Anti-rabies virus mutant antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-rabies virus mutant antibody as well as a preparation method and application thereof, and belongs to the field of antibodies. The technical problem to be solved is to provide an anti-rabies virus mutant antibody with longer in-vivo half-life period and longer effective antibody concentration maintenance time. According to the key point of the technical scheme, the anti-rabies virus mutant antibody comprises a heavy chain and a light chain, and the nucleotide sequence of the heavy chain is as shown in SEQ ID NO: 1. According to the invention, a YTE mutation technology is adopted to carry out long-acting modification on the recombinant human anti-rabies virus monoclonal antibody R8. Through synthesis of heavy chain and light chain gene sequences of the mutated antibody, a mutation expression plasmid is constructed, and pharmacokinetic verification is carried out after transient transfection expression purification, so that long-acting transformation and preparation of the R8 antibody are realized, and technical support is provided for clinical application.

Description

A mutant antibody against rabies virus, its preparation method and application Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to a mutant antibody against rabies virus, its preparation method, and its application. Background Technology

[0002] For understanding the technical content of this invention:

[0003] Rabies is a highly contagious and acute zoonotic infectious disease caused by the rabies virus (RV). Currently, the core strategy in the post-exposure prophylaxis system for rabies is the combined use of active rabies vaccination and passive immunization agents. Passive immunization agents can rapidly neutralize the virus at the wound site and in the bloodstream, providing a critical window for the vaccine to induce an active immune response, and are an important component of post-exposure prophylaxis.

[0004] In the development of passive immunization agents, traditional anti-rabies serum (such as equine anti-rabies serum) has faced numerous safety concerns, including high immunogenicity of heterologous proteins and a tendency to induce allergic reactions, which have limited its clinical application. With breakthroughs in monoclonal antibody technology, fully human anti-rabies virus monoclonal antibodies, with their advantages of low immunogenicity, high safety, and strong specificity, have gradually become a research hotspot for replacing traditional serum preparations, showing broad application prospects in the field of post-exposure prophylaxis for rabies.

[0005] Various long-term modification techniques for monoclonal antibodies have been developed in this field, mainly including polyethylene glycol (PEG) modification, site-directed mutagenesis of the antibody Fc fragment, albumin fusion, and nanoparticle encapsulation. Among these, site-directed mutagenesis of the Fc fragment has received widespread attention and application due to its advantages such as high structural uniformity and complete preservation of biological activity in the modified antibody. Among the many Fc fragment mutation strategies, YTE mutation is a highly efficient long-term modification scheme that has been validated through extensive practice: this mutation significantly enhances the binding affinity of the antibody to the Neonatal Fc Receptor (FcRn) by altering the amino acid composition and spatial conformation of the Fc fragment. FcRn is a key receptor regulating antibody metabolism in vivo; it can bind to the antibody Fc fragment and mediate the intracellular recycling and reuse of the antibody, reducing the proportion of antibody degraded by lysosomes. Studies have confirmed that monoclonal antibodies modified by YTE mutation can have their in vivo half-life significantly extended without affecting their antigen-binding activity and biological neutralizing activity. This technology has been successfully applied in the long-term modification of various monoclonal antibodies, such as anti-respiratory syncytial virus antibodies and anti-human immunodeficiency virus antibodies.

[0006] Relevant patent literature retrieved: Publication country: China, Publication number: CN104193823A, Publication date: December 10, 2014. This literature discloses a neutralizing antibody against rabies virus, particularly a humanized or fully human monoclonal antibody, to meet the clinical needs for rabies diagnosis and / or treatment. This invention employs phage antibody library technology, using 32 high-titer peripheral blood samples from healthy individuals vaccinated against rabies as raw materials to prepare a phage antibody library. This antibody library underwent three rounds of screening, yielding 7 ELISA-positive antibodies. Further analysis using the RFFIT method determined their neutralizing activity; four of these, R5, R7, R8, and R9, exhibited strong neutralizing activity. This invention's high-affinity humanized anti-rabies virus antibody can be used to replace ERIG and HRIG for active and / or passive immunization therapy in individuals severely exposed to rabies virus.

[0007] This document, published in China (CN117771365B) on March 29, 2024, discloses a combination monoclonal antibody preparation against rabies virus, relating to the field of biomedical technology. The preparation comprises R92 and R71 antibodies; the light chain sequence of the R92 antibody is shown in SEQ ID NO:1, the heavy chain sequence in SEQ ID NO:2, the light chain sequence in SEQ ID NO:3, and the heavy chain sequence in SEQ ID NO:4. The ratio of R92 to R71 antibodies in the preparation is also disclosed as (1-5):(1-5). This invention's combination monoclonal antibody preparation exhibits high affinity for rabies virus glycoproteins, good neutralizing ability and thermostability, and excellent neutralizing ability against various street strains. The two antibodies target different epitopes, providing broader protection.

[0008] Relevant non-patent literature retrieved: Journal title: CNKI China Excellent Master's Thesis Full-text Database; Article title: Screening and Evaluation of Rabies Virus G Protein Monoclonal Antibodies; Volume No.: 2025-10; Publication date: September 16, 2025. This article discloses the screening and evaluation of rabies virus G protein monoclonal antibodies. This research focuses on the development of nanobodies for rabies treatment and carries out a series of works: First, an insect cell expression system was constructed to successfully obtain recombinant protein of the extracellular region of the RABVERA strain G protein. Based on this, three monoclonal antibodies (C2, E6, and F8) were screened. Western blot, ELISA, and indirect immunofluorescence experiments verified that these antibodies can specifically recognize the target recombinant protein and inactivated virus particles. Through sequencing of the antibody variable region gene and molecular docking simulation, the specific binding site of E6 monoclonal antibody to the G protein was clarified, laying a key molecular foundation for subsequent antibody modification. Subsequently, using computer-aided design strategies, the reported complementarity-determining region (CDR) of 17C7 antibodies was grafted onto the nanobody backbone to construct the VHH17C7 model, and its affinity was optimized through virtual mutation. Based on previous findings, the E6 monoclonal antibody was modified into a nanobody form (VHHE6), and mutants M1 (with enhanced affinity than the non-mutated type) and M2 (with no binding activity) were screened. This confirmed the feasibility of functional transplantation of the traditional antibody CDR region into the nanobody backbone, as well as the synergistic mechanism of backbone modification and CDR region optimization. The final study confirmed that the three screened monoclonal antibodies can serve as detection tools for RABV laboratory diagnosis and basic research, while the affinity-enhanced M1 nanobody provides a candidate molecule for treating RABV central nervous system infections. Further evaluation of its neutralizing activity, blood-brain barrier penetration efficiency, and protective effect in animal models is needed to promote its translation into practical applications.

[0009] The aforementioned literature represents existing technologies with at least the following unresolved technical problems or defects: The recombinant human anti-rabies virus monoclonal antibody R8 has a short in vivo retention time. Evidence suggests that R8 antibodies can efficiently block the interaction between the virus and host cell surface receptors, thereby achieving neutralizing activity against the virus. However, preclinical and preliminary clinical studies have shown that this R8 antibody has a short in vivo half-life: the half-life of conventional human monoclonal antibodies in humans is typically 1-2 weeks, while the in vivo retention time of R8 antibodies is roughly the same as or even slightly lower than this conventional level, resulting in a short duration of effective blood drug concentration. To ensure preventative efficacy, multiple doses are required in clinical applications, which not only increases the complexity of clinical procedures and medical costs but also places a heavy burden on patients and increases compliance pressure, greatly limiting the clinical application value and market potential of R8 antibodies. Therefore, long-acting modification of R8 antibodies to extend their in vivo half-life and the duration of effective blood drug concentration has become a key technical problem urgently needing to be solved in this field.

[0010] This invention employs YTE mutation technology to modify the recombinant human anti-rabies virus monoclonal antibody R8 for long-term efficacy. By synthesizing the mutated antibody heavy and light chain gene sequences, constructing a mutant expression plasmid, transiently transfecting and purifying it, and then performing pharmacokinetic verification, the long-term efficacy modification and preparation of the R8 antibody is achieved, providing technical support for clinical applications. Summary of the Invention

[0011] The purpose of this invention is to provide: an anti-rabies virus mutant antibody, its preparation method and application, and related technologies, to solve technical problems such as providing an anti-rabies virus mutant antibody with a longer in vivo half-life and a longer effective antibody concentration maintenance time, or combinations thereof.

[0012] Terminology: Unless otherwise defined, all technical terms in this document have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] Definitions of standard chemical terms can be found in the reference books “Molecular Cloning: A Laboratory Manual”, Science Press, authors: (US) MR. Green and J. Sambrook, 2013-4, “Biotechnology Pharmaceuticals”, Higher Education Press, author: Xia Huanzhang, 2022-4, and “Principles of Medical Virology”, Chemical Industry Press, author: Li Jing, 2020-1.

[0015] Unless otherwise stated, conventional methods within the scope of this art, such as intramuscular injection and plasmid transformation, shall be used. Unless specifically defined, the use of all commercially available products used herein shall employ standard techniques. For example, they may be performed using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally performed according to conventional methods well known in the art, based on the descriptions in the various general and more specific documents cited and discussed in this specification.

[0016] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0017] The term "rabies virus (RV)" as used in this article refers to a single-stranded negative-sense RNA virus belonging to the genus *Rabiesvirus* of the family Rhabdoviridae. It primarily infects the central nervous system, can infect humans and various animals, and causes rabies with a near 100% mortality rate. Its viral particles are bullet-shaped, and its genome encodes structural proteins such as nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). Among these, the glycoprotein is a key protein mediating viral adsorption, invasion of host cells, and induction of neutralizing antibody production.

[0018] The term "active immunity" used in this article refers to the process by which the body's immune system is stimulated to produce specific antibodies and sensitized lymphocytes through artificial vaccination (such as inactivated vaccines, live attenuated vaccines, recombinant protein vaccines, etc.) or natural infection with pathogens, thereby acquiring long-term immunity against specific pathogens. Its core feature is that the body's own immune system is activated, the immune protection is durable, and the immune memory response can be quickly initiated upon re-exposure to pathogens.

[0019] The term "passive immunization" as used in this article refers to the process by which the body immediately acquires immunity against a specific pathogen through the direct introduction of exogenous immunologically active substances (such as specific antibodies, immunoglobulins, antitoxins, etc.). This immunization method does not require activation of the body's own immune system, has a rapid onset of action but a short duration of action (usually several weeks to several months), and is mainly used for emergency prevention or treatment (such as post-exposure prophylaxis with anti-rabies immunoglobulin).

[0020] The term "heterologous protein immunogenicity" used in this article refers to the ability of proteins derived from non-host organisms (such as recombinant protein drugs, exogenous antigen proteins in vaccines, and xenobiotic animal proteins) to stimulate the host's immune system to produce a specific immune response (including humoral and cellular immunity) after entering the host body. Its strength depends on the structural characteristics of the protein (such as amino acid sequence and spatial conformation), the host's immune status, and the protein delivery method. Excessive immunogenicity may trigger allergic reactions or antibody-mediated drug clearance.

[0021] The term "polyethylene glycol (PEG) modification technology" as used in this article refers to the technique of covalently linking polyethylene glycol (a water-soluble, biocompatible polymer) to the surface of biomolecules such as proteins, peptides, antibodies, or nanocarriers using chemical methods. This technique can improve the physicochemical properties of the modified molecules (such as increasing water solubility and reducing hydrophobicity), reduce the probability of them being recognized and cleared by the body's immune system, prolong their half-life in vivo, and reduce the immunogenicity of heterologous proteins. It is widely used in the research and development of biopharmaceuticals (such as PEGylated interferon and PEGylated antibodies).

[0022] The term "antibody Fc fragment site-directed mutagenesis" used in this article refers to a technique based on genetic engineering that involves targeted mutations (such as substitution, insertion, or deletion of amino acids) at specific amino acid sites in the antibody Fc fragment (a crystallizable segment responsible for interacting with Fc receptors, complement, etc.). This technique can regulate the effector function of antibodies (such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), prolong the in vivo half-life of antibodies (such as enhancing binding to FcRn receptors), or reduce immunogenicity, and is one of the core technologies for optimizing the performance of therapeutic antibodies.

[0023] The term "albumin fusion technology" used in this article refers to the technique of recombining the coding gene of a target protein (such as cytokines, peptides, or antibody fragments) with the coding gene of albumin (such as human serum albumin HSA) and obtaining a fusion protein through genetic engineering expression. Albumin fusion can take advantage of its good biocompatibility, long in vivo half-life, and non-immunogenicity (human albumin) to prolong the in vivo circulation time of the target protein, improve its stability, and reduce the immunogenicity of the target protein. It is often used in the development of long-acting biological drugs.

[0024] The term "nanoparticle encapsulation technology" as used in this article refers to the technology of encapsulating active substances such as drugs, antigens, and nucleic acids within or adsorbing them onto the surface of nanoscale (1-1000 nm) carriers (such as liposomes, polymer nanoparticles, inorganic nanoparticles, dendritic macromolecules, etc.) using physical or chemical methods. This technology can protect the encapsulated substances from enzymatic degradation or immune clearance in vivo, achieve targeted delivery (such as targeting tumor cells or immune cells), improve bioavailability, and optimize their in vivo behavior by controlling the particle size, surface charge, and material properties of nanoparticles. It has wide applications in fields such as vaccine delivery and targeted drug therapy.

[0025] The term "293 cell" used in this article refers to an immortalized cell line obtained by transforming human embryonic kidney cells with adenovirus type 5 DNA. This cell line has adherent growth characteristics, is easy to culture and transfect, can efficiently express exogenous genes, and can be suspension-acclimated. It is widely used in the production of recombinant proteins (such as antibodies and cytokines), viral vectors (such as adenoviruses and lentiviruses), and in basic biological research.

[0026] The term "SPF-grade guinea pig" used in this article refers to guinea pigs that are specific pathogen-free (SPF), meaning they are raised in a barrier environment and do not carry specified zoonotic pathogens, pathogens of highly contagious animal diseases, or major opportunistic pathogens. They have a clear microbial background, high genetic stability, and are suitable for experiments such as drug safety evaluation, vaccine efficacy testing, and immunological research. They are commonly used model animals in the biomedical field.

[0027] The term "HEK-293F cell" used in this article refers to a derivative of the 293 cell line, which is a human embryonic kidney cell line adapted for serum-free suspension culture. This cell line has a fast growth rate and high transfection efficiency, and can efficiently express recombinant proteins (especially glycosylated eukaryotic proteins) in large-scale suspension culture systems. It is one of the core engineered cell lines in the biopharmaceutical field for the production of recombinant antibodies, viral vectors and recombinant protein drugs.

[0028] The term "PEI" used in this article refers to polyethyleneimine, a water-soluble polymer with a large number of positive charges. Based on molecular weight, it can be divided into low molecular weight PEI and high molecular weight PEI. Its positive charge can form a complex (i.e., PEI-nucleic acid complex) with negatively charged nucleic acids (DNA, RNA) through electrostatic interaction, realizing the intracellular delivery of nucleic acids. It is commonly used as a non-viral vector for gene transfection and can also be used for the construction of nanomedicine carriers.

[0029] The term "Fc fragment" used in this article refers to the crystallizable fragment of an antibody molecule, which is a region formed by the C-terminal portions of the two heavy chains of an antibody linked by disulfide bonds. It does not participate in antigen binding but is responsible for the antibody's effector function and in vivo metabolism. Its main functions include: binding to Fc receptors on the surface of macrophages, NK cells, etc., to mediate ADCC, CDC, and other effects; binding to FcRn receptors to regulate the antibody's in vivo half-life; binding to complement C1q to activate the complement system; in addition, the Fc fragment is also a key target for antibody modification (such as site-directed mutagenesis, PEGylation, and fusion proteins).

[0030] The term "ELISA" used in this article refers to Enzyme-Linked Immunosorbent Assay, an immunological technique based on the specific binding reaction of antigens and antibodies, and utilizing enzyme-labeled catalytic substrate color development for quantitative or qualitative detection. Its basic principle involves immobilizing antigens or antibodies on a solid-phase carrier, forming immune complexes through specific antigen-antibody binding, and then adding enzyme-labeled secondary antibodies or substrates. The intensity of the color produced by the enzymatic reaction reflects the concentration of the analyte. ELISA is widely used in disease diagnosis, vaccine efficacy testing, and protein quantification.

[0031] In a first aspect, the present invention provides: a mutant antibody against rabies virus.

[0032] Among its technical features is the use of mutant antibodies.

[0033] Among them, the technical feature mutation antibody is selected from: mutation antibodies including heavy chain and light chain.

[0034] Specifically, the nucleotide sequence of the heavy chain is shown in SEQ ID NO:1.

[0035] Specifically, the amino acid sequence of the heavy chain is shown in SEQ ID NO:2.

[0036] Specifically, the nucleotide sequence of the light chain is shown in SEQ ID NO:3.

[0037] Specifically, the amino acid sequence of the light chain is shown in SEQ ID NO:4.

[0038] Specifically, the mutation refers to a YTE mutation in the Fc region of the original antibody heavy chain.

[0039] Specifically, the amino acid sequence of the original antibody heavy chain is shown in SEQ ID NO:7.

[0040] Specifically, the amino acid sequence of the Fc region of the original antibody heavy chain is shown in SEQ ID NO:5. Specifically, the YTE mutation is to mutate amino acids at positions 255, 257, and 259 of the original antibody heavy chain to tyrosine, threonine, and glutamic acid, respectively.

[0041] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: a first preferred solution: a mutant antibody against rabies virus, comprising a heavy chain and a light chain, wherein the nucleotide sequence of the heavy chain is shown in SEQ ID NO:1. This technical solution, based on solving the technical problem of "providing a mutant antibody against rabies virus with a longer in vivo half-life and a longer effective antibody concentration maintenance time," further solves the technical problem of "providing the nucleotide sequence of the heavy chain of the mutant antibody against rabies virus."

[0042] Secondly, the present invention provides: an isolated polynucleotide.

[0043] Among them, the technical feature is: polynucleotides.

[0044] The technical feature polynucleotide is selected from the polynucleotide encoding the above-mentioned mutant antibody.

[0045] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: a first preferred solution: a polynucleotide encoding the above-mentioned mutant antibody. This technical solution, based on solving the technical problem of "providing a mutant antibody against rabies virus with a longer in vivo half-life and a longer effective antibody concentration maintenance time," further solves the technical problem of "providing a polynucleotide encoding a mutant antibody against rabies virus."

[0046] Thirdly, the present invention provides: a recombinant expression vector.

[0047] Among its technical features is the recombinant expression vector.

[0048] The recombinant expression vector with technical characteristics is selected from recombinant expression vectors containing the above-mentioned polynucleotides.

[0049] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes: a first preferred solution: a recombinant expression vector containing the above-mentioned polynucleotides. This technical solution, based on solving the technical problem of "providing an anti-rabies virus mutant antibody with a longer in vivo half-life and a longer effective antibody concentration maintenance time," further solves the technical problem of "providing a recombinant expression vector containing polynucleotides encoding an anti-rabies virus mutant antibody."

[0050] Fourthly, the present invention provides: a host cell.

[0051] Among them, the technical feature is the host cell.

[0052] The host cell for the technical feature is selected from host cells containing the above-mentioned mutant antibody, polynucleotide, or recombinant expression vector.

[0053] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the fourth aspect of the present invention includes: a first preferred solution: a host cell comprising the above-mentioned mutant antibody, polynucleotide, or recombinant expression vector. This technical solution, in addition to solving the technical problem of "providing a mutant antibody against rabies virus with a longer in vivo half-life and a longer effective antibody concentration maintenance time," further solves the technical problem of "providing a host cell comprising a mutant antibody against rabies virus, polynucleotide, or recombinant expression vector."

[0054] Fifthly, the present invention provides: a rabies virus detection kit.

[0055] Among its technical features is the rabies virus detection kit.

[0056] The rabies virus detection kit with technical characteristics is selected from rabies virus detection kits containing the above-mentioned mutant antibodies, polynucleotides, recombinant expression vectors or host cells.

[0057] Specifically, the rabies virus detection kit also includes any one or more of the following: detection buffer, protein standard, negative control, and positive control.

[0058] Specifically, the rabies virus test kit also includes an instruction manual.

[0059] Specifically, the instruction manual describes the usage method, result interpretation criteria, and storage conditions of the test kit.

[0060] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the fifth aspect of the present invention includes: a first preferred solution: a rabies virus detection kit comprising the above-mentioned mutant antibody, polynucleotide, recombinant expression vector, or host cell. This technical solution, while solving the technical problem of "providing a rabies virus mutant antibody with a longer in vivo half-life and a longer effective antibody concentration maintenance time," further solves the technical problem of "providing a rabies virus detection kit."

[0061] Sixthly, the present invention provides: an application.

[0062] This includes technical features: applications.

[0063] The application of the technical features is selected from: the application of the above-mentioned mutant antibodies in the preparation of drugs for the prevention and / or treatment of rabies.

[0064] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the sixth aspect of the present invention includes: a first preferred solution: the application of the above-mentioned mutant antibody in the preparation of a drug for the prevention and / or treatment of rabies. This technical solution, based on solving the technical problem of "providing a mutant antibody against rabies virus with a longer in vivo half-life and a longer duration of effective antibody concentration," further solves the technical problem of "providing the application of a mutant antibody against rabies virus."

[0065] In a seventh aspect, the present invention provides: a drug.

[0066] Among them, the technical feature is: drug.

[0067] Among them, the technically characteristic drugs are selected from drugs for the prevention and / or treatment of rabies.

[0068] Specifically, the drug contains the aforementioned mutated antibody.

[0069] Specifically, the drug also includes pharmaceutically acceptable carriers, diluents, or excipients.

[0070] Specifically, the dosage form of the drug is an injection.

[0071] Specifically, the injection is administered via intramuscular injection, intravenous injection, or subcutaneous injection.

[0072] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the seventh aspect of the present invention includes: a first preferred solution: a drug comprising the above-mentioned mutant antibody for the prevention and / or treatment of rabies. This technical solution, based on solving the technical problem of "providing an anti-rabies virus mutant antibody with a longer in vivo half-life and a longer duration of effective antibody concentration," further solves the technical problem of "providing a drug comprising an anti-rabies virus mutant antibody."

[0073] The present invention has at least the following beneficial effects: compared with the prior art, the present invention has better technical effects in terms of the long-term in vivo effect of anti-rabies virus monoclonal antibody.

[0074] According to experimental tests, the present invention increases the effective serum concentration in guinea pigs after 56 days of administration from 0.25 μg / mL in the prior art to over 0.77 μg / mL.

[0075] Considering the possibility of this invention entering other countries, this invention also provides the following technical solution: a method for preventing or treating rabies, the method comprising using the anti-rabies virus mutant antibody of this invention, the isolated polynucleotide of this invention, the recombinant expression vector of this invention, the host cell of this invention, the rabies virus detection kit of this invention, and the drug of this invention; wherein, the technical feature method comprises treating a subject with rabies by administering the drug of this invention via intramuscular injection.

[0076] Among them, the technical characteristics of the subjects included mammals.

[0077] Among these, the preferred subjects for technical characteristics are human beings. Attached Figure Description

[0078] Figure 1 shows the serum drug concentration-time curves of guinea pigs after a single dose in the R8 group and the R8LT group. Detailed Implementation

[0079] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0080] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0081] 1. Experimental materials (1) Antibody genes: Heavy chain and light chain encoding genes of recombinant human anti-rabies virus monoclonal antibody R8 (preserved by the laboratory of Lanzhou Institute of Biological Products Co., Ltd.); Control antibody recombinant human anti-rabies virus monoclonal antibody R8 was prepared by the laboratory of the above company.

[0082] (2) Vectors and strains: PCDNA3.1(-) vector (purchased from Invitrogen), Escherichia coli DH5α competent cells, HEK-293F cells (purchased from Thermo Fisher Scientific); (3) Enzymes and reagents: restriction endonucleases (BamHI, XhoI, purchased from NEB), T4 DNA ligase (purchased from Takara), plasmid extraction kit (purchased from QIAGEN, catalog number: 12943), 293 transient transfection medium (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.), 293 transient transfection medium feed (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.), PEI40K transfection reagent (purchased from Seville Biotechnology Co., Ltd.), ampicillin sodium (purchased from Seville Biotechnology Co., Ltd.), penicillin-streptomycin sodium (purchased from Seville Biotechnology Co., Ltd.), Protein A affinity chromatography packing material (purchased from Suzhou Nanomicro Technology Co., Ltd.), rabies virus standard strain (CVS-11, purchased from the National Institutes for Food and Drug Control); (4) experimental animals: SPF grade guinea pigs (weight 250-300g, half male and half female, provided by the experimental animal room of Lanzhou Institute of Biological Products Co., Ltd.).

[0083] 2. Major equipment and instruments: Operetta CLS high-content cell analysis system purchased from PerkinElmer; ST8R centrifuge purchased from Thermo Fisher Scientific; XDS-B digital inverted microscope purchased from Chongqing Optoelectronic Instrument Co., Ltd.; IX71S1F-3 fluorescence microscope purchased from Olympus Corporation; VS-1300L-U clean bench purchased from Suzhou Antai Air Technology Co., Ltd.; Class A2 biosafety cabinet purchased from Thermo Fisher Scientific; ÄKTA avant protein purification system purchased from GE Life Sciences.

[0084] Example 11: Determination of YTE mutation sites in the R8 antibody heavy chain: Site-directed mutagenesis was performed on amino acids 255, 257, and 259 of the original R8 antibody heavy chain. The sequences before and after mutation are shown in Table 1: Table 1

[0085]

[0086] 2. The light chain gene is not mutated; the light chain gene sequence of the R8 antibody developed by Lanzhou Institute of Biological Products Co., Ltd. is used.

[0087] (1) The nucleotide sequence of the light chain is as shown in SEQ ID NO:3; SEQ ID NO:3: CAGAGCGTGCTGACTCAGCCTCCTAGCGTGAGCGAAGCGCCTAGGCAGAGGGTGACTATCAGCTGTAGCGGCAGCAGCAGCAATATCGGCGAGAATGCCGTGAATTGGTACCAGCAGGTGCCTGGCAAAGCCCCTAGGCTGCTGATCTACAGCGATGATCAGCTGAGCAGCGGCATCAGCGATAGGTTCAGCGGCAGCAAGAGCGGCACTAGCGCCAGCCTGGCCATCAGCGGCCTGCTGAGCGAGGATGAGGCCGATTACTTCTGTGCCGCCTGGGATGATAGCCTGGATGGCGTGGTGTTCGGCGGCGGCACTAAGCTGACTGTGCTGGGCCAGCCTAAGGCCGCCCCTAGCGTGACTCTGTTCCCTCCTAGCAGCGAGGAGCTGCAGGCCAATAAGGCCACTCTGGTGTGTCTGATCAGCGATTTCTACCCTGGCGCCGTGACTGTGGCCTGGAAGGCCGATAGCAGCCCTGTGAAGGCCGGCGTGGAGACTACTACTCCTAGCAAGCAGAGCAATAATAAGTACGCCGCCAGCAGCTACCTGAGCCTGACTCCTGAGCAGTGGAAAAGCCACAGGAGCTACAGCTGTCAGGTGACTCACGAGGGCAGCACTGTGGAGAAGACTGTGGCCCCTACTGAGTGTAGCTGA; (2) The amino acid sequence of the light chain is as shown in SEQ ID NO:4; SEQ ID NO:4: QSVLTQPPSVSEAPRQRVTISCSGSSSNIGENAVNWYQQVPGKAPRLLIYSDDQLSSGISDRFSGSKSGTSASLAISGLLSEDEADYFCAAWDDSLDGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0088] 3. Construction of expression plasmids: The above-mentioned heavy chain gene and light chain gene were synthesized and constructed into the PCDNA3.1(-) vector, respectively. The above part was completed by Genscript Biotech Co., Ltd. The plasmids were transformed into DH5α competent cells, plated on LB plates containing ampicillin (final concentration 100 μg / mL), positive clones were screened, and sequenced to verify, obtaining recombinant expression plasmids: PCDNA3.1-R8-L and pCDNA3.1-R8-H-YTE.

[0089] 4. Antibody expression and purification (1) Transient transfection: HEK-293F cells were cultured to a density of 1×10⁻⁶. 6 Cells / mL were transfected using the PEI method. The recombinant expression plasmid pCDNA3.1-R8-L and pCDNA3.1-R8-H-YTE were mixed at a mass ratio of L:H=2:1 (66 μg of light chain plasmid and 33 μg of heavy chain plasmid per 100 mL cell culture system). The plasmid mixture was added to 2.5% of the total cell culture volume in serum-free medium. PEI was added at three times its concentration to the 2.5% transfection volume of medium. The PEI suspension was then added to the plasmid suspension, gently mixed, and allowed to stand for 15 min. The PEI-DNA suspension was added to the cells, and the cells were cultured at 37℃, 5% CO2, and 125 rpm for 24 h. Then, 7.5% of 293 transient transfection medium was added as feed, and the cells were cultured for another 5-7 days before harvesting the cell culture supernatant.

[0090] (2) Antibody purification: The antibody in the culture supernatant was purified by Protein A affinity chromatography: ① Equilibration: The Protein A chromatography column was equilibrated with 100mM PBS buffer at pH 7.4; ② Loading: The cell culture supernatant was loaded onto the chromatography column; ③ Washing: The chromatography column was washed with 100mM PBS buffer at pH 7.4 until A280 < 50mAu; ④ Elution: The bound antibody was eluted with 2mM acetate-sodium acetate buffer at pH 3.7, the elution peak was collected, and neutralized to pH 7.0 with 1M Tris (pH 8.0) to obtain R8-YTE mutant antibody with a purity of ≥95% (concentration: 1.056mg / mL).

[0091] Experiment Example 1: Pharmacokinetic Experiment 1. Experimental Grouping: Two guinea pigs were randomly selected from 14 guinea pigs to serve as a blank control, and the rest were randomly divided into two groups. The specific grouping is shown in Table 2: Table 2

[0092] The original R8 antibody was prepared according to Example 1. The nucleotide sequence of its light chain is shown in SEQ ID NO:3 and the amino acid sequence is shown in SEQ ID NO:4. The nucleotide sequence of its heavy chain is shown in SEQ ID NO:6 and the amino acid sequence is shown in SEQ ID NO:7.

[0093]

[0094] 2. Drug administration and blood collection: The drugs were administered via intramuscular injection according to the above grouping. Blood was collected from the heart on days 0 (before drug administration), 7, 14, 28, 42 and 56 after drug administration (0.5 mL each time). The serum was separated by centrifugation at 3000 rpm for 10 min and stored at -80℃ for later use.

[0095] 3. Blood drug concentration detection: The concentration of R8 antibody in serum was detected by ELISA: ① Coating: Rabies virus CVS-11 glycoprotein (1 μg / mL) was coated onto the ELISA plate and incubated overnight at 4°C; ② Blocking: 5% skim milk was used for blocking at 37°C for 1 h; ③ Sample addition: Serum samples were serially diluted and added to the wells, and incubated at 37°C for 1 h; ④ Secondary antibody binding: HRP-labeled anti-human IgG secondary antibody (1:5000) was added and incubated at 37°C for 1 h; ⑤ Color development: TMB chromogenic solution was used for color development in the dark for 15 min, and the reaction was terminated with 2M H2SO4; ⑥ Reading: The absorbance at 450 nm was measured using an ELISA reader, and the antibody concentration in serum was calculated according to the standard curve (prepared with R8 antibody gradient dilution buffer).

[0096] 4. Pharmacokinetic Experiment Results: Serum antibody concentrations at different time points after administration are shown in Table 3 (n=6, mean) and Figure 1. From 14 to 56 days after administration, the concentration of the R8-YTE mutant antibody prepared in Example 1 in guinea pigs was significantly higher than that of the original antibody, and an effective concentration (>0.5 μg / mL) could still be detected 56 days after administration, while the original R8 antibody was close to the detection limit after 56 days.

[0097] Experimental results show that the R8 antibody modified by YTE mutation (Example 1) has a significantly longer retention time in guinea pigs, and its half-life is much better than that of the original R8 antibody, demonstrating outstanding long-acting effect.

[0098] Table 3

[0099] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A mutant antibody against rabies virus, characterized in that, It includes a heavy chain and a light chain, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO:

1.

2. The mutant antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:

2.

3. The mutant antibody according to claim 1, characterized in that, The nucleotide sequence of the light chain is shown in SEQ ID NO:3, and the amino acid sequence of the light chain is shown in SEQ ID NO:

4.

4. The mutant antibody according to claim 1, characterized in that, The mutation refers to a YTE mutation in the Fc region of the original antibody heavy chain; the amino acid sequence of the original antibody heavy chain is shown in SEQ ID NO:

7.

5. The mutant antibody according to claim 4, characterized in that, The YTE mutation is described as the mutation of amino acids 255, 257, and 259 of the original antibody heavy chain into tyrosine, threonine, and glutamic acid, respectively.

6. An isolated polynucleotide, characterized in that, The polynucleotide encodes the mutant antibody according to any one of claims 1-5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide of claim 6.

8. A host cell, characterized in that, The cells comprise the mutant antibody of any one of claims 1-5, the polynucleotide of claim 6, or the recombinant expression vector of claim 7.

9. A rabies virus detection kit, characterized in that, It includes the mutant antibody according to any one of claims 1-5, the isolated polynucleotide according to claim 6, the recombinant expression vector according to claim 7, or the host cell according to claim 8.

10. The rabies virus detection kit according to claim 9, characterized in that, The rabies virus detection kit also includes any one or more of the following: detection buffer, protein standard, negative control, and positive control.

11. The rabies virus detection kit according to claim 9 or 10, characterized in that, The rabies virus test kit also includes an instruction manual, which describes how to use the test kit, the criteria for interpreting results, and the storage conditions.

12. The use of the mutant antibody according to any one of claims 1-5 in the preparation of a medicament for the prevention and / or treatment of rabies.

13. A drug for the prevention and / or treatment of rabies, characterized in that, It includes the mutant antibody according to any one of claims 1-5.

14. The medicament according to claim 13, characterized in that, The drug also includes pharmaceutically acceptable carriers, diluents, or excipients.

15. The medicament according to claim 13 or 14, characterized in that, The drug is in the form of an injection.

16. The medicament according to claim 15, characterized in that, The injection can be administered via intramuscular injection, intravenous injection, or subcutaneous injection.

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