Neutralizing antibody targeting abrus precatorius toxin and application thereof

Abstract

The invention discloses a neutralizing antibody targeting abrus precatorius toxin and application of the neutralizing antibody. Specifically disclosed is a monoclonal antibody or an antigen-binding fragment thereof targeting an abrine toxin, comprising a heavy chain variable region (SEQ ID NO: 1) and a light chain variable region (SEQ ID NO: 2). The monoclonal antibody has high affinity, can specifically target an A chain of Abrin-a toxin, can significantly inhibit Abrin-a induced cytotoxicity, has a significant protection effect on cells attacked by abrus precatorius toxin, can provide long-acting protection, and has good neutralizing ability. The monoclonal antibody can be prepared into products such as a therapeutic drug and a diagnostic drug for abrus precatorius toxin poisoning, a detection kit for abrus precatorius toxin and the like clinically. Based on a unique action mechanism and a remarkable treatment effect, the invention provides a novel biological preparation for preventing and treating Abrin poisoning, and has a wide clinical application prospect in the fields of first-aid medicine and biological defense.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to neutralizing antibodies targeting abreast toxin and their applications. Background Technology

[0002] Abrin is a toxin derived from the legume Abrus precatorius (also known as the aralia elata). Abrus precatorius The type II ribosome-inactivating protein (RIP-II) extracted from L. seeds has a molecular weight of approximately 65 kDa and consists of an A chain (30 kDa) and a B chain (35 kDa) linked by disulfide bonds. The A chain irreversibly inhibits ribosome function, blocking protein synthesis in cells; while the B chain, via a galactose-binding site, mediates toxin internalization into cells. Together, they induce cell death. Studies have shown that abrin is extremely toxic, with a median lethal dose (LD50) in mice. 50 The lethal dose of abrin is only 0.04 μg / kg, with an adult lethal dose as low as 5-7 μg / kg, making it approximately 70 times more toxic than ricin. This toxin can enter the human body through ingestion, inhalation, or injection. The incubation period after poisoning can be as long as several hours to several days, with clinical manifestations including mucosal damage, multiple organ failure, and a high mortality rate. Abrin is widely available, easily purified, highly toxic, and lacks an effective antidote, and has been listed as a potential important toxin warfare agent and bioterrorism pathogen. Currently, there are no approved antidotes for abrin poisoning worldwide.

[0003] In the field of abrin-based disease control, existing antibody drugs have significant limitations. Although monoclonal antibodies have been used for toxin detection, they generally suffer from insufficient neutralizing activity (e.g., neutralization efficiency of less than 30% for subtypes such as Abrin-B) and cross-reactivity issues (e.g., cross-binding rate with ricin exceeding 15%), severely restricting the specificity of detection and treatment. Furthermore, polyclonal antibodies, due to poor batch-to-batch stability and the risk of heterologous immunogenicity, struggle to meet clinical application needs. Notably, the A chain of abrin, due to its potent cytotoxicity, has been explored for targeted cancer therapy in recent years, for example, by constructing immunotoxins through antibody-drug conjugates to specifically kill tumor cells. However, existing antibodies suffer from insufficient targeting and stability defects, resulting in a narrow therapeutic window and severe off-target toxicity, greatly limiting their translational medical value.

[0004] Given the aforementioned technological bottlenecks, developing a highly specific and high-affinity anti-Abrin antibody has become an urgent need in the fields of biosafety and precision medicine. The successful development of such antibodies will overcome existing technological limitations, providing not only novel defense mechanisms against bioterrorism threats but also, through engineering modifications (such as constructing immunodrug conjugates), expanding into the field of tumor-targeted therapy, thus achieving a full-chain technological upgrade from basic research to clinical translation. Summary of the Invention

[0005] One objective of this invention is to provide a neutralizing antibody targeting abreast toxin and its application. The technical problem to be solved by this invention is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To achieve the above objectives, the present invention first provides an antibody or antigen-binding fragment targeting abrinus toxin, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises amino acid sequences as shown in positions 26-35 of SEQ ID NO:1, CDR2 as shown in positions 50-65 of SEQ ID NO:1, and CDR3 as shown in positions 96-113 of SEQ ID NO:1, respectively. The light chain variable region comprises amino acid sequences as shown in positions 24-34 of SEQ ID NO:2, CDR2 as shown in positions 50-56 of SEQ ID NO:2, and CDR3 as shown in positions 89-97 of SEQ ID NO:2, respectively.

[0007] The antigen-binding fragments may include, but are not limited to, Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide bond-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, i.e., nanobody), minibody, and minimum recognition unit (MRU).

[0008] The sequence of the complementary determination region (CDR) is defined according to the Kabat numbering system.

[0009] Both the heavy chain variable region and the light chain variable region contain a framework region (FR, which is the region outside the CDR in the variable region, including FR1, FR2, FR3 and FR4). The framework region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, horses, monkeys, camels, alpacas, etc.).

[0010] Furthermore, the heavy chain variable region may include a frame region comprising at least one of the following: 1) Frame region FR1, whose amino acid sequence is the first 25th position of SEQ ID NO:1 or has more than 80% identity with the first 25th position of SEQ ID NO:1; 2) Frame region FR2, whose amino acid sequence is the third 49th position of SEQ ID NO:1 or has more than 80% identity with the third 49th position of SEQ ID NO:1; 3) Frame region FR3, whose amino acid sequence is the sixth 95th position of SEQ ID NO:1 or has more than 80% identity with the sixth 95th position of SEQ ID NO:1; 4) Frame region FR4, whose amino acid sequence is the 114th 120th position of SEQ ID NO:1 or has more than 80% identity with the 114th 120th position of SEQ ID NO:1.

[0011] Furthermore, the light chain variable region may include a frame region comprising at least one of the following: 1) Frame region FR1, whose amino acid sequence is the first 23rd position of SEQ ID NO:2 or has more than 80% identity with the first 23rd position of SEQ ID NO:2; 2) Frame region FR2, whose amino acid sequence is the third 35th 49th position of SEQ ID NO:2 or has more than 80% identity with the third 35th 49th position of SEQ ID NO:2; 3) Frame region FR3, whose amino acid sequence is the fifth 57th 88th position of SEQ ID NO:2 or has more than 80% identity with the fifth 57th 88th position of SEQ ID NO:2; 4) Frame region FR4, whose amino acid sequence is the ninth 98th 107th position of SEQ ID NO:2 or has more than 80% identity with the ninth 98th 107th position of SEQ ID NO:2.

[0012] The frame region typically does not come into contact with the antigen or has very little contact, and it can provide structural support for the CDR. Part or all of the frame region of the antibody or its antigen-binding fragment described in this invention can be replaced with any suitable frame region, such as a human frame region, a monkey frame region, etc.

[0013] Further, the amino acid sequence of the heavy chain variable region may be SEQ ID NO:1, or an amino acid sequence with more than 80% identity to SEQ ID NO:1 obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region may be SEQ ID NO:2, or an amino acid sequence with more than 80% identity to SEQ ID NO:2 obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:2.

[0014] Furthermore, the substitution may be a substitution of a conserved amino acid.

[0015] In some embodiments, insertions, deletions, and / or substitutions may occur within one or more complementarity-determining regions or frame regions of any of the antibodies or their antigen-binding fragments described in this invention, provided that such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as is well known to those skilled in the art, where conserved substitution of amino acids does not alter the properties and function of the protein) may be made to the complementarity-determining regions and / or frame regions without materially reducing binding affinity. For example, such changes may occur outside of antigen-contacting residues in the complementarity-determining regions.

[0016] Furthermore, the antibody or its antigen-binding fragment may further include a heavy chain constant region (CH) and a light chain constant region (CL). The heavy chain constant region may be selected from the heavy chain constant regions of IgG, IgA, IgM, IgD, or IgE. The heavy chain constant region may also be selected from the CH1, Fc, and CH3 domains. The light chain constant region may be selected from Kappa (κ) or lambda (λ) type light chain constant regions. The heavy chain constant region and light chain constant region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, horses, monkeys, camels, alpacas, etc.).

[0017] Furthermore, the heavy chain constant region can be selected from the heavy chain constant regions of human IgG subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant region can also be selected from the heavy chain constant regions of mouse IgG subclasses such as IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgG5, and IgG6.

[0018] In some embodiments, the heavy chain amino acid sequence of the antibody targeting abreast toxin described herein is SEQ ID NO:5; the light chain amino acid sequence is SEQ ID NO:6.

[0019] The present invention also provides biomaterials, which may include any of the following: A1) Nucleic acid molecules that encode the heavy chain variable region and light chain variable region of the antibody or its antigen-binding fragment described herein; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), or recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant vector described in A3); A5) A recombinant host cell containing the nucleic acid molecule described in A1), or a recombinant host cell containing the expression cassette described in A2), or a recombinant host cell containing the recombinant vector described in A3).

[0020] In the aforementioned biological materials, the recombinant vector can be either a cloning vector or an expression vector.

[0021] The recombinant vector can be constructed using a cloning vector. The construction of cloning vectors is typically for purposes such as large-scale amplification of the target gene, restriction enzyme digestion, sequencing, and facilitating long-term preservation and immediate use of the target gene. Common cloning vectors include the pUC series, pGEM series, pMD18-T, pBluescript, and pBR322. Those skilled in the art can select a suitable cloning vector as needed.

[0022] The recombinant vector can be constructed using expression vectors (such as prokaryotic and eukaryotic expression vectors). The structure of expression vectors is well known to those skilled in the art. Expression vectors typically contain elements required for target gene expression, such as promoters, multiple cloning sites, terminators, and ribosome binding sites. They may also contain selection marker genes (such as kanamycin resistance gene kanr, neomycin resistance gene neo, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, streptomycin resistance gene str, bleomycin resistance gene ble, etc.). Expression vectors can be constructed using any method known in the art (such as recombination technology, synthetic technology, etc.) or can be commercially purchased. Those skilled in the art can choose a suitable expression vector as needed.

[0023] The prokaryotic expression vector can be selected from Escherichia coli expression vectors (such as pET series vectors, pGEX series vectors, pMAL series vectors, pTrxFus, etc.), Bacillus subtilis expression vectors (such as pHT01, pHT43, pWB980, etc.) and Streptomyces expression vectors (such as pIJ702, pHJL197, pSET152, etc.).

[0024] The eukaryotic expression vector can be selected from: (1) yeast expression vectors (such as pYES2, pPICZaA, pUG6, etc.). (2) insect cell expression vectors (such as plasmid vectors such as pMT-Bip-V5-HisA, pAc5.1, etc., and baculovirus expression vectors); wherein, the baculovirus expression vector can be selected from Bac-to-Bac expression system (including baculovirus expression vectors such as pFastBac1) and MultiBac expression system (including baculovirus expression vectors such as pFBDM). (3) mammalian cell expression vectors (such as plasmid vectors such as pVAX1, pGX0001, pCAGGS, pCMV3, pCMVp-NEO-BAN, pEGFP, pcDNA3.1, pcDNA3.4, etc., and animal virus expression vectors). The animal virus expression vector can be selected from adeno-associated virus (AAV) vectors, adenovirus vectors, herpes simplex virus (HSV) vectors, lentivirus (LV) vectors, poxvirus vectors, retrovirus vectors, rhabdovirus vectors, papillomavirus vectors, Sendai virus vectors, and simianvirus expression vectors.

[0025] The microorganisms described herein may include bacteria, viruses, fungi, actinomycetes, algae, etc. Genes that can be used to clone or express the antibodies or antigen-binding fragments described in this invention include, such as *Escherichia coli*, *Corynebacterium glutamicum*, *Brevibacterium lactis*, *Pseudomonas*, *Bacillus subtilis*, *Agrobacterium tumefaciens*, yeasts (e.g., *Saccharomyces cerevisiae*, *Candida albicans*, *Methanolobacterium methylprednisolone*, *Pichia pastoris*), *Streptomyces*, as well as rotaviruses, baculoviruses, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, influenza viruses, papillomaviruses (e.g., SV40), and herpesviruses (e.g., herpes simplex virus).

[0026] The host cell (also referred to as the recipient cell) mentioned herein can refer to any type of cell (including plant cells and animal cells) used to introduce the vector. Suitable host cells are those known in the art; for example, the animal cell can be a mammalian cell, including but not limited to Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell subline (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), mouse breast cancer cells (C127 cells), young hamster kidney cells (BHK cells), human HeLa cells, human embryonic kidney cells (HEK293 cells), fibroblasts, etc.

[0027] In the above-mentioned biological materials, the nucleic acid molecule may be the DNA molecule shown in SEQ ID NO:3 and SEQ ID NO:4.

[0028] The DNA molecule shown in SEQ ID NO:3 encodes the amino acid sequence of the heavy chain variable region of SEQ ID NO:1. The DNA molecule shown in SEQ ID NO:4 encodes the amino acid sequence of the light chain variable region of SEQ ID NO:2.

[0029] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequences shown in SEQ ID NO:3 and / or SEQ ID NO:4. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in specific species as needed.

[0030] Those skilled in the art can readily mutate the nucleotide sequences encoding the antibodies or antigen-binding fragments described herein using known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis) or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random recombination). Artificially modified nucleotide sequences that possess more than 75% identity with the nucleotide sequences encoding the antibodies or antigen-binding fragments described herein (such as SEQ ID NO:3 and / or SEQ ID NO:4), as long as they encode the antibodies or antigen-binding fragments described herein, are derived from and equivalent to the sequences of this invention.

[0031] The present invention also provides the use of the antibody or its antigen-binding fragment, or the biological material, in any of the following: B1) Use in the preparation of products for the prevention or treatment of absinthecetine poisoning; B2) Application in the preparation of products for inhibiting or neutralizing the activity of abrinogen toxins; B3) Application in the preparation of products for detecting abrinogen toxins; B4) Application in the preparation of products for diagnosing abrin poisoning; B5) Application in the preparation of products for the separation or purification of abrinogen toxins.

[0032] In this article, the abrin toxin mentioned may be abrin-a.

[0033] The products described herein may include reagents, kits, chips, test strips, test cards, immunosensors (such as electrochemical immunosensors), formulations, pharmaceuticals, and pharmaceutical compositions.

[0034] The detection of abrinogen toxins described in B3) may include the detection of any in vivo or in vitro abrinogen toxin based on the principle of antigen-antibody specific reaction. The detection of abrinogen toxins may be the detection of whether a sample contains abrinogen toxins and / or the detection of the content of abrinogen toxins in the sample.

[0035] The method for diagnosing abrin poisoning as described in B4) may include the following steps: obtaining a sample from a subject, then using the antibody or its antigen-binding fragment described in this invention to detect whether abrin poisoning or the content of abrin poisoning in the sample, and making a diagnosis or auxiliary diagnosis of abrin poisoning based on the detection results.

[0036] The subjects can be humans or non-human animals (such as pigs, cows, sheep, rabbits, cats, horses, deer, monkeys, chickens, dogs, etc.).

[0037] (B5) The application may include, based on the principle of antigen-antibody specific reaction, using the antibody or its antigen-binding fragment described in this invention as an affinity ligand, immobilized on a solid-phase support to prepare a product capable of specifically capturing, enriching, and / or purifying abrinogen toxin. For example, the antibody or its antigen-binding fragment described in this invention can be prepared into an immunoaffinity chromatography column. Based on the principle that the antigen can be captured by the antibody when passing through the chromatography column, and that environmental conditions such as changing the pH value can cause the antigen to dissociate from it, abrinogen toxin can be screened and separated.

[0038] The principle of inhibiting or neutralizing the activity of abrinogen toxin as described in B2) includes binding abrinogen toxin with antibodies, thereby causing the abrinogen to lose its ability to bind to receptors, and subsequently lose its ability to enter cells and mediate toxic effects, thereby achieving the purpose of inhibiting or neutralizing the activity of abrinogen toxin.

[0039] The prevention or treatment of abrinogen poisoning described in B1) further includes long-term prevention or treatment of abrinogen poisoning.

[0040] The present invention also provides antibody conjugates comprising an antibody portion and a conjugation portion, wherein the antibody portion comprises the antibody described herein or an antigen-binding fragment thereof.

[0041] Furthermore, the antibody portion and the conjugated portion can be directly connected or covalently connected through a connector (such as a hydrazone bond, disulfide bond, thioether bond, or peptide bond).

[0042] Furthermore, the coupling portion may be selected from a detectable tag.

[0043] Furthermore, the detectable markers include enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, etc.), chemiluminescent reagents (such as acridine esters, acridine sulfonamides, luminol and its derivatives, ruthenium derivatives, etc.), and fluorescent dyes (such as AMCA, FITC, CFSE, GFP, DAPI, 7-AAD, Hoechst 33342, Pacific Blue, PE, PE-TR, PE-Cy7, PE-Cy5, PI, PerCP-Cy5.5, APC, APC-CY7, APC-H7, V500, Alexa). 700, BV605, BV480, BV785, BV510, BV711, BV421, etc.), near-infrared dyes (such as cyanine dyes, BODIPY dyes, rhodamine dyes, squaric acid dyes, porphyrin dyes, etc.), radionuclides (such as 125I, 18F, 11C, 99mTc, 123I, etc.), biotin, nanoparticles for magnetic resonance imaging, quantum dots for magnetic resonance imaging, magnetic materials (such as magnetic beads, nanoparticles containing gadolinium complexes, superparamagnetic iron oxide nanoparticles), and colloidal gold, but not limited to these.

[0044] Furthermore, the detectable markers also include tag proteins. To facilitate the isolation, purification, detection, and / or localization of the antibodies or their antigen-binding fragments described herein, tag proteins may be attached to the amino or carboxyl termini of the antibodies or their antigen-binding fragments. These tag proteins include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, nitrogen utilization substrate A (NusA) tag protein, His tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA (influenza hemagglutinin) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose-binding domain) tag protein, phage T7 protein kinase (T7PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein. Those skilled in the art know how to select appropriate tag proteins according to the desired purpose. The use of tags does not alter the function of the target protein; its purpose is to separate, purify, detect, or trace the protein. Therefore, the tagged proteins applicable to this invention are not limited to specific types. Tags can be separated from the target protein using chemical cleavage methods or enzymatic methods known in the art (such as introducing protease cleavage sites and using TEV protease to remove the tag).

[0045] The present invention also provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof described herein, the biological material or the antibody-drug conjugate, and one or more pharmaceutically acceptable carriers.

[0046] The pharmaceutical composition may have at least one of the following uses: (1) for the prevention or treatment of abrin poisoning; (2) for the inhibition or neutralization of abrin poisoning activity; (3) for the diagnosis of abrin poisoning; (4) for the inhibition of A-chain glycosidase activity of Abrin-a; and (5) for the inhibition of Abrin-a-induced cell death.

[0047] The pharmaceutically acceptable carrier may be selected from excipients, preservatives, protective agents, solubilizers, diluents (such as water, physiological saline, PBS (phosphate-buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, croscarmellose, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), binders (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), and penetration enhancers (such as Brij-78). The carrier includes pH adjusters, stabilizers (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactants (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption enhancers (such as chitosan), thickeners (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidants (such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, etc.), plasticizers (such as glycerin, sorbitol, phthalates, etc.), propellants (such as hydrofluorocarbons, dimethyl ether, etc.), atomizing agents, suspending agents, dispersants, colorants (such as TiO2, pigments, etc.), and flavoring agents. Those skilled in the art will recognize that a carrier typically has multiple functions; for example, starch can act as both a disintegrant and a binder. Those skilled in the art can routinely select the above-mentioned carriers based on the properties of the drug and the route of administration.

[0048] The dosage forms of the pharmaceutical compositions include, but are not limited to, injections (including solutions for injection and powders for injection), gels, eye drops (including eye drops and intraocular injection solutions), oral solutions, suppositories, effervescent tablets, capsules, ointments, creams, sprays, aerosols, topical solutions, tablets, powders, pills, granules, scratch inhibitors, drops, ointments, patches, and long-acting sustained-release formulations. Those skilled in the art will recognize that the above-mentioned dosage forms can be prepared using the active ingredient (such as the antibody or its antigen-binding fragment of the present invention), combined with a suitable pharmaceutically acceptable carrier, and following conventional pharmaceutical manufacturing processes.

[0049] Furthermore, the dosage form of the pharmaceutical composition may be an injectable formulation.

[0050] Injectable preparations generally refer to solutions, emulsions, and sterile powders prepared from original drugs through extraction and purification for injection into the body. These include injectable solutions and powders for injection. The preparation methods for injectable preparations are well-known to those skilled in the art; for example, powders for injection can be prepared using vacuum freeze-drying, spray drying, or spray freeze-drying techniques. Alternatively, injectable solutions can be prepared by reconstituted the drug with suitable diluents, solubilizers, and / or wetting agents using concentrated or diluted methods, followed by filtration (such as surface filtration and / or depth filtration), filling, and sterilization.

[0051] To formulate the pharmaceutical composition into injectable preparations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, commonly used diluents in the art, such as water, physiological saline, PBS (phosphate-buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, and dimethyl sulfoxide, can be used as solvents to prepare the injection solution. Additionally, to prepare isotonic injections, appropriate amounts of carriers such as sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, and other carriers can also be added.

[0052] The administration methods of the pharmaceutical composition include, but are not limited to, injection (e.g., administration via injection), mucosal administration (e.g., administration via spray, aerosol, tablet, eye drops, suppository, granule, capsule, etc.), and transdermal administration (e.g., administration via gel, ointment, patch, film, etc.).

[0053] The administration methods of the pharmaceutical composition include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, mucosal administration, oral administration, oral / nasal spray, nebulized inhalation, implantation, and external delivery via a portable device.

[0054] The active ingredient of the pharmaceutical composition may be any of the antibodies or antigen-binding fragments described herein.

[0055] The present invention also provides a kit comprising the antibody or antigen-binding fragment thereof described herein.

[0056] The kit may have at least one of the following uses: (1) for detecting abrin toxin; (2) for detecting abrin-a; (3) for detecting the A-chain protein of abrin-a; (4) for diagnosing abrin toxin poisoning; (5) for isolating or purifying abrin toxin; (6) for isolating or purifying abrin-a; (7) for isolating or purifying the A-chain protein of abrin-a.

[0057] The test samples for the kit can be environmental samples, blood samples (such as whole blood, plasma, serum), sputum samples, tissue samples, cell samples, fecal samples, etc., but are not limited to these. Furthermore, the environment can include soil, water, air, objects (such as medical devices, ground, walls, sinks, containers, packaging, fabrics, food, feed, etc.), but are not limited to these.

[0058] The kit may be a chemiluminescent immunoassay kit, enzyme-linked immunosorbent assay kit, immunoprecipitation assay kit, immunoblotting assay kit, immunochromatographic assay kit, flow cytometry assay kit, immunohistochemistry assay kit, colloidal gold immunoassay kit, or fluorescent immunoassay kit, but is not limited thereto.

[0059] Furthermore, the kit may also include reagents required for immunoassay, such as labeled antibodies or antigens, magnetic microparticles, blocking solution, diluent, washing solution, chromogenic solution, stop solution, etc., but not limited to these.

[0060] The various reagent components of the kit may be present in separate containers, or may be pre-assembled into a reagent mixture, either wholly or partially.

[0061] The components of the kit may be provided in solution form, such as an aqueous solution. When present in aqueous solution, the concentration or content of these components can be readily determined by those skilled in the art according to different needs. For example, for storage purposes, the components may be present at a higher concentration, which can be reduced to the working concentration by diluting the higher concentration solution when in operation or for use.

[0062] The present invention also provides a method for preparing an antibody or antigen-binding fragment thereof targeting abrin toxin, the method comprising expressing the antibody or antigen-binding fragment thereof described herein in a host cell and recovering or separating the antibody or antigen-binding fragment thereof.

[0063] Further, the preparation method may include the following steps: cloning a nucleic acid molecule encoding the antibody or its antigen-binding fragment of the present invention into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector) to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell expressing the antibody or its antigen-binding fragment; culturing the recombinant host cell, and recovering or separating the antibody or its antigen-binding fragment from the cultured recombinant host cell culture.

[0064] Furthermore, the recovery or separation can be achieved by precipitation methods (such as salting out, organic solvent precipitation, octanoic acid-saturated ammonium sulfate precipitation, isoelectric point precipitation) or chromatographic techniques (such as ion exchange chromatography, gel filtration chromatography, affinity chromatography) from the culture (including all substances within the culture vessel).

[0065] The methods of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria through chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria through phage transduction. (3) directly transferring the target gene into plant recipient cells through physical or chemical methods, such as chemical stimulation, electroporation, liposome-mediated transformation, microinjection, gene gun, laser microbeam, pollen tube pathway, ultrasound, air gun, and eddy current methods. (4) transferring the target gene into plant recipient cells using a vector as a medium, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems) mediated transformation. (5) The target gene is introduced into isolated animal cells (transfection) by calcium phosphate coprecipitation, cationic polymer method (such as DEAE-dextran transfection), cationic liposome method, electroporation method (i.e. electrotransfection), microinjection, gene gun method or virus-mediated method (such as adenovirus infection method, lentivirus infection method).

[0066] The present invention also provides a method for detecting abrinogen, the method comprising using the antibody or antigen-binding fragment thereof described herein, the antibody-drug conjugate or the kit described herein to detect abrinogen.

[0067] Furthermore, the method can be a method for in vitro detection of abrinogen toxins in samples. The method can be for non-disease diagnostic purposes. These non-disease diagnostic purposes may include: testing food samples (including grains, legumes, and processed foods, etc.) for the prevention of poisoning incidents and to ensure food safety; testing environmental water samples (such as surface water, groundwater, and wastewater) to monitor environmental pollution and bioterrorism; testing plant extracts (such as crude extracts of abrinogen seeds and other related plants) for drug development or toxicity studies; testing pharmaceutical raw materials (such as components in traditional Chinese medicine or biological agents) to ensure drug safety and efficacy; testing cosmetic ingredients (such as natural plant additives) to meet the compliance requirements of cosmetic safety regulations; testing feed products (such as plant components in animal feed) to prevent livestock poisoning and ensure livestock safety; and testing industrial raw materials (such as raw materials in biotechnology products) to ensure industrial process safety and compliance with quality management standards, etc.

[0068] Furthermore, the method may include the following steps: (1) Contact the sample with the antibody or its antigen-binding fragment described herein, or the antibody-drug conjugate, under conditions that allow the antibody or its antigen-binding fragment, or the antibody-drug conjugate, to form a complex with abrinogen toxin; (2) Detect the formation of the complex to indicate the presence or content of abrinogen toxin in the sample.

[0069] The methods for detecting abrin toxin can be immunological assays, such as magnetic microparticle chemiluminescence detection, precipitation reaction, agglutination test, Western blotting, enzyme immunoassay (e.g., ELISA), double-antibody sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), chemiluminescence immunoassay, fluorescence immunoassay, radioimmunoassay (RIA), colloidal gold immunochromatography (GIC), colloidal gold immunochromatography (GICA), immunohistochemistry (IHC), complement fixation reaction, multivariate immunoassay, and fluorescence immunochromatography.

[0070] The present invention also provides an antibody binding epitope having the following characteristics: recognizing the A chain (toxic subunit) of Abrin-a.

[0071] The antibody targeting abrin toxin described in this invention can be a monoclonal antibody, named Abr-IME-7, which can specifically bind to the A chain of Abrin-a.

[0072] The monoclonal antibody Abr-IME-7 of this invention may have at least one of the following functions: (1) prevention or treatment of abrin toxin poisoning; (2) inhibition or neutralization of abrin toxin activity; (3) detection of abrin toxin; (4) diagnosis of abrin toxin poisoning; (5) isolation or purification of abrin toxin; (6) inhibition of Abrin-a A-chain glycosidase activity; (7) inhibition of Abrin-a-induced cell death; (8) detection of Abrin-a A-chain protein; (9) isolation or purification of Abrin-a A-chain protein. Further, the abrin toxin may include abrin toxin-a (Abrin-a).

[0073] This invention utilizes hybridoma technology to screen and obtain the neutralizing monoclonal antibody Abr-IME-7 targeting Abrin-a toxin. This antibody exhibits high affinity and specifically targets the A chain of Abrin-a toxin, neutralizing it by blocking its RNA N-glycosidase activity. In vitro experimental data show that this antibody significantly inhibits Abrin-a-induced cytotoxicity. When the toxin concentration is 10 ng / mL, the cell viability pretreated with 10 μg / mL Abr-IME-7 antibody increased from 36.26% in the control group to 63.48%; and it provides long-lasting protection to the cells.

[0074] The antibody Abr-IME-7, targeting abrin toxin, significantly inhibits abrin-a-induced cytotoxicity, provides significant protection against toxin-attacked cells, and exhibits good neutralizing ability. It can be expressed and produced in prokaryotic cells, eukaryotic cells, and various recombinant systems, and can be formulated into clinical products such as therapeutic drugs, diagnostic drugs, and abrin toxin detection kits for abrin toxin poisoning. Based on its unique mechanism of action and significant therapeutic effect, this invention provides a novel biological agent for the prevention and treatment of abrin poisoning, and has broad clinical application prospects in emergency medicine and biological defense. Attached Figure Description

[0075] Figure 1 The image shows the in vitro neutralizing activity (CCK-8) results of antibody Abr-IME-7.

[0076] Figure 2 The graph shows the results of the in vitro neutralizing activity (RTCA) of antibody Abr-IME-7.

[0077] Figure 3 The image shows the binding results of antibody Abr-IME-7 and Abrin.

[0078] Figure 4 The image shows the results of the affinity test (biomembrane interference method) between antibody Abr-IME-7 and Abrin A chain. Detailed Implementation

[0079] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0081] Example 1: Preparation of neutralizing antibodies targeting abrinogen toxin 1. Animal immunization Six- to eight-week-old female BALB / c mice were used. Abrin-a inactivated protein was used as the immunogen for three immunizations. The first immunization was performed by subcutaneous injection of 100 μg of Abrin-a (with complete Freund's adjuvant). The second immunization was performed every two weeks by subcutaneous injection of 100 μg of Abrin-a (with incomplete Freund's adjuvant) for three immunizations. The third immunization was performed by intraperitoneal injection of 100 μg of Abrin-a (without Freund's adjuvant) three days before fusion.

[0082] 2. Cell fusion and screening and cloning of positive hybridoma cells BALB / c mice that had completed full immunization were euthanized by cervical dislocation, and the abdominal cavity was exposed under aseptic conditions to isolate the spleen. The spleen was placed in a 200-mesh stainless steel sieve and homogenized at low speed using a glass homogenizer to prepare a single-cell suspension. The suspension was collected by centrifugation at 1000 rpm for 5 min. Logarithmic growth phase SP2 / 0 myeloma cells were taken and mixed with spleen cells at a ratio of 5:1. The mixture was centrifuged again and the supernatant was discarded. PEG1500 fusion agent (final concentration 50%) pre-warmed to 37℃ was added, and the mixture was stirred continuously in a 37℃ constant temperature water bath for 3 min to induce cell fusion. Serum-free culture medium was immediately added to terminate the reaction. After centrifugation and discarding the supernatant, the cells were resuspended in DMEM complete culture medium containing 2% HAT, 1% penicillin-kanamycin, 1% L-glutamine, and 20% fetal bovine serum, and the volume was adjusted to 50 mL. The fusion cell suspension was seeded at 100 μL / well in 96-well plates pre-coated with feeder cells (mitomycin C-treated mouse peritoneal macrophages) and selectively cultured at 37°C in a 5% CO2 incubator. The medium was replaced at half volume every 3 days to maintain nutrition. Clonal growth was observed after 10 days of culture. The supernatant from wells containing clearly visible cell clones was used to detect specificity using an indirect ELISA method: Inactivated Abrin-a antigen (10 μg / mL) diluted in coating buffer (0.05 M carbonate buffer, pH 9.6) was used to coat the ELISA plate overnight at 4°C, followed by blocking with 0.1% casein-PBS for 2 h. The supernatant to be tested was added and incubated at 37°C for 30 min, followed by washing 5 times with PBST. HRP-labeled goat anti-mouse IgG (1:1500 dilution) was incubated at 37°C for 20 min, followed by washing again, adding TMB substrate for color development for 10 min, and stopping the reaction with 2 M H2SO4. OD was then measured. 450 Value. Positive clones were continuously screened using the limiting dilution method: For cell clones that reacted positively, the limiting dilution method was used three times until a 100% positive well rate was achieved.

[0083] 3. Preparation of monoclonal antibodies 7-8 week old BALB / c mice were selected for the preparation of monoclonal antibody ascites fluid. Seven days prior to the experiment, the mice were aseptically sensitized with 1 mL of liquid paraffin via intraperitoneal injection. Seven days later, antibody-positive monoclonal cell lines were collected for activity counting at a concentration of 2.5 × 10⁻⁶. 6Adjust the cell suspension density to 1 mL / mL and inject 1 mL of the cell suspension intraperitoneally under aseptic conditions. Observe the abdominal condition of mice 7-14 days after injection: if the abdomen is significantly distended, ascites fluid is collected aseptically; if the distension is not significant, continue feeding. During collection, mice are euthanized by cervical dislocation and fixed to a dissecting board. The skin and peritoneum are cut along the midline of the abdomen. If white nodular cell clusters are seen in the peritoneal cavity, the ascites fluid is aspirated using a pipette. After collection, first aspirate the residual fluid in the peritoneal cavity, then inject 5 mL of PBS and gently shake to rinse. Aspirate the rinsing fluid again. Combine the two fluids, centrifuge at 1000×g for 10 min, discard the precipitate, aliquot and label the supernatant, and store at -20℃.

[0084] 4. Purification of monoclonal antibodies Ascites fluid was centrifuged at 4°C and 10,000×g for 10 min to remove impurities, and the supernatant was collected. It was mixed with acetate buffer at a 1:2 ratio, and caprylic acid solution was added dropwise to a final concentration of 33% while stirring at room temperature. Stirring was continued for 30 min, followed by incubation at 4°C for 1 h to promote precipitation. The precipitate was discarded after centrifugation at 4°C and 10,000×g for 30 min. The supernatant was adjusted to pH 7.4 by adding 10% (v / v) of 0.1 M PBS (pH 7.4). Saturated ammonium sulfate (SAS) was slowly added dropwise to a final concentration of 50%, and the mixture was incubated at 4°C for 3 h. The supernatant was then discarded after centrifugation at 4°C and 10,000×g for 30 min. The precipitate was dissolved in physiological saline and transferred to 50-100 times its volume of physiological saline. Salting-out was performed overnight at 4°C. The process of centrifugation at 4°C and 10,000×g for 30 min followed by discarding the supernatant was repeated, followed by incubation at 4°C for 3 h and centrifugation again. The precipitate was resuspended in an appropriate amount of dialysis buffer, placed in a dialysis bag, and dialyzed with running water for 5 h, then transferred to 4℃ for 12 h. The precipitate was discarded by centrifugation at 10000×g for 10 min at 4℃, and the antibody concentration was finally determined by ultraviolet spectrophotometry.

[0085] 5. The sequence of a monoclonal antibody Through screening using biolayer interference technology, high affinity (binding dissociation constant Kd < 10) was ultimately obtained. -12 A neutralizing antibody targeting abrinogen toxin with good specificity was named Abr-IME-7. The sequence of antibody Abr-IME-7 obtained by sequencing is as follows: The amino acid sequence of the variable region of the heavy chain of antibody Abr-IME-7 (the three underlined parts are CDR1, CDR2, and CDR3 in order): QVQLLQSGAELVRPGVSVKISCKGS GYTFTDYTMH WVKQSHAKSLEWIG VISPYYGDVDYNQKFK GKATMTVDKSSSTAYMELARLTSEDSAIYY CARGGRLYGPWFAYWGQG TLVTVSA (SEQ ID NO:1).

[0086] The nucleotide sequence of the gene encoding the variable region of the antibody Abr-IME-7 heavy chain is shown in SEQ ID NO:3.

[0087] The amino acid sequence of the variable region of the light chain of antibody Abr-IME-7 (the three underlined parts are CDR1, CDR2, and CDR3 in order): DVQMIQSPSSLSASLGDIVTMTC QASQGTSLNLN WFQQKPGKAPKLLIY GTSNLED GVPSRFSGSRYGTDFTLTISSLEDEDMATYFC LQHSYLPYS FGGGTKLEIK (SEQ ID NO:2).

[0088] The nucleotide sequence of the gene encoding the variable region of the antibody Abr-IME-7 light chain is shown in SEQ ID NO:4.

[0089] The sequence of complementary determinant regions is defined according to the Kabat numbering system.

[0090] The amino acid sequence of the heavy chain of antibody Abr-IME-7 is SEQ ID NO:5. The amino acid sequence of the light chain of antibody Abr-IME-7 is SEQ ID NO:6.

[0091] Example 2: In vitro cytotoxicity assay of antibody Abr-IME-7 1. Culture of HeLa cells HeLa cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 units / mL streptomycin added to the culture medium. The cells were incubated at 37°C and 5% CO2 until they reached a good cell condition before proceeding with subsequent experiments.

[0092] 2. Antibody neutralization activity assay (CCK-8) (1) Solution preparation: Abrin-a standard solution was prepared into working solutions with final concentrations of 0.01 ng / mL, 0.03 ng / mL, 0.1 ng / mL, 0.3 ng / mL, 1 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, and 100 ng / mL using serum-free RPMI-1640 medium. Abr-IME-7 antibody was prepared into a working solution with a final concentration of 10 μg / mL using serum-free RPMI-1640 medium. The blank control group consisted of wells containing only cells and serum-free medium, used to assess cell viability under normal culture conditions. The negative control group consisted of wells containing 10 ng / mL toxin, cells, and serum-free medium, used to assess the toxicity of abrin-a toxin to cells. The antibody neutralization activity assay contained wells containing 10 μg / mL Abr-IME-7, 10 ng / mL toxin, cells, and serum-free culture medium. This assay was used to evaluate the neutralizing and protective effect of the Abr-IME-7 antibody against abrin-a toxin, i.e., to test whether the antibody could neutralize the cytotoxicity of the toxin and thus maintain cell viability.

[0093] (2) Toxin-antibody pre-incubation: According to step (1), the working solution of Abrin-a and the working solution of antibody Abr-IME-7 were obtained. The different concentrations of Abrin-a and antibody Abr-IME-7 were mixed at a ratio of 1:1 (volume ratio) and placed in a cell culture incubator (37℃, 5% CO2) for 1 h.

[0094] (3) Detection of neutralizing activity of antibody Abr-IME-7: HeLa cells were serially diluted to 2×10⁻⁶ cells / mL using RPMI-1640 medium. 5 Cells at a concentration of 100 μL / well were seeded into 96-well plates, with three replicates per well. After incubation for 10 min until cells settled, the plates were placed in a cell culture incubator (37℃, 5% CO2) to allow adherence. After 24 h, the 96-well plates were removed, the old culture medium was discarded, and pre-incubated toxin-antibody mixture (100 μL / well) was added. The plates were cultured for another 24 h, and then 10 μL of LCK-8 dye was added to each well. The plates were incubated for 1-2 h, and the absorbance (OD) at 450 nm was measured using a microplate reader. 450 The formula for calculating cell viability is as follows:

[0095] The toxin concentration-effect curve was fitted using GraphPad Prism.

[0096] 3. Antibody neutralization activity assay (RTCA) (1) Fix the detection baseline and add cells: Add 50 µL of cell culture medium containing serum but without antibiotics to each well of E-Plate 16 and place it on RTCA Station. Ensure that the Cell Index of all wells is below 0.063 and start baseline detection. After the detection is completed, add 100 µL of HeLa cells to each well, that is, the number of cells in each well is 5000. Place it in a constant temperature incubator for 30 min to allow the cells to adhere to the wall, and then start detecting the cell proliferation curve for 10 h. (2) Detection of neutralizing activity of antibody Abr-IME-7: Take out E-Plate 16, add Abrin-a at a working concentration of 10 ng / mL and Abr-IME-7 at a working concentration of 10 µg / mL to the corresponding wells, and continue to detect cell proliferation curves; after the cell curves stabilize, stop the measurement, normalize the time points of adding interfering substances, export the results, and use GraphPad Prism to plot and analyze the results.

[0097] In vitro CCK-8 neutralization assay results showed that ( Figure 1 The antibody Abr-IME-7 significantly inhibited abrin-a-induced HeLa cell cytotoxicity. Under a toxin concentration gradient (0.01-100 ng / mL), the Abr-IME-7 antibody (10 μg / mL) exhibited a protective effect: at a toxin concentration of 10 ng / mL, the cell survival rate of cells pretreated with Abr-IME-7 increased from 36.26% in the control group to 63.48%, a significant difference (p<0.0001). Even at a toxin concentration of 30 ng / mL, Abr-IME-7 maintained neutralizing activity (survival rate >58%), indicating its broad-spectrum antitoxic potential. In vitro RTCA neutralization assays showed... Figure 2 When the toxin concentration is 10 ng / mL, the 10 μg / mL Abr-IME-7 antibody can show a protective effect for up to 17 hours, with the best protective effect occurring 5 to 11 hours after the addition of the toxin and antibody, indicating that the Abr-IME-7 antibody can provide long-lasting protection against Abrin-a at concentrations up to 10 μg / mL.

[0098] Example 3: Binding assay of antibody Abr-IME-7 with Abrin-a 1. Solution preparation (1) BSA protein standard: Dilute the 25 mg / mL protein standard solution with PBS to a final concentration of 0.5 mg / mL; (2) BCA working solution: Mix BCA reagent A and BCA reagent B at a volume ratio of 50:1; (3) 10% separating gel: 4 mL ultrapure water, 3.3 mL 30% Acrylamide (Arc-Bis), 2.5 mL 1.5 M Tris-HCl, 0.1 mL 10% SDS, 0.1 mL 10% APS and 0.01 mL TEMED, vortex to mix; (4) 5% stacking gel: 2.1 mL ultrapure water, 0.5 mL 30% Acrylamide (Arc-Bis), 0.38 mL 1 M Tris-HCl, 0.03 mL 10% SDS, 0.03 mL 10% APS and 0.003 mL TEMED, vortex to mix; (5) Electrophoresis buffer (5×): Dissolve 15.1 g Tris and 94 g glycine in 900 mL of ultrapure water, add 50 mL of 10% SDS, and add deionized water to 1 L to make 5× buffer. Dilute to 1× buffer before use. (6) Transfer buffer: Add 3.03 g Tris and 14.4 g glycine to 200 mL CH3OH, and add ultrapure water to 1 L; (7) PBST buffer: Measure 955 mL of 1×PBS, add 0.5 mL of 10% Tween-20 solution, and mix well; (8) 5% skim milk: Dissolve 5 g of skim milk powder in 100 mL of PBST buffer.

[0099] 2. Western-Blot (1) BCA quantification: Prepare 20 μL / well BSA protein standard solutions of 0.5 mg / mL BSA protein solution using PBS, with final concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL respectively. Add an appropriate volume of Abrin-a to the 96-well plate. If the volume is less than 20 μL, add PBS to make up the difference. Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes. Measure the absorbance of each well at 550 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and sample volume.

[0100] (2) SDS-PAGE: Prepare a 10% SDS polyacrylamide gel according to step 1. Separate 100 ng of Abrin-a under reducing and non-reducing conditions. The voltage is initially 80V (30 min), then switched to 120V (60 min). Electrophoresis is terminated when bromophenol blue reaches the bottom of the gel.

[0101] (3) Transfer and blocking: Prepare transfer buffer and 5% skim milk as in step 1. Place the transfer clamp in the tray containing the transfer buffer, and place a sponge pad and three layers of filter paper on both sides. Pry open the glass plate and scrape off the stacking gel. Peel the separating gel onto the black side of the filter paper. Soak the PVDF membrane in methanol for 30 seconds, then place it on the gel and remove air bubbles. Close the clamp and place it in the transfer tank. Transfer at 100 V for 100 min. Block with 5% skim milk for 2 h at room temperature.

[0102] (4) Antibody incubation and development: The primary antibody Abr-IME-7 (1 mg / mL) was diluted with PBST at a ratio of 1:1000 and incubated overnight at 4°C. The secondary antibody rabbit anti-mouse IgG was diluted with 5% skim milk at a ratio of 1:5000 and incubated at room temperature for 2 h. After each incubation, the membrane was washed three times with PBST buffer at room temperature for 5 min each time. Under light-protected conditions, equal volumes of ECL chemiluminescent reagent A and B were mixed, spread on the target protein area, and observed in a developing instrument.

[0103] The binding assay results of antibody Abr-IME-7 to Abrin-a showed that ( Figure 3 Under non-reducing conditions, antibody Abr-IME-7 specifically recognizes intact Abrin-a (65 kDa), indicating that its epitope binding depends on the native spatial conformation of the toxin. When the toxin dissociates into A chain (30 kDa) and B chain (35 kDa) under reducing conditions, antibody Abr-IME-7 specifically binds only to the A chain. This finding suggests that antibody Abr-IME-7 influences the cytotoxic effect of the toxin by targeting functional domains of the A chain, such as glycosidase active sites.

[0104] Example 4: Affinity of antibody Abr-IME-7 to Abrin-a A chain 1. Solution preparation: PBST (containing 0.02% Tween).

[0105] 2. Recombinant Expression and Purification of the A Chain of Abrin-a Toxin: The gene for abrin-a toxin (Uniprot ID: P11140, gene number ABRA_ABRPR) was obtained using chemical synthesis methods and cloned into the expression vector pET21a to construct a recombinant plasmid. The recombinant plasmid was then transformed into *Escherichia coli* Rosetta strain to achieve heterologous expression of the toxin protein. The recombinant strain was cultured, and its OD... 600When the concentration of the bacterial cell reached 0.6–0.8, 0.5 mM IPTG was added, and the cells were incubated at 18°C ​​and 150 rpm for 15–20 h. The cells were collected, sonicated, and centrifuged at 8000 rpm for 5 min to collect the supernatant. Purification was performed using nickel affinity chromatography, with washings of 20, 40, and 80 mM imidazole sequentially to remove contaminating proteins, followed by elution with 250 mM imidazole.

[0106] 3. Biotin-labeled antibody antigen: The A chain of abrin-a toxin was labeled with biotin using the Elabscience® Biotin Labeling Kit. 4 μg / mL of biotin-labeled abrin-a toxin A chain protein was used, and its binding dissociation constant with different concentrations of monoclonal antibody Abr-IME-7 was analyzed using biolayer interferometry (Sartorius' Octet molecular interaction technology). The test concentrations of monoclonal antibody Abr-IME-7 were 0, 0.1, 1, 5, 10, 50, 100, and 500 nM.

[0107] The results are as follows Figure 4 As shown, standard curves were plotted for the reaction of 133 nM (4 μg / mL) biotin-labeled Abrin-a A chain protein with 0.1, 1, 5, 10, 50, 100, and 500 nM monoclonal antibody Abr-IME-7. The goodness of fit R of the curves was [value missing]. 2 =0.9991, the dissociation constant Kd is calculated to be <10. -12 M indicates that the antigen and antibody have extremely high binding efficiency.

[0108] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An antibody or its antigen-binding fragment targeting abrin toxin, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDR1, as shown in positions 26-35 of SEQ ID NO:1; CDR2, as shown in positions 50-65 of SEQ ID NO:1; and CDR3, as shown in positions 96-113 of SEQ ID NO:

1. The light chain variable region comprises CDR1, as shown in positions 24-34 of SEQ ID NO:2; CDR2, as shown in positions 50-56 of SEQ ID NO:2; and CDR3, as shown in positions 89-97 of SEQ ID NO:

2.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is SEQ ID NO:1, or an amino acid sequence with more than 80% identity to SEQ ID NO:1 obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region is SEQ ID NO:2, or an amino acid sequence with more than 80% identity to SEQ ID NO:2 obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:

2.

3. A biomaterial, characterized in that, The biomaterial includes any of the following: A1) A nucleic acid molecule that encodes the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof as described in claim 1 or 2; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), or recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant vector described in A3); A5) A recombinant host cell containing the nucleic acid molecule described in A1), or a recombinant host cell containing the expression cassette described in A2), or a recombinant host cell containing the recombinant vector described in A3).

4. The biomaterial according to claim 3, characterized in that, The nucleic acid molecule is the DNA molecule shown in SEQ ID NO:3 and SEQ ID NO:

4.

5. The use of the antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, or the biological material as claimed in claim 3 or 4, in any of the following: B1) Use in the preparation of products for the prevention or treatment of absinthecetine poisoning; B2) Application in the preparation of products for inhibiting or neutralizing the activity of abrinogen toxins; B3) Application in the preparation of products for detecting abrinogen toxins; B4) Application in the preparation of products for diagnosing abrin poisoning; B5) Application in the preparation of products for the separation or purification of abrinogen toxins.

6. An antibody conjugate comprising an antibody portion and a conjugation portion, characterized in that, The antibody portion comprises the antibody or antigen-binding fragment thereof as described in claim 1 or 2.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof as described in claim 1 or 2, the biological material as described in claim 3 or 4 or the antibody conjugate as described in claim 6, and one or more pharmaceutically acceptable carriers.

8. A reagent kit, characterized in that, The kit contains the antibody or its antigen-binding fragment as described in claim 1 or 2.

9. A method for preparing an antibody or antigen-binding fragment targeting abrinus toxin, characterized in that, The preparation method includes expressing the antibody or antigen-binding fragment of claim 1 or 2 in a host cell and recovering or separating the antibody or antigen-binding fragment.

10. A method for detecting abrin toxin, characterized in that, The method includes detecting abrinogen toxin using the antibody or antigen-binding fragment thereof as described in claim 1 or 2, the antibody-drug conjugate as described in claim 6, or the kit as described in claim 8.

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