS_2
By comparing the detection data of ELISA method performed on hybridoma cell line culture supernatants, cell lines stably secreting anti-vWF/PF 4 monoclonal antibodies and having higher antibody titers, respectively labeled as H4E2, etc., can be further selected therefrom. And (5) performing liquid nitrogen freezing after amplification culture on the cells with the positive rate reaching 100% after cloning.
6. Preparation and purification of ascites
Hybridoma cell line H4E2 was isolated at 1X 10 6 The abdominal cavity of 8-10 week old BALB/c female mice pretreated with liquid paraffin is injected into the amount of the liquid paraffin, and the ascites is extracted when the abdomen of the mice expands after 10-14 days of feeding observation. Purifying the monoclonal antibody by adopting an affinity chromatography Protein G Sepharose Fast Flow, and determining the purity of the monoclonal antibody by SDS PAGE, wherein the purity reaches more than 95%.
Example 2 characterization of monoclonal antibodies
1. Determination of antibody concentration: ascites prepared by hybridoma H4E2 and the like is purified to obtain an anti-VWF/PF 4 monoclonal antibody, and the concentration of the anti-VWF/PF 4 monoclonal antibody is >1mg/mL by using a BCA method.
2. Identification of antibody subtypes: subtype of hybridoma cell strain is identified by using a mouse monoclonal antibody subtype identification kit of Thermofisher, subtype of H4E2 and other secretion antibodies is IgG1 type, and light chain is kappa chain.
3. Identification of titers of purified antibodies: the VWF/PF4 complex was diluted to 50. Mu.g/mL with 50mM PBS and added to polystyrene 96-well plates at 100. Mu.L/well and coated overnight at 4 ℃. After the coating solution was discarded, 200. Mu.L of blocking solution (PBS containing 1% fetal bovine serum) was added to a polystyrene 96-well plate per well, and blocked at 37℃for 2 hours. The plates are washed three times by PBST, dried by an air drying oven at 37 ℃ for 4 hours, sealed by an aluminum plastic sealing bag and preserved at 4 ℃ for standby. Murine immune serum was subjected to 10 with a dilution (PBS containing 1% fetal bovine serum) 2 ～10 6 After dilution by a factor of 100. Mu.L/well in 96-well plates followed by incubation at 37℃for 1h, followed by three washes with PBST, goat anti-mouse IgG was labeled with horseradish peroxidase by 1:50000 dilution by a factor of 100. Mu.L/well, followed by incubation at 37℃for 30min, followed by three washes with PBST. TMB was then added to each well to develop 100. Mu.L/well, and the wells were protected from light at room temperature for 10min, after which 1M H was added in an amount of 50. Mu.L/well 2 SO 4 The reaction was terminated.
And measuring the 450nm absorption value, taking PBS as a negative control, and taking the positive judgment value that the ratio of the measured value to the control value is more than or equal to 2.1 to determine the titer. The titer of the purified antibody was > 500000.
4. Monoclonal antibody sequencing
The sequencing results of the anti-VWF/PF 4 monoclonal antibodies prepared from the purified hybridoma cells H4E2 on the light chain variable region and the heavy chain variable region are shown in Table 3.
Figure SMS_3
The antibody with the corresponding variable region sequence has high affinity and good specificity; the corresponding antibody variable regions can thus be used for the development of recombinant antibodies, single chain antibodies, bispecific antibodies, for diagnostic or therapeutic use in the development of related products.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. A monoclonal antibody against vWF/PF4 protein, or an antigen-binding fragment thereof, comprising the amino acid sequence of SEQ ID NO:1, a heavy chain variable region CDR1, SEQ ID NO:2, a heavy chain variable region CDR2, SEQ ID NO:3, a heavy chain variable region CDR3 as set forth in SEQ ID NO:4, the light chain variable region CDR1, SEQ ID NO:5, the light chain variable region CDR2, SEQ ID NO:6, and a light chain variable region CDR3.
2. A nucleotide molecule encoding the monoclonal antibody or antigen binding fragment thereof of claim 1.
3. A vector comprising the nucleotide molecule of claim 2.
4. An engineered host cell or population of host cells comprising the same, wherein the engineered host cell or population of host cells comprising the same comprises the monoclonal antibody or antigen-binding fragment thereof of claim 1, the nucleotide molecule of claim 2, or the vector of claim 3.
5. An antibody derivative of a monoclonal antibody or antigen-binding fragment thereof directed against vWF/PF4 protein, comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1 coupled directly or indirectly to a detectable label or drug-forming complex.
6. A product for detecting vWF/PF4 protein, comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, the engineered host cell or population of host cells comprising the same of claim 4, or the antibody derivative of claim 5.
7. A product for detecting heparin-induced thrombocytopenia, comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, the engineered host cell or host cell population comprising the same of claim 4, or the antibody derivative of claim 5.
8. A composition comprising the monoclonal antibody or antigen-binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, the engineered host cell of claim 4, or a population of host cells comprising the same.
9. The method of any one of the following:
1) A method of producing the monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising the steps of: transforming the nucleotide molecule of claim 2 or the vector of claim 3 into a host cell; culturing the transformed host cell under suitable conditions; separating and purifying in a host cell culture solution to obtain the monoclonal antibody or the antigen binding fragment thereof according to claim 1;
2) A method of inhibiting vWF/PF4 protein in vitro, comprising the steps of: introducing the monoclonal antibody or antigen-binding fragment thereof of claim 1, the nucleotide molecule of claim 2, or the vector of claim 3 into a cell of an organism, inhibiting vWF/PF4 protein activity by expressing the monoclonal antibody or antigen-binding fragment thereof of claim 1;
3) A method for the non-diagnostic and non-therapeutic destination detection of vWF/PF4 protein or a nucleic acid molecule encoding the same in a test sample, characterized in that the method comprises the steps of: contacting a test sample with the monoclonal antibody or antigen-binding fragment thereof of claim 1, or contacting a test sample with the antibody derivative of claim 5; detecting the formation of complexes of vWF/PF4 protein or a nucleic acid molecule encoding the same with the monoclonal antibody or antigen-binding fragment thereof according to claim 1, or with the antibody derivative according to claim 5.
10. Use of any one of the following:
1) Use of the monoclonal antibody or antigen binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, the engineered host cell or host cell population comprising the same of claim 4, or the antibody derivative of claim 5 for detecting vWF/PF4 protein or a nucleic acid fragment encoding the same;
2) Use of the monoclonal antibody or antigen binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, or the engineered host cell of claim 4 or host cell population comprising the same for the preparation of a product for detecting vWF/PF4 protein;
3) Use of the monoclonal antibody or antigen binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, or the engineered host cell of claim 4 or host cell population comprising the same for the preparation of a product for detecting heparin-induced thrombocytopenia;
4) Use of the monoclonal antibody or antigen binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, or the engineered host cell or host cell population comprising the same of claim 4 in the manufacture of a medicament for the prevention or treatment of heparin-induced thrombocytopenia;
5) Use of the monoclonal antibody or antigen binding fragment thereof of claim 1, the nucleotide molecule of claim 2, the vector of claim 3, or the engineered host cell or host cell population comprising the same of claim 4 for the preparation of a medicament for modulating vWF/PF4 protein activity or expression level.
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