CN116925230B - Anti-human PLA2R antibody and antigen binding fragment thereof and application thereof - Google Patents
Anti-human PLA2R antibody and antigen binding fragment thereof and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-human PLA2R antibody and an antigen binding fragment thereof and application thereof, and relates to the technical field of bioengineering. The antibody and the antigen binding fragment thereof have higher affinity with PLA2R, can specifically bind with PLA2R protein, and can target fragments of PLA2R containing CysR, fnII, CTLD 1. Therefore, the method can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like; can also be used for developing products for detecting, diagnosing, prognosing, evaluating curative effect or monitoring recurrence of membranous nephropathy.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-human PLA2R antibody, an antigen binding fragment thereof and application thereof.
Background
Membranous nephropathy (membranous nephropathy, MN), membranous glomerulonephritis, in particular nephrotic syndrome caused by excessive deposition of immune complexes in glomerular capillaries, is one of the most common pathological types in adult nephrotic syndrome. Clinically MN is mainly manifested by massive proteinuria, hypoproteinemia, edema and hyperlipidemia.
Depending on the etiology, MN can be classified into idiopathic membranous nephropathy (idiopathic membranous nephropathy, IMN) and secondary membranous nephropathy (secondary membranous nephropathy, SMN). Among them, IMN is a major type of MN, and its cause is not clear, but is also called primary membranous nephropathy (primary membranous nephropathy, PMN), with a ratio of about 70% to 80%. Most scholars consider PMN as an autoimmune glomerular disease mediated by antibodies, and target antigens located on podocytes are recognized by autoantibodies and combined with the autoantibodies to form immune complexes deposited under basal membrane podocytes, and activation of the complement system causes podocyte injury and shedding, resulting in increased basal membrane permeability and thus occurrence of a large amount of proteinuria.
The phospholipase A2 receptor (PLA 2R) was first reported by Beck et al to be one of the major antigens responsible for PMN development in 2009, and an anti-phospholipase A2 receptor antibody (phospholipase-A2 receptor antibody, PLA 2R-Ab) was detected in 70% of PMN patient blood and kidney tissue eluate, which has attracted widespread medical attention to PLA 2R. PLA2R-Ab can be specifically combined with target antigen (namely PLA 2R) on podocyte in Kidney tissue, can be expressed in 70% -80% of serum of PMN patients (Bobart SA, de Vriese AS, pawar AS, et al Kidney int.2019; 95 (2): 429-438), and has extremely low PLA2R-Ab antibody positive expression rate in serum of healthy people, SMN and other glomerular disease patients, so that the specificity of diagnosing MN by PLA2R-Ab is higher. A recent Meta analysis shows (J.Ind. Lu Rong, zhang Jiaqin, et al. J.clinical laboratory, 2017, 35 (7): 545-549) that serum PLA2R-Ab has a total sensitivity of 69% and a total specificity of 97% for diagnosing PMNs, further demonstrates that serum PLA2R-Ab has better sensitivity and very high specificity for PMNs, and can be used as a specific biomarker for diagnosing PMNs.
PLA2R is composed of multiple domains (FIG. 1), and studies have shown that the domains responsible for PLA2R-Ab production consist essentially of three domains, cysR, fnII, CTLD (Liyo Kao, vinson Lam, et al J Am Soc Nephrol.2015 Feb; 26 (2): 291-301). Therefore, monoclonal antibodies targeting these three domains are of great significance for cell and animal model establishment, mechanism research, diagnosis and drug development of PLA2R type membranous nephropathy. However, no commercially available mouse monoclonal antibodies can target these three domains.
In view of this, the present invention has been made.
Disclosure of Invention
The present invention is directed to an antibody against human PLA2R and antigen binding fragments thereof to solve the above-mentioned problems. The anti-human PLA2R monoclonal antibody provided by the invention can target fragments of PLA2R containing CysR, fnII, CTLD1 domains, and can be applied to detection and screening fields such as Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like. The invention also provides a nucleic acid molecule for encoding the anti-human PLA2R antibody and an antigen binding fragment thereof, a recombinant vector and application of the antibody in a PLA2R protein detection method or product.
The invention is realized in the following way:
in a first aspect, the invention provides an antibody against human PLA2R and antigen binding fragments thereof, comprising a heavy chain complementarity determining region and a light chain complementarity determining region;
the heavy chain complementarity determining regions include CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NO.3-5, respectively;
the light chain complementarity determining region includes CDR-L1 having the amino acid sequence shown in SEQ ID NO.6, CDR-L2 having the amino acid sequence WAS, and CDR-L3 having the amino acid sequence shown in SEQ ID NO. 7.
The amino acid sequence SEQ ID NO.3 of CDR-H1 is GYSFTGYY;
the amino acid sequence SEQ ID NO.4 of CDR-H2 is FNPYNGAT;
the amino acid sequence of CDR-H3, SEQ ID NO.5, is AKEGPYYGNYGYFDY;
the amino acid sequence of CDR-L1 is SEQ ID NO.6 as QSLLNSRTRKNY;
the amino acid sequence of CDR-L2 is WAS;
the amino acid sequence SEQ ID NO.7 of CDR-L3 is KQSYNLYNT.
Through screening, antibodies and antigen binding fragments thereof with specific heavy chain complementarity determining regions and light chain complementarity determining regions are found to have higher affinity with PLA2R, can specifically bind to PLA2R proteins, and can target fragments of PLA2R comprising three domains of CysR, fnII, CTLD. Therefore, the method can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like; can also be used for developing products for detecting, diagnosing, prognosing, evaluating curative effect or monitoring recurrence of membranous nephropathy.
In the present invention, "PLA2R" and "phospholipase A2 receptor" are all referred to in the same sense.
In a preferred embodiment of the invention for use, the antibody and antigen-binding fragment thereof further comprises a heavy chain framework region comprising FR-VH1, FR-VH2, FR-VH3 and FR-VH4 shown in sequence as SEQ ID NO.17-20 and/or a light chain framework region; and/or; the light chain framework region includes FR-VL1, FR-VL2, FR-VL3 and FR-VL4 shown in SEQ ID NO.21-24 in that order.
The structure of the heavy chain variable region and the heavy chain framework region is as follows:
FR-VH1-CDR1-FR-VH2-CDR2-FR-VH3-CDR3-FR-VH4。
the structure of the light chain variable region and the light chain framework region is as follows:
FR-VL1-CDR1-FR-VL2-CDR2-FR-VL3-CDR3-FR-VL4。
in a preferred embodiment of the invention, the antigen binding fragment is selected from any one of the group consisting of Fab ', fab, F (ab') 2, scFv and Fv of an antibody.
The above antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived, and are selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody, so long as they exhibit the desired antigen-binding activity. It will be readily appreciated by those skilled in the art from the teachings herein that the antigen binding fragments described above may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The antigen binding fragments described above are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
The above antibodies or antigen binding fragments may also be obtained synthetically by recombinant genetic techniques also known to those skilled in the art or by automated peptide synthesizers such as those sold for example as Applied BioSystems.
In a preferred embodiment of the invention, the antibody is selected from at least one of a multispecific antibody, a fusion antibody, and a chimeric antibody. In other embodiments, the antibody is selected from a monoclonal antibody.
Noun interpretation:
multispecific antibodies are polymers of monoclonal antibodies that recognize different epitopes, which can bind different targets or different epitopes of the same target, and have higher antigen recognition capability than a single monoclonal antibody.
Fusion type antibody: the antibody or antigen binding fragment thereof provided by the invention can be easily combined with other structures (such as BSA, igG-Fc and the like) to form a novel fusion molecule, such as an enzyme, an antibacterial peptide or a developing substance and the like for prolonging the half-life period of the fusion molecule. In the novel fusion molecule, the antibody is bound to its target antigen in a targeted manner, and the moiety fused to the antibody performs the corresponding function. In clinicians, they want the drug to stay in the body long enough, however, the blood clearance of the antibody is fast, which is detrimental to the drug it carries. Therefore, the antibody and the long-life molecule are fused together by the gene technology, so that the existence time of the antibody in blood can be prolonged, namely the half life of the antibody is prolonged, and a better therapeutic effect is achieved.
The "chimeric antibody" according to the present invention includes, but is not limited to, an antibody in which a variable region of a humanized antibody is fused to a constant region or a framework region of a human antibody (or an antibody derived from another source), and can reduce an immune response induced by a non-humanized antibody. In other embodiments, the constant regions may be adaptively recombined or optimized from different sources or types as desired.
In a preferred embodiment of the use of the invention, the antibody or antigen binding fragment thereof further comprises a constant region comprising a light chain constant region and a heavy chain constant region, the type of light chain constant region being either kappa-type or lambda-type; the heavy chain constant region is of the type IgM, igD, igG, igA or IgE heavy chain constant region.
IgG is selected from the heavy chain constant region of any one of IgG1, igG2, igG3 and IgG 4.
In a preferred embodiment of the invention, the constant region of the antibody is derived from human, mouse, rat, bovine, equine, ovine, rabbit or canine species.
In a preferred embodiment of the present invention, the amino acid sequences of the light chain constant region and the heavy chain constant region are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively.
Further, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.
In a second aspect, the invention also provides the use of an antibody or antigen-binding fragment thereof against human PLA2R in the preparation of a PLA2R assay product;
the PLA2R detection product is selected from a reagent, a kit or a chip;
the detection product comprises an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof is labeled with a detectable label.
In a preferred embodiment of the invention, the kit comprises a carrier, and the carrier carries the antibody or antigen-binding fragment thereof; in a preferred embodiment of the present invention, the carrier is a nanoparticle, a magnetic bead, an agarose gel microsphere, a silica gel microsphere, a latex microsphere or a porous material.
The carrier is coupled and/or physically linked to the antibody. Physical attachment includes, but is not limited to, electrostatic attraction, and the like.
In other embodiments, the porous material is selected from ELISA plates or other solid supports.
For example, the activated beads are incubated with the antibody such that the beads are coated with the antibody. For example, the corresponding immunoaffinity adsorption material is prepared by coupling antibodies on agarose gel microspheres or silica gel microspheres.
In an alternative embodiment, the nanoparticles are selected from the group consisting of organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
The magnetic beads are also called magnetic microspheres, and the magnetic microspheres are magnetic composite microspheres with a certain structure, wherein organic polymers and inorganic magnetic nanoparticles are combined by a proper method to form the magnetic composite microspheres with a special structure. Magnetic beads include, but are not limited to, nano magnetic beads and micro magnetic microspheres. In an alternative embodiment, the magnetic beads include, but are not limited to: carboxyl magnetic beads, amino magnetic beads, oleylamine modified magnetic beads, silicon hydroxyl magnetic beads, sulfonic acid magnetic microspheres, sulfhydryl magnetic microspheres, PEG modified magnetic beads, non-modified ferroferric oxide magnetic beads, monodisperse silicon-coated magnetic beads, epoxy magnetic beads, monodisperse mesoporous silicon-coated magnetic beads, gold-coated magnetic nanoparticles, streptavidin modified magnetic beads, polylysine modified magnetic beads, nickel magnetic beads, magnetic polystyrene microspheres and silicon dioxide magnetic microspheres.
Latex microspheres are spherical polymer particles in the colloidal size range formed by an amorphous polymer, typically polystyrene, for example particles having a diameter of less than 100nm, or particles having a particle size of 0.3-0.5 μm, or particles having a particle size exceeding 1 μm.
The latex microspheres include, but are not limited to, latex microspheres having at least one functional group of hydroxyl, carboxyl or tostal modified on the surface.
In an alternative embodiment, the kit further comprises a buffer solution, a positive control, and a washing solution.
In a preferred embodiment of the use of the invention, the detectable label is selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent and a colloid.
Fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-photoprotein (FAM), tetrachlorophotoprotein (TET), and the like, or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof, for example, including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazoxanthin (chlorophyll), and the like, for example.
In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in an alternative embodiment, the chemiluminescent reagent is selected from at least one of acridinium esters, luminol, lucigenin, crustacean fluorescein, ruthenium bipyridine, dioxane, lomustine, isoluminol, and peroxyoxalate.
Colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In a third aspect, the invention also provides the use of an antibody or antigen-binding fragment thereof against human PLA2R in the manufacture of a product for the detection, diagnosis, prognosis, efficacy assessment or recurrence monitoring of membranous nephropathy, the marker of which is PLA2R;
membranous nephropathy is primary membranous nephropathy or secondary membranous nephropathy.
By detecting the blood or serum PLA2R level of the target sample, it is possible to diagnose whether the target sample has or assess the risk of membranous nephropathy, as well as to provide a reference for a poor prognosis of membranous nephropathy.
In addition, PLA2R changes also allow for knowledge of the patient's state of disease progression. A decrease in PLA2R concentration in the patient's blood after treatment is indicative of successful treatment, while an increase in concentration is indicative of the residual or recurrence of membranous nephropathy. Therefore, the anti-PLA 2R antibody provided by the invention can be used for efficacy evaluation or recurrence monitoring of membranous nephropathy.
The detection, diagnosis, prognosis, efficacy assessment or recurrence monitoring product of membranous nephropathy is selected from reagents, kits or chips;
the detection, diagnosis, prognosis, efficacy assessment or recurrence monitoring product of membranous nephropathy comprises an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof is labeled with a detectable label.
In a preferred embodiment of the invention, the kit comprises a carrier, and the carrier carries the antibody or antigen-binding fragment thereof; in a preferred embodiment of the present invention, the carrier is a nanoparticle, a magnetic bead, an agarose gel microsphere, a silica gel microsphere, a latex microsphere or a porous material.
The carrier is coupled and/or physically linked to the antibody. Physical attachment includes, but is not limited to, electrostatic attraction, and the like.
In other embodiments, the porous material is selected from ELISA plates or other solid supports.
For example, the activated beads are incubated with the antibody such that the beads are coated with the antibody. For example, the corresponding immunoaffinity adsorption material is prepared by coupling antibodies on agarose gel microspheres or silica gel microspheres.
In an alternative embodiment, the nanoparticles are selected from the group consisting of organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
The magnetic beads are also called magnetic microspheres, and the magnetic microspheres are magnetic composite microspheres with a certain structure, wherein organic polymers and inorganic magnetic nanoparticles are combined by a proper method to form the magnetic composite microspheres with a special structure. Magnetic beads include, but are not limited to, nano magnetic beads and micro magnetic microspheres. In an alternative embodiment, the magnetic beads include, but are not limited to: carboxyl magnetic beads, amino magnetic beads, oleylamine modified magnetic beads, silicon hydroxyl magnetic beads, sulfonic acid magnetic microspheres, sulfhydryl magnetic microspheres, PEG modified magnetic beads, non-modified ferroferric oxide magnetic beads, monodisperse silicon-coated magnetic beads, epoxy magnetic beads, monodisperse mesoporous silicon-coated magnetic beads, gold-coated magnetic nanoparticles, streptavidin modified magnetic beads, polylysine modified magnetic beads, nickel magnetic beads, magnetic polystyrene microspheres and silicon dioxide magnetic microspheres.
Latex microspheres are spherical polymer particles in the colloidal size range formed by an amorphous polymer, typically polystyrene, for example particles having a diameter of less than 100nm, or particles having a particle size of 0.3-0.5 μm, or particles having a particle size exceeding 1 μm.
The latex microspheres include, but are not limited to, latex microspheres having at least one functional group of hydroxyl, carboxyl or tostal modified on the surface.
In an alternative embodiment, the kit further comprises a buffer solution, a positive control, and a washing solution.
In a preferred embodiment of the use of the invention, the detectable label is selected from at least one of a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent and a colloid.
In a fourth aspect, the invention also provides a detection reagent or kit comprising an antibody or antigen-binding fragment thereof directed against human PLA 2R.
In a fifth aspect, the invention also provides immunoconjugates, bispecific molecules, chimeric and antigen receptors or pharmaceutical compositions comprising an antibody or antigen binding fragment thereof directed against human PLA 2R.
The immunoconjugate is for example selected from antibody-drug conjugates, and the corresponding antibody-drug conjugate is prepared by reacting an antibody with a drug having a specific linker. It is within the scope of the invention for a person skilled in the art to engineer antibodies such that a certain site on the antibody serves as a drug conjugation site.
The bispecific molecule is for example selected from bispecific antibodies.
The chimeric antigen receptor also includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.
In a sixth aspect, the invention also provides a vector comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described above against human PLA 2R.
The nucleic acid molecule comprises a DNA sequence shown as SEQ ID No.8 and is used for encoding a heavy chain of an anti-human PLA2R antibody; and the DNA sequence shown as SEQ ID No.9 is used to encode the light chain of an anti-human PLA2R antibody.
Considering the degeneracy of codons, the sequence of the genes encoding the above antibodies may be modified in the coding region thereof without changing the amino acid sequence to obtain genes encoding the same antibodies; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.
The vector is an expression vector or cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can be used to introduce the DNA fragment of interest directly or indirectly (e.g., packaged into a virus) into a host cell by transformation, transfection or transduction for expression of the gene of interest.
One type of vector is a plasmid, i.e., a circular double stranded DNA molecule, into which a DNA fragment of interest can be ligated into a plasmid loop.
In a seventh aspect, the invention also provides a recombinant cell comprising the vector described above.
The mammalian cells are selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, vero, BHK cells, CV1 cells, heLa cells, MDCK cells, hep-2 cells, and Per6 cells. Among them, 293 series cells, per6 cells and CHO cells are common mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.
In an eighth aspect, the invention also provides the use of an antibody or antigen-binding fragment thereof directed against human PLA2R for detecting the level of human PLA2R or the detection of pharmaceutical activity in a cellular model expressing PLA2R, for non-diagnostic and therapeutic purposes.
The drug activity detection comprises the steps of constructing cells which stably express human PLA2R or express membrane proteins comprising three domains CysR, fnII, CTLD1, inducing antibody-induced cell killing effect (including CDC, ADCC, ADCP) by anti-human PLA2R antibodies to establish a cell damage model, and detecting the inhibition of the killing effect by drugs, thereby being used as a quantitative activity detection and drug quality control method. The anti-human PLA2R antibodies provided by the invention can be used in two aspects, namely, determining that cells stably expressing human PLA2R or expressing membrane proteins comprising three domains CysR, fnII, CTLD1 are successfully established and detecting the expression level. And secondly, triggering antibody-induced cell killing to establish a damage model.
The invention has the following beneficial effects:
through screening, antibodies and antigen binding fragments thereof with specific heavy chain complementarity determining regions and light chain complementarity determining regions are found to have higher affinity with PLA2R, can specifically bind to PLA2R proteins, and can target fragments of PLA2R comprising three domains of CysR, fnII, CTLD. Therefore, the method can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like; can also be used for developing products for detecting, diagnosing, prognosing, evaluating curative effect or monitoring recurrence of membranous nephropathy.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic structural diagram of PLA 2R.
FIG. 2 is a schematic view showing the structure of an expression cassette in example 1 of the present invention.
FIG. 3 is a CC1H SDS-PAGE analysis in example 2 of the present invention; lane 1 (labeled M in the figure) molecular weight reference; lane 2 (labeled CC 1H-non-reduced in the figure) purified CC1H is non-reduced; lane 3 (labeled CC 1H-reduction in the figure) purified CC1H reduction.
FIG. 4 is a SDS-PAGE identification of the antibodies of example 3 of the present invention; lane 1 (M in the figure) marker; lane 2 (labeled 1 in the figure) antibody non-reduced; lane 3 (marker 2 in the figure) antibody reduction.
FIG. 5 is a graph showing the fitting of murine anti-human monoclonal antibody in example 4 of the present invention.
Fig. 6 is a graph of the protein standard curve (y= 0.6849x+0.6325, r=0.9925) measured by Bradford method in example 5 of the present invention.
FIG. 7 is a diagram showing the purification effect of SDS-PAGE for detecting CC1H-Biotin in example 5 of the present invention, lane 1 is marker; lane 2, panel 1: CC1H-Biotin containing DTT reduction loading buffer; lane 3, panel 2: CC1H-Biotin in non-reducing loading buffer.
FIG. 8 shows measured or fitted curves of different concentrations of murine anti-human in example 5 of the present invention.
FIG. 9 is a plasmid map of the expression of membrane protein mCC1 in example 6 of the present invention.
FIG. 10 is a plasmid map of the expression of membrane protein mCC2 in example 6 of the present invention.
FIG. 11 is an electrophoresis chart of CC1H, mCC, mCC2 and plasmid HEK293 in example 6 of the present invention; lane 1 (M in the figure) marker; lane 2, CC1H; lane 3, mCC2; lane 4: mCC1; lane 5: plasmid HEK293.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols inMolecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a construction method of PLA2R main antigen region recombinant protein
The plasmid vector adopts pcDNA3.4 transient expression vector, the vector comprises a native full-length CMV promoter and WPRE elements downstream of cloning sites, the vector based on the CMV promoter generally has good expression level for a CHO cell transient expression system, the WPRE elements are positioned downstream of multiple cloning sites, the transcription and the expression of genes can be effectively improved, and the vector has ampicillin resistance genes in escherichia coli. Then, the vector gene pcDNA3.4 was amplified by PCR technique, and the gene sequences linked to the target gene (gene of interest) and signal peptide (signal peptide) were ligated. The PCR template is obtained through gene synthesis, pcDNA3.4 carrier fragments and synthetic genes are connected through a homologous recombination kit (the gene fragments obtained after connecting target genes and signal peptide sequences are called synthetic genes for short), DH5 alpha competence is transformed to obtain monoclone, sequencing is carried out after monoclone amplification, and plasmid extraction is carried out after sequencing confirmation, so as to obtain the synthetic gene recombination expression plasmid. Wherein the synthetic gene was cloned between the CMV promoter and WPRE genes of plasmid pcDNA3.4, see in particular FIG. 2.
Wherein the amino acid sequence of the signal peptide (SEQ ID NO. 10) is:
MLLSPSLLLLLLLGAPRGCA；
the nucleotide sequence of the signal peptide (SEQ ID NO. 11) is:
ATGCTGCTGTCGCCGTCGCTGCTGCTGCTGCTGCTGCTGGGGGCGCCGCGGGGCTGCGCC。
the structure of PLA2R is schematically shown in FIG. 1, wherein the target gene is the gene sequence corresponding to the amino acid sequence from 21 to 367 (Glu) of the PLA2R full-length protein sequence (UniProt sequence accession number: Q13018). The constructed transient expression plasmid was named pcDNA3.4-CC1H.
The amino acid sequence of the synthetic gene is shown as SEQ ID NO.12, and the nucleotide sequence of the synthetic gene is shown as SEQ ID NO. 13.
Example 2
This example is directed to the transient expression plasmid pcDNA3.4-CC1H of example 1 for transient expression, purification and identification of CC1H.
(1) Transient expression of CC1H
About 24 hours before transfection, freeStyle ™ CHO-S cube cells (cell density 5-6X10) 5 /mL, from Gibco corporation, freeStyle ™ MAX CHO expression System, cat: k900020 At 37 ℃, 5% CO 2 And culturing at 120-135 rpm.
On the day of transfection, cells were diluted to a cell density of 1X 10 6 Per mL, each shake10 mL cells (cell viability above 95%) were added to the flask.
Gently mix FreeStyle ™ MAX Reagent several times, note that vortex shaking is not possible.
pcDNA3.4-CC1H 12.5. Mu.g was taken, optiPRO ™ SFM was added to a total volume of 0.2 mL and gently mixed.
12.5. Mu.L of FreeStyle ™ MAX Reagent was taken, opti-Pro ™ SFM was added to a total volume of 0.2 mL and gently mixed. After mixing well, add to plasmid mix, mix gently to obtain 0.4 mL of DNA-FreeStyle ™ MAX mixture and incubate at room temperature for 10 minutes to allow complex formation.
The 0.4 mL DNA-FreeStyle ™ MAX complex described above was slowly added to the cells while the flask was slowly rotated.
At 37 ℃, 5% CO 2 Cells were cultured at 120-135 rpm without the need to replace or supplement the medium, and the cell culture supernatant was harvested after 7 days.
(2) Purification and identification of CC1H
Sample: cell culture supernatant 25 mL in step (1). The sample was centrifuged at 7000 rpm at 4℃for 10 min and passed through a 0.22 μm needle filter. And loading on a Ni sepharose 6FF column at 2 mL/min. Washing with 20mM phosphate buffer, 500mM NaCl,20mM imidazole, pH 6.8. 5 times the column volume of 0-100% 20mM phosphate buffer, 1M NaCl,500mM imidazole, pH 6.8. The fraction was collected 2 mL/tube.
The elution peak 30 kD was ultrafiltered 3 times in an ultrafiltration centrifuge tube and the solution was replaced with 50mM phosphate buffer, 150mM NaCl, pH7.4. Quantification was performed using the Bradford method. The expression level of CC1H was found to be 37mg/L. The protein sizes of the CC1H recombinant proteins are shown in Table 1.
Table 1: molecular weight of CC1H recombinant protein
SDS-PAGE analysis is carried out on the CC1H, and the analysis result is shown in figure 3, so that the CC1H protein has correct molecular weight and no impurity band, and the protein is purer.
Example 3
Mice were immunized with the CC1H protein purified in the examples, screened for monoclonal antibodies, and sequenced.
Immunized mice, hybridoma screening and sequencing were performed by Beijing Yiqiao Shenzhou technologies, inc.
Specifically, 5 mice were immunized with antigen CC1H. Antigen CC1H was coated and serum titers were measured by ELISA with serum dilutions of 8000 and 16000. And (5) fusing after the serum titer ELISA is qualified.
According to the serum titer result, selecting the optimal 1 mouse to carry out hybridoma fusion screening and limiting dilution, taking the immunized mouse spleen cells, and carrying out the following steps of 1: 1. mixing the mixture with SP2/0 myeloma cells in proportion, and fusing by using an electrofusion method to obtain hybridoma cells. Whole spleen is plated, immunogen is used as screening antigen, cell supernatant is measured by an indirect ELISA method, positive holes combined with the immunogen are selected as positive main clones, subcloning is carried out through limiting dilution until hybridoma cell strains which stably secrete monoclonal antibodies are obtained, positive hybridoma cells are obtained, and cell freezing and antibody production are carried out.
Positive clones obtained by screening were cultured on appropriate scale, and the supernatant was purified with ProteinA to obtain 0.1-0.5mg of antibodies, which were identified by UV, SDS-PAGE (FIG. 4).
Sequencing the positive hybridoma cells to obtain the mouse monoclonal antibody sequence.
Table 2: molecular weight of murine monoclonal antibody protein
The amino acid sequence of murine monoclonal antibody is as follows:
the heavy chain is shown as SEQ ID NO.1, and the light chain is shown as SEQ ID NO. 2.
Wherein the heavy chain comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 and the light chain comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3.
The amino acid sequence SEQ ID NO.3 of CDR-H1 is GYSFTGYY;
the amino acid sequence SEQ ID NO.4 of CDR-H2 is FNPYNGAT;
the amino acid sequence of CDR-H3 is SEQ ID NO.5 as AKEGPYYGNYGYFDY;
the amino acid sequence of CDR-L1 is SEQ ID NO.6 as QSLLNSRTRKNY;
the amino acid sequence of CDR-L2 is WAS;
the amino acid sequence SEQ ID NO.7 of CDR-L3 is KQSYNLYNT.
The amino acid sequence of FR-VH1 is SEQ ID NO.17 EVQLQQSGPELVKPGASVKISCKAS;
the amino acid sequence of FR-VH2 is SEQ ID NO.18 MHWVRQSHVKSLEWIGR;
the amino acid sequence of FR-VH3 is shown as SEQ ID NO.19 as NYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYC;
the amino acid sequence of FR-VH4 is shown as SEQ ID NO.20 as WGQGTTLTVSS;
the amino acid sequence SEQ ID NO.21 of FR-VL1 is DIVMSQSPSSLAVSAGEKVTMSCKSS;
the amino acid sequence SEQ ID NO.22 of FR-VL2 is LAWYQQKPGQFPKLLIY;
the amino acid sequence of FR-VL3 is SEQ ID NO.23 TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYFC;
the amino acid sequence of FR-VL4 SEQ ID NO.24 is FGGGTKLEIK.
Sequencing a monoclonal antibody gene to obtain the DNA sequence of the mouse monoclonal antibody as follows:
the heavy chain is shown as SEQ ID NO.8, and the light chain is shown as SEQ ID NO. 9.
Example 4
The binding effect of the antibodies obtained by the screening of example 3 on human PLA2R CC1H was examined by ELISA.
The reagents of this example were from solebao, unless specified otherwise.
10. Mu g/mL CC1H is plated, mouse anti-human PLA2R monoclonal antibody (prepared in example 3) which is diluted in a gradient way is added, goat anti-mouse HRP secondary antibody is added, and color development detection is carried out;
[1] 100. Mu.L/well of a coated liquid seal plate containing 10. Mu.g/mL CC1H, overnight at 4 ℃;
[2] discarding liquid, washing a plate by using PBST, washing 4 wheels by using a plate washer, spin-drying by using a plate slinger, and beating the plate;
[3] adding 100 mu L/hole sealing liquid, and incubating at 37 ℃ for 60 min;
[4] discarding the solution, adding 100 mu L/hole mouse anti-human PLA2R monoclonal antibody (PBST dilution), sealing the plate, and incubating for 30 min at room temperature;
[5] repeating the step [2] of washing the plate;
[6] mu.L of detection antibody (EASYBIO, BE0102,1:2000 diluted with PBST) was added to each well, the plates were closed and incubated for 30 min at room temperature;
[7] repeating the step [2] of washing the plate;
[8] after adding 100. Mu.L of TMB substrate chromogenic solution to each well and incubating for 5 min at 25℃in the dark, 100. Mu.L of stop solution was added to each well and OD450 was measured.
A murine anti-human mab fit curve was drawn by the measurements, as shown with reference to figure 5.
ELISA experimental results show that the purified antibody prepared in the example 3 is positive to the immunogen ELISA.
Example 5
This example was directed to the antibodies screened in example 3 for kinetic, thermodynamic analysis of interactions with CC1H.
The binding constant (Ka), dissociation constant (Kd) and affinity constant (KD) of the interaction of murine anti-human PLA2R mab with Biotin (Biotin) labeled CC1H were detected using a Sartorius (Sartorius) Octet 2 molecular interaction instrument based on the biological membrane interference technique (Bio-Layer Interferometry, BLI).
The specific experimental steps are as follows:
(1) Biotin labelling of CC1H
The purified CC1H protein was labeled with Biotin (Biotin) using the EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, 21335) kit:
equilibrate the kit to room temperature; then 10 mM biotin reagent solution was prepared. Adding 90 [ mu ] L of sterile double distilled water into each 0.5mg biotin reagent for full dissolution; adding 20 times mol number of biotin reagent solution of protein to the CC1H protein solution; the system was then incubated on ice for 2 hours, with several inversions to allow adequate mixing.
(2) Purification of CC1H-Biotin
Purification of Biotin-labeled CC1H-Biotin using a G-25 prefilled desalting column (Bogurone, EG 001):
20mM PBS buffer, pH 7.2, was prepared according to Table 3;
table 3 PBS buffer formulation table
Balance: the PBS buffer was withdrawn with a 10 mL syringe, each time a "drop-to-drop" column and syringe, to prevent air bubbles; then cutting off the outlet end; a total of 25 mL PBS buffer, 5 mL/min (120 drops/min) was used to remove ethanol from the column, and the column effluent was discarded.
Loading: 1.5 mL (less than 1.5 mL supplemented with PBS buffer) was loaded at 5 mL/min using a3 mL syringe, and the column effluent was discarded; continuing to inject 3 mL of PBS buffer; together about 2 mL.
Column cleaning and preservation: 25 mL of PBS buffer was passed through the column using a 10 mL syringe, followed by 25 mL water, and the column effluent was discarded; stored at 4 ℃ with a 10 mL syringe over 25 mL of 20% ethanol.
Sample storage: and (5) performing suction filtration sterilization on the collected sample by using a 0.2 mu m pore size filter, and subpackaging and preserving.
(3) Measurement of CC1H-Biotin protein concentration by Bradfod method (Biyun, P0060):
protein standard (5 mg/ml BSA) was completely thawed and mixed, diluted with PBS buffer, and 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5 mg/ml protein standard was formulated and thoroughly mixed;
mu.L of protein standard with different concentration is added into standard wells of a 96-well immune plate (Thermo Scientific, 468667);
taking 5 μl of sample into the sample wells of a 96-well immunization plate (less than 5 μl supplemented with PBS buffer); adding 250 mu LG250 staining solution into each well; measuring A595 by using an enzyme-labeled instrument;
the protein concentration in the sample was calculated from a standard curve, which is shown in fig. 6. Where y=0.6849x+0.6325, r=0.9925.
The concentration of CC1H-Biotin protein was calculated to be 2.36 mg/mL.
(4) Sample purity, concentration were identified by reducing/non-reducing SDS-PAGE:
sample purity, concentration were identified by reducing/non-reducing SDS-PAGE. The purification effect of the CC1H-Biotin is shown in FIG. 7, wherein lanes from left to right are the CC1H-Biotin containing the DTT reduction loading buffer and the CC1H-Biotin containing the non-reduction loading buffer in sequence.
The molecular weight of CC1H-Biotin is about 41 kDa, and the purity is more than 98%.
(5) Biotin incorporation levels were detected using the Pierce ™ Biotin Quantitation Kit (HABA assay) (Thermo Scientific ™, 28005) kit:
balancing an ABA/Avidin premix to room temperature; then 100. Mu.L of ultra pure water was added to the HABA/Avidin premix tube and blown up with the gun head.
160 μl of PBS buffer was added to wells of 96-well immunoplates (Thermo Scientific, 468667); then 20. Mu.L of HABA/Avidin premix solution was added to the wells containing PBS buffer, and after shaking and mixing, A500 was measured.
Add 20. Mu.L of biotinylated sample to wells containing HABA/Avidin and measure A500 for at least 15 seconds after shaking and mixing.
Biotin incorporation levels were calculated from beer's law. The results are shown in Table 4. The Biotin incorporation level of CC1H-Biotin was calculated to be 1.80 mol Biotin/mol protein.
TABLE 4 Biotin incorporation levels
(6) Interaction assay of CC1H-Biotin with murine anti-human PLA2R monoclonal antibodies screened in example 3.
SA sensors (Sartorius, octet square Streptavidin (SA) Biosensor, 18-5019) were selected using a Sartorius (Sartorius) Octet square N1 molecular interaction instrument:
immersing the sensor in PBSTB buffer (PBS buffer with 0.02% Tween-20 and 0.1% BSA) for pre-wetting for >10 min, and loading onto a molecular interaction instrument;
the sensor was immersed in different buffers sequentially and tested using a 5-step method:
baseline 1 (300 μl of PBSTB buffer, 1 min) - > cured (CC 1H-biotin,20 μg/mL 300 μl,2 min) - > baseline 2 (PBSTB buffer, 3 min) - > bound (murine anti-human PLA2R mab 300 μl,2 min) - > dissociated (PBSTB buffer 300 μl,5 min) with a gradient of PBSTB buffer);
different concentrations of murine anti-human measured or fitted curves were plotted (fig. 8).
The binding constant (Ka), dissociation constant (Kd) and affinity constant (KD) were calculated and the results are shown in Table 5. The results in Table 5 show that CC1H-Biotin has high affinity with murine anti-human PLA2R monoclonal antibodies.
TABLE 5 Ka, kd, KD values for CC1H-Biotin and murine anti-human PLA2R monoclonal antibody
Example 6
The membrane proteins containing the three domains CysR, fnII, CTLD1 were detected using western blot.
(1) Construction of plasmid stable expression of membrane proteins comprising the CysR, fnII, CTLD1 domain.
Constructing two membrane proteins, wherein a mCC1 protein coding region comprises a human PLA2R CysR, fnII, CTLD structural domain and a transmembrane region, namely an amino acid sequence of 1 to 367 (Glu) of a PLA2R full-length protein sequence (UniProt sequence accession number: Q13018) and an amino acid sequence of 1386 to 1463 transmembrane regions of the PLA2R full-length protein sequence; the mCC protein coding region comprises the human PLA2R CysR, fnII, CTLD, CTLD2 domains and transmembrane regions, i.e., amino acid sequences comprising amino acid sequences 1 to 510 (Asp) of the PLA2R full-length protein sequence (UniProt sequence accession number: Q13018) and the 1386 to 1463 transmembrane region of the PLA2R full-length protein sequence.
Wherein, the plasmid map of the expression membrane protein mCC1 is shown in figure 9, and the plasmid map of the expression membrane protein mCC2 is shown in figure 10.
pNEO as plasmid vector
mCC1 and mCC signal peptide (SEQ ID NO. 14);
Atgctgctgtcgccgtcgctgctgctgctgctgctgctgggggcgccgcggggctgcgcc。
mCC1 sequence (SEQ ID NO. 15): 48.5kD.
The nucleotide sequence of the code mCC1 sequence is shown as SEQ ID NO. 27.
mCC2 sequence (SEQ ID NO. 16): 65.0kD.
The nucleotide sequence of the code mCC2 sequence is shown as SEQ ID NO. 28.
(2) Screening for cell lines stably expressing mCC1 or mCC2
HEK293 cells were transfected with pNeo-mCC or pNeo-mCC, and screened with G418 (Biyun Tian, ST 081-1G) to be substantially free of floating cells and >95% cell viability.
(3) Mouse anti-human PLA2R monoclonal antibody western blot detection mCC1 or mCC2
HEK293 cells stably transfected with pNeo-mCC1 or pNeo-mCC2, and untransfected HEK293 cells were lysed with RIPA lysate (Bio, C500005-0050), SDS-PAGE was performed and transferred to PVDF membrane. Conventional western blot. Antibodies were added as in table 6.
TABLE 6 antibody addition Table
Western blot (FIG. 11) shows that lanes CC1H, mCC, mCC all appear striped at the expected molecular weight positions, and that the negative control untransfected plasmid HEK293 cell lanes are striped, showing that murine anti-human PLA2R monoclonal antibodies can be used for Western blot detection of proteins comprising the CysR, fnII, CTLD1 domain.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. An anti-human PLA2R antibody and antigen-binding fragments thereof, characterized in that it comprises a heavy chain complementarity determining region and a light chain complementarity determining region;
the heavy chain complementarity determining region comprises CDR-H1, CDR-H2 and CDR-H3 with amino acid sequences shown in SEQ ID NO.3-5 respectively;
the light chain complementarity determining region includes CDR-L1 with the amino acid sequence shown in SEQ ID NO.6, CDR-L2 with the amino acid sequence WAS and CDR-L3 with the amino acid sequence shown in SEQ ID NO. 7.
2. The anti-human PLA2R antibody and antigen-binding fragment thereof according to claim 1, wherein the antibody and antigen-binding fragment thereof further comprises a heavy chain framework region and/or a light chain framework region, said heavy chain framework region comprising FR-VH1, FR-VH2, FR-VH3 and FR-VH4 as shown in sequence in SEQ ID No. 17-20; and/or; the light chain framework region comprises FR-VL1, FR-VL2, FR-VL3 and FR-VL4 shown in SEQ ID NO.21-24 in sequence.
3. The anti-human PLA2R antibody and antigen-binding fragments thereof of claim 2, wherein the antigen-binding fragments are selected from any one of Fab ', fab, F (ab') 2, scFv, and Fv of the antibody.
4. The anti-human PLA2R antibody and antigen-binding fragments thereof of claim 2, wherein the antibody or antigen-binding fragment thereof further comprises constant regions including a light chain constant region and a heavy chain constant region, the type of light chain constant region being either a kappa-type or a lambda-type light chain constant region; the heavy chain constant region is IgM, igD, igG, igA or IgE heavy chain constant region.
5. The anti-human PLA2R antibody and antigen binding fragments thereof according to claim 4, wherein the constant region of the antibody is of human, mouse, rat, bovine, equine, ovine, rabbit or canine in species origin.
6. The anti-human PLA2R antibody and antigen-binding fragment thereof according to claim 4, wherein the amino acid sequences of the light chain constant region and the heavy chain constant region are shown as SEQ ID No.25 and SEQ ID No.26, respectively.
7. Use of an antibody against human PLA2R as defined in any of claims 1-6 and an antigen binding fragment thereof for the preparation of a PLA2R assay product;
the PLA2R detection product is selected from a reagent, a kit or a chip;
the detection product comprises the antibody or antigen binding fragment thereof, and the antibody or antigen binding fragment thereof is labeled with a detectable label, which is used for non-diagnostic and therapeutic purposes.
8. The use of an antibody against human PLA2R and antigen-binding fragments thereof according to any one of claims 1-6 for the manufacture of a product for the detection, diagnosis, prognosis, efficacy assessment or recurrence monitoring of membranous nephropathy, characterized in that the marker for the detection, diagnosis, prognosis, efficacy assessment or recurrence monitoring of membranous nephropathy is PLA2R;
the membranous nephropathy is primary membranous nephropathy.
9. A detection reagent or kit comprising an antibody against human PLA2R according to any one of claims 1 to 6, and antigen binding fragments thereof.
10. A vector comprising a nucleic acid molecule encoding the anti-human PLA2R antibody of any one of claims 1-6 and antigen-binding fragments thereof.
11. A recombinant cell comprising the vector of claim 10.
12. Use of an antibody against human PLA2R and antigen binding fragments thereof according to any one of claims 1-6 for the detection of human PLA2R levels or the detection of pharmaceutical activity in a cellular model expressing PLA2R, wherein said use is for non-diagnostic and therapeutic purposes, said detection of pharmaceutical activity is a detection: inhibitory activity of the drug against antibody induced cell killing by human PLA 2R.
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