Fully human neutralizing antibody against rabies virus and use thereof

Abstract

Provided are a fully human neutralizing antibody against rabies virus and a combination thereof, comprising any one of the following light chain variable regions (VL) comprising SEQ ID NOs: 6-10 and heavy chain variable region (VH) comprising SEQ ID NOs: 1-5. The neutralizing antibody can neutralize rabies virus.

Description

A fully human neutralizing antibody against rabies virus and its application

[0001] Cross-reference to related applications

[0002] This invention claims priority to the earlier application filed on July 22, 2024, with patent application number 202410984481.8 and entitled "A Fully Human Anti-Rabies Virus Neutralizing Antibody and Its Application". The entire contents of that earlier application are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and in particular to a fully human neutralizing antibody against rabies virus, an antibody combination, and their applications. Background Technology

[0004] Rabies is an acute zoonotic infectious disease caused by the rabies virus, most commonly found in carnivores such as dogs, wolves, and cats. When a person is bitten or scratched by an infected animal, the virus in the animal's saliva enters the body through the wound, causing the disease. A small number of patients also contract the disease due to contamination of the conjunctiva with the saliva of an infected animal. Rabies is currently the deadliest zoonotic infectious disease, with 99% of human rabies cases occurring in developing countries, primarily in Asia, Africa, Latin America, and the Caribbean. Asia has the highest number of rabies cases globally, with an estimated 30,000 deaths annually. India currently has the most severe rabies epidemic, and China has the second highest incidence of human rabies cases after India, with 3,300 reported cases at the peak of the epidemic in 2007. Rabies deaths have consistently ranked among the top three infectious disease-related deaths in my country. Furthermore, surveys indicate that the underreporting rate of rabies in some areas may be as high as 35%, suggesting that the disease burden of rabies in my country may be underestimated.

[0005] The World Health Organization (WHO) recommends that for individuals with rabies virus exposure at level III and those with immunocompromised level II exposure, vaccination should be combined with thorough wound cleaning and infiltration injection of passive immunization agents around the wound. Currently, the main emergency prophylactic treatments for rabies are blood-derived immunoglobulins, equine anti-rabies serum (ERA), and human rabies immunoglobulin (HRIG). In China, the majority of equine anti-rabies serum used is equine-derived. Because ERA serum is equine-derived, it is heterologous to humans and can easily cause severe side effects, including serum sickness, angioedema, and anaphylactic shock. A few companies also produce human rabies serum, but because it requires serum from individuals already immunized, its production is very limited, it is expensive, and like all serum products, it carries the potential to transmit other diseases.

[0006] Monoclonal antibodies, especially human antibodies, can overcome the shortcomings of current active and passive immunization agents. Firstly, regarding PEP (post-exposure prophylaxis) vaccination, it generally takes 7-14 days for the body to produce sufficient antibodies after vaccination. During this period, monoclonal antibodies can provide immediate immune protection within the window between vaccination and the production of sufficient antibodies. Secondly, human monoclonal antibodies have higher specificity than current passive immunization agents and reduce the risks associated with heterologous origins, making them easier to mass-produce and meet current market demands. However, current monoclonal antibody technologies, such as cell fusion, phage surface presentation, antibody engineering, and B-cell immortalization, result in human antibodies whose sequences are not 100% derived from humans. Natural, fully human antibodies would possess better affinity and safety.

[0007] Furthermore, due to the high mutation rate and extremely high mortality rate after infection with rabies virus (RABV), the case fatality rate is almost 100% once symptoms appear. For example, the antibody drug CL184 (produced by Crucell) has had its two human antibodies, CR57 and CR4098, discontinued due to a lack of broad coverage. Experiments have shown that antibody CR57 cannot neutralize EBLV-1, and antibody CR4098 cannot neutralize DUVV, EBLV-1, EBLV-2, IRKV, and KHUV viruses. Therefore, to maximize the neutralization of naturally occurring rabies virus strains and prevent post-infection rabies, it is essential to develop broad-spectrum anti-rabies virus antibodies to provide effective immunoprophylaxis and protection against multiple types of rabies viruses. Summary of the Invention

[0008] This invention obtains PBMC samples with neutralizing activity against multiple rabies virus strains by screening plasma cells from donor PBMC samples. Plasma cells with broad neutralizing activity are then sorted, and monoclonal antibodies are isolated from these plasma cells. The resulting monoclonal antibodies can selectively bind to G protein epitopes in their natural form and inhibit infection from multiple rabies virus strains, and can be used for the treatment, diagnosis, and monitoring of rabies.

[0009] This invention relates to a method for isolating fully human anti-rabies virus neutralizing antibodies with high affinity, high binding activity, and broad neutralizing activity. The method involves obtaining peripheral blood mononuclear cells (PBMCs) from rabies virus-infected donor plasma, sorting individual plasma cells from samples containing specific anti-rabies virus antibodies, and isolating and detecting antibody genes.

[0010] In one embodiment of the present invention, the method includes obtaining peripheral blood mononuclear cells (PBMCs) from rabies virus-infected donor plasma, selecting PBMCs with high antibody neutralizing titers in the plasma as donors, and screening single plasma cells from the donor PBMC samples. Antibody variable region genes are isolated from the single plasma cells using single-cell RT-PCR, sequenced, and screened for fully human antibodies specifically targeting the rabies G protein and the vaccine, including five antibodies: TRN1040, TRN1041, TRN1042, TRN1043, and TRN1044. Furthermore, five antibody expression systems are constructed for antibody expression and detection.

[0011] Another aspect of the present invention relates to a fully human neutralizing antibody against rabies virus, comprising a complementation-determining region of any of the following heavy chain variable regions and a complementation-determining region of the light chain variable region:

[0012] The complementarity-determining region (CDR) sequence of the heavy chain variable region includes any of the following:

[0013] (1) H1CDR1: SEQ ID NO: 11; H1CDR2: SEQ ID NO: 12; H1CDR3: SEQ ID NO: 13;

[0014] (2) H2CDR1: SEQ ID NO: 14; H2CDR2: SEQ ID NO: 15; H2CDR3: SEQ ID NO: 16;

[0015] (3) H3CDR1: SEQ ID NO: 17; H3CDR2: SEQ ID NO: 18; H3CDR3: SEQ ID NO: 19;

[0016] (4) H4CDR1: SEQ ID NO: 20; H4CDR2: SEQ ID NO: 21; H4CDR3: SEQ ID NO: 22;

[0017] (5) H5CDR1: SEQ ID NO: 23; H5CDR2: SEQ ID NO: 24; H5CDR3: SEQ ID NO: 25;

[0018] Or an amino acid sequence that has at least 70% identity with the complementarity-determining region of the heavy chain variable region;

[0019] The complementarity-determining region (CDR) sequence of the light chain variable region includes any of the following:

[0020] (1') L1CDR1: SEQ ID NO: 26; L1CDR2: SEQ ID NO: 27; L1CDR3: SEQ ID NO: 28;

[0021] (2') L2CDR1: SEQ ID NO: 29; L2CDR2: SEQ ID NO: 30; L2CDR3: SEQ ID NO: 31;

[0022] (3') L3CDR1: SEQ ID NO: 32; L3CDR2: SEQ ID NO: 33; L3CDR3: SEQ ID NO: 34;

[0023] (4') L4CDR1: SEQ ID NO: 35; L4CDR2: SEQ ID NO: 36; L4CDR3: SEQ ID NO: 37;

[0024] (5') L5CDR1: SEQ ID NO: 38; L5CDR2: SEQ ID NO: 39; L5CDR3: SEQ ID NO: 40;

[0025] Or an amino acid sequence that has at least 70% identity with the complementarity-determining region of the light chain variable region;

[0026] In one embodiment of the present invention, the neutralizing antibody includes any of the following complementarity-determining regions of the heavy chain variable region and the light chain variable region:

[0027] The complementarity-determining region (CDR) sequences of the heavy chain variable region include: H1CDR1: SEQ ID NO:11; H1CDR2: SEQ ID NO:12; H1CDR3: SEQ ID NO:13; The complementarity-determining region (CDR) sequences of the light chain variable region include: L1CDR1: SEQ ID NO:26; L1CDR2: SEQ ID NO:27; L1CDR3: SEQ ID NO:28;

[0028] Alternatively, the complementarity-determining region (CDR) sequences of the heavy chain variable region include: H2CDR1: SEQ ID NO:14; H2CDR2: SEQ ID NO:15; H2CDR3: SEQ ID NO:16; the complementarity-determining region (CDR) sequences of the light chain variable region include: L2CDR1: SEQ ID NO:29; L2CDR2: SEQ ID NO:30; L2CDR3: SEQ ID NO:31;

[0029] Alternatively, the complementarity-determining region (CDR) sequences of the heavy chain variable region include: H3CDR1: SEQ ID NO:17; H3CDR2: SEQ ID NO:18; H3CDR3: SEQ ID NO:19; the complementarity-determining region (CDR) sequences of the light chain variable region include: L3CDR1: SEQ ID NO:32; L3CDR2: SEQ ID NO:33; L3CDR3: SEQ ID NO:34;

[0030] Alternatively, the complementarity-determining region (CDR) sequences of the heavy chain variable region include: H4CDR1: SEQ ID NO:20; H4CDR2: SEQ ID NO:21; H4CDR3: SEQ ID NO:22; the complementarity-determining region (CDR) sequences of the light chain variable region include: L4CDR1: SEQ ID NO:35; L4CDR2: SEQ ID NO:36; L4CDR3: SEQ ID NO:37;

[0031] Alternatively, the complementarity-determining region (CDR) sequences of the heavy chain variable region include: H5CDR1: SEQ ID NO:23; H5CDR2: SEQ ID NO:24; H5CDR3: SEQ ID NO:25; and the complementarity-determining region (CDR) sequences of the light chain variable region include: L5CDR1: SEQ ID NO:38; L5CDR2: SEQ ID NO:39; L5CDR3: SEQ ID NO:40;

[0032] Alternatively, it may have an amino acid sequence that is at least 70% identical to the complementarity-determining regions of the heavy chain variable region and the light chain variable region.

[0033] In one embodiment of the present invention, the neutralizing antibody comprises any one of the following heavy chain variable regions (VH) and light chain variable regions (VL):

[0034] Heavy chain variable region: the sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:1, the sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:2, the sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:3, the sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:4, and the sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:5;

[0035] Light chain variable region: the sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:6; the sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:7; the sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:8; the sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:9; the sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:10.

[0036] In one embodiment of the present invention, a fully human neutralizing antibody against rabies virus of any of the following sequences is provided.

[0037] 1) The heavy chain variable region shown in SEQ ID NO:1 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:1, and the light chain variable region shown in SEQ ID NO:6 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:6;

[0038] 2) The heavy chain variable region shown in SEQ ID NO:2 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:2, and the light chain variable region shown in SEQ ID NO:7 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:7;

[0039] 3) The heavy chain variable region shown in SEQ ID NO:3 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:3, and the light chain variable region shown in SEQ ID NO:8 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:8;

[0040] 4) The heavy chain variable region shown in SEQ ID NO:4 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:4, and the light chain variable region shown in SEQ ID NO:9 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:9;

[0041] 5) The heavy chain variable region shown in SEQ ID NO:5 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:5; the light chain variable region shown in SEQ ID NO:10 or an amino acid sequence having at least 70% sequence identity with SEQ ID NO:10.

[0042] In one embodiment of the present invention, the anti-rabies virus neutralizing antibody of the present invention is isolated from plasma cells of a human donor infected with rabies virus. In one embodiment of the present invention, the neutralizing antibody is a fully human neutralizing antibody. In one embodiment of the present invention, the neutralizing antibody can broadly and effectively neutralize multiple strains of rabies virus. Exemplarily, the neutralizing antibody of the present invention can neutralize 100% of the street rabies virus strains listed in Tables 6-7. The neutralizing antibody of the present invention exhibits superior neutralizing efficacy against most viral strains compared to known broadly effective neutralizing antibodies.

[0043] In one embodiment of the present invention, the neutralizing antibodies all exhibit high neutralizing activity. For example, against rabies virus CVS-11 cytotoxicity, TRN1043 showed a neutralizing titer as high as 17045.97 IU / mg, significantly superior to known neutralizing antibodies such as human immunoglobulin, TRN073, and TRN006.

[0044] In one embodiment of the present invention, the neutralizing antibody includes a full-length antibody, an antibody fragment, a single-chain antibody, a bispecific antibody, a domain antibody, an antibody fusion compound, ScFv, Fab, Fab′, F(ab′)2, or Fv.

[0045] In one embodiment of the present invention, the neutralizing antibody may be an IgG, IgA, IgD, IgE, or IgM antibody. In another embodiment of the present invention, the constant region of the neutralizing antibody includes any one of the constant regions of IgG, IgA, IgD, IgE, or IgM, preferably the IgG constant region.

[0046] In one embodiment of the present invention, the neutralizing antibody includes a modified or conjugated neutralizing antibody.

[0047] In one embodiment, the modification includes disulfide bond formation, glycosylation, esterification, acetylation, methylation, phosphorylation, etc.

[0048] In one implementation, the coupling includes a detectable tag connection.

[0049] In one embodiment, the detectable label includes labels such as radioactive substances, enzymes, coenzymes, fluorescent agents, chemiluminescent agents, bioluminescent agents, chromogenic agents, enzyme substrates or cofactors, enzyme inhibitors, complexes, and dyes. The enzymes include horseradish peroxidase, alkaline phosphatase, galactosidase, or acetylcholinesterase; the complexes include streptavidin / biotin and avidin / biotin; the fluorescent agents include fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin; the chemiluminescent agents include luminol; the bioluminescent agents include luciferase and fluorescein; and the radioactive substances include 1251, 35S, or 3H. The detection label can be detected using methods such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), and fluorescence immunoassay.

[0050] In one embodiment, the connection method is a conventional method in this invention, such as coupling through a linking group, which includes hydrazone groups, disulfide groups, thioether groups, amide groups, short peptide groups, etc., preferably disulfide groups and thioether groups.

[0051] In another aspect, the present invention provides a neutralizing antibody composition comprising at least two of the above-described fully human anti-rabies virus neutralizing antibodies.

[0052] In a specific embodiment of the present invention, the antibody composition includes a combination of 3) and 4), a combination of 3) and 2), a combination of 3) and 1), a combination of 3) and 5), and more preferably a combination of 3) and 4) or a combination of 3) and 2). It can also be represented as including TRN1042+TRN1043, TRN1042+TRN1041, TRN1042+TRN1040, and TRN1042+TRN1044; preferably TRN1042+TRN1043 and TRN1042+TRN1041.

[0053] In a specific embodiment of the present invention, antigen competition analysis of five neutralizing antibodies revealed that TRN1044 and TRN1040 have similar or identical epitopes; TRN1043 partially overlaps with TRN1041, TRN1044, and TRN1040; TRN1042 has different epitopes from the other four antibodies; TRN1041 and TRN1043 have partially overlapping epitopes, but different epitopes from the other three antibodies. By combining neutralizing antibodies with different antigenic epitopes, the coverage of different virus strains can be maximized, and when one antibody develops resistance, the other antibody can still provide protection, effectively preventing immune escape from rabies virus.

[0054] In another aspect, the present invention provides a polynucleotide encoding the above-mentioned neutralizing antibody.

[0055] In one specific embodiment of the present invention, the polynucleotide includes a nucleotide sequence encoding part or all of the variable region of the antibody heavy chain and / or a nucleotide sequence encoding part or all of the variable region of the antibody light chain.

[0056] Furthermore, the nucleic acid molecule also includes nucleic acid molecules having conserved nucleotide sequence variants of the aforementioned nucleotide sequence. These conserved nucleotide sequence variants originate from genetic code degeneracy and silencing variants, and nucleotide substitutions, deletions, and additions are also included. In a specific embodiment of the invention, the polynucleotide includes polynucleotide variants having sequence identity with TRN1044, TRN1041, TRN1043, TRN1040, or TRN1042, for example, containing at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.

[0057] Another aspect of the present invention relates to an expression vector comprising the above-mentioned polynucleotides.

[0058] In a specific embodiment of the present invention, the expression vector includes plasmids, bacteriophages, or viruses. The plasmids include, but are not limited to, PcDNA3.1 and PcDNA3.3. The bacteriophages include, but are not limited to, λ phage or M13 phage. The viruses include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.

[0059] In one specific embodiment of the present invention, the expression vector further includes untranslated regions, such as enhancers, promoters, and 5' and 3' untranslated regions that interact with host cell proteins to perform transcription and translation. Depending on the vector system and host used, any number of transcriptional and translational elements can be used, including promoters and inducible promoters (such as SV40).

[0060] Another aspect of the present invention relates to a host cell comprising the aforementioned polynucleotide or expression vector.

[0061] In one specific embodiment of the present invention, any suitable host cell / vector system can be used to express the DNA sequence encoding the antibody molecule or a fragment thereof of the present invention. In one specific embodiment of the present invention, the host cell includes bacteria, fungi, or animal cells. The bacteria include, but are not limited to, *Escherichia coli* and *Bacillus subtilis*. For example, *Escherichia coli* can be used in part to express antibody fragments, such as Fab and F(ab')2 fragments, particularly Fv fragments and single-chain antibody fragments, such as single-chain Fvs. The fungi include, but are not limited to, yeast cells and *Aspergillus*. The animal cells include, but are not limited to, fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and 293 cells. In a further specific embodiment of the present invention, the host cell is a 293T cell.

[0062] Another aspect of the present invention relates to a pharmaceutical composition comprising the above-described neutralizing antibody, neutralizing antibody composition, polynucleotide, expression vector, or host cell.

[0063] In one specific embodiment of the invention, the pharmaceutical composition further includes one or more other therapeutic agents. These other therapeutic agents can be used to treat specific indications, such as treating infections or preventing side effects. Preferably, they include secondary antibodies, antiviral agents, anti-infective agents, and / or cardioprotective agents.

[0064] In one specific embodiment of the present invention, the one or more other therapeutic agents are administered in combination with the anti-rabies virus neutralizing antibody. The combined administration includes administration in any order or at any time interval, such that the two or more therapeutic agents exert their biological activity simultaneously. Preferably, the combined administration produces a synergistic therapeutic effect. Preferably, the anti-rabies virus neutralizing antibody is administered in combination with other antibodies targeting other antigens of the rabies virus.

[0065] Another aspect of the present invention relates to a rabies virus detection product comprising the above-described antibody or antibody composition, polynucleotide, expression vector or host cell.

[0066] In one specific embodiment of the present invention, the detection product includes, but is not limited to, detection reagents, kits, chips, or test strips. All rabies virus detection products prepared using the aforementioned fully human anti-rabies virus neutralizing antibodies are included within the scope of this invention.

[0067] Another aspect of the present invention relates to a formulation comprising the above-described antibody or antibody composition.

[0068] In one specific embodiment of the present invention, the formulation further includes a pharmaceutically acceptable carrier.

[0069] In one embodiment of the present invention, the formulation includes dosage forms such as lyophilized formulations, solutions, and suspensions.

[0070] In one embodiment of the present invention, the preparation is administered via intravenous, intramuscular, intraperitoneal, cerebrospinal, subcutaneous, intramedullary, intrathecal, oral, local, or inhalation routes. Intravenous injection or subcutaneous administration is preferred.

[0071] Another aspect of the present invention relates to a method for stimulating an immune response, treating or preventing symptoms of rabies virus infection by administering rabies virus neutralizing antibodies to a subject.

[0072] Another aspect of the invention relates to a method of administering the rabies virus neutralizing antibody of the present invention to a subject after exposure to rabies virus. For example, the rabies virus neutralizing antibody of the present invention is used to treat or prevent rabies virus infection. The dose of the rabies virus neutralizing antibody is sufficient to promote viral clearance or eliminate rabies virus-infected cells.

[0073] Another aspect of the present invention relates to a method for detecting whether the biological sample contains rabies virus, wherein the biological sample and the above-mentioned antibody or antibody composition, polynucleotide, expression vector, host cell or detection product are subjected to qualitative or quantitative detection.

[0074] In one embodiment of the present invention, the following steps are specifically included:

[0075] (1) Obtain a sample of rabies virus;

[0076] (2) Contact the sample obtained in step (1) with the above-mentioned neutralizing antibody or neutralizing antibody composition;

[0077] (3) Detect the immune response of the sample to the neutralizing antibody.

[0078] Another aspect of the present invention relates to a method for determining the presence of rabies virus infection in a patient by contacting a biological sample obtained from the patient with a rabies virus monoclonal antibody.

[0079] In another aspect, the present invention also provides the use of the above-described antibody or antibody composition in any of the following:

[0080] 1) Preparation of drugs to inhibit rabies virus;

[0081] 2) To prepare drugs for the prevention of diseases caused by rabies virus infection;

[0082] 3) Prepare rabies virus detection reagents.

[0083] Another aspect of the present invention relates to a method for generating the neutralizing antibody, comprising culturing a host cell containing the vector of the present invention under suitable conditions and isolating antibody molecules.

[0084] In one embodiment of the present invention, the antibody molecule may contain only heavy chain or light chain polypeptides. In this case, only the sequence encoding the heavy chain or light chain needs to be used to transfect the host cell. To produce a product containing both heavy and light chains, the host cell can be transfected using two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector can be used, comprising sequences encoding both the light and heavy chain polypeptides.

[0085] In one embodiment of the present invention, the method may include a step of purifying antibodies.

[0086] Compared with the prior art, the present invention has the following beneficial effects:

[0087] 1) The fully human anti-rabies virus neutralizing antibody of the present invention can effectively neutralize rabies virus, has anti-infective effect, and plays a role in prevention and treatment.

[0088] 2) The antibody of the present invention can be used to prepare rabies virus detection reagents for efficient detection of rabies virus.

[0089] 3) The antibodies of this invention have good broad-spectrum activity and can effectively neutralize multiple RABV strains circulating both domestically and internationally, such as CQ-I isolated in Chongqing in 2006, GD-II isolated in Guangdong in 2016, JS-I isolated in Jiangsu in 2018, Afghanistan I isolated from dogs in Afghanistan in 2011, and USA-2 isolated from skunks in the United States. They also have good neutralizing effects on other strains, such as ABLV, BBLV, and GBLV strains. Among these, TRN1043, TRN1042, and TRN1041 have even better broad-spectrum activity, neutralizing not only the aforementioned strains but also ARAV, DUVV, EBLV-1, EBLV-2, IRKV, and KHUV strains.

[0090] 4) The antibody combination of the present invention is a dual antibody cocktail therapy. The FDA's industry guidance "FDA Rabies, mAb cocktails for the passive immunization components of post exposure prophylaxis" announced in 2021 aims to promote the development of anti-rabies virus monoclonal antibody cocktails.

[0091] Compared to single antibodies such as bispecific antibodies, dual-antibody cocktail therapy has several advantages. Composed of antibodies that bind to different antigenic epitopes, it can maximally cover different rabies strains, significantly reducing the risk of infection from different strains. Furthermore, once the rabies virus enters the human body, it continuously replicates and spreads. Bispecific antibody drugs may face drug resistance issues during use. With cocktail therapy, different antibodies target different antigenic epitopes. Even if one antibody develops resistance, the other antibody can still provide protection, effectively preventing immune escape from the rabies virus.

[0092] 5) The antibody of the present invention is a fully human monoclonal antibody, the amino acid antibody sequence of which is 100% derived from humans. It has undergone human immune surveillance / tolerance selection and provides a method for isolating antibody genes from a single B lymphocyte based on gene cloning technology. It retains the natural pairing of light chain and heavy chain variable regions, and is a truly fully human antibody. Therefore, it has the advantages of good gene diversity, low immunogenicity, high specificity, and high safety. It can also be mass-produced and its quality is controllable, which effectively increases the accessibility of the product. Attached Figure Description

[0093] Figure 1 shows the results of flow cytometry sorting.

[0094] Figure 2 shows the detection results of SDS-PAGE and Western Blot (WB), where 1: Marker; 2: TRN1044; 3: TRN1041; 4: TRN1043; 5: TRN1040; 6: TRN1042;

[0095] Figure 3. Study strategy for neutralizing broad-spectrum antirabies monoclonal antibodies.

[0096] Figure 4. Results of antigen competition analysis Detailed Implementation

[0097] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0098] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0099] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule containing four polypeptide chains (two heavy chains (H) and two light chains (L)), or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding characteristics of an Ig molecule. This includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0100] The term "monoclonal antibody" or "mAb" refers to an antibody derived from a single copy or clone of, for example, a eukaryotic, prokaryotic, or phage clone. That is, the individual antibodies constituting the group are identical and / or bind to the same epitopes, except that they are typically present in small amounts as possible variant antibodies (e.g., those containing natural mutations or variant antibodies generated during the production of monoclonal antibody articles). The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic techniques such as CDR grafting, or combinations of such or other techniques known in the art.

[0101] The term "neutralizing antibody" refers to an antibody or antibody fragment that can bind to a pathogen and eliminate or significantly reduce the pathogen's virulence (e.g., its ability to infect cells). Such neutralizing antibodies typically function by killing pathogens outside the cell, preventing them from invading the cell.

[0102] The term “modification” includes any naturally modified or intervened neutralizing antibody; such as disulfide bond formation, glycosylation, lipolysis, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a marker or bioactive component.

[0103] The term “deletion, substitution and / or insertion of amino acids” refers to an amino acid alteration of a target antibody region (e.g., heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region) by deletion, substitution and / or insertion of at least one, such as 1-30, or 1-20 or 1-10, such as 1, 2, 3, 4 or 5 amino acids, wherein the antibody substantially retains the biological properties of the antibody molecule before the alteration, and the antibody retains at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% of the biological activity (e.g., antigen binding capacity) of the antibody before the alteration.

[0104] The term "identity" refers to the fact that a sequence or molecule has X% "identity" with another sequence or molecule if X% of the amino acids in the sequence are the same and in the same relative positions. For example, a neutralizing antibody derivative having at least 70% or 80% identity with the neutralizing antibody will have at least 70% or 80% of the same amino acids in the same relative positions as the neutralizing antibody. Sequence identification methods include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0105] The terms “activity” or “bioactivity” or “biological property” or “biocharacteristic” are used interchangeably in this disclosure and include, but are not limited to, epitope or antigen affinity, specificity, ability to neutralize or antagonize toxin activity in vivo or in vitro, half-inhibitory concentration (IC50), in vivo stability of the antibody, and immunogenicity of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (such as cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high levels of protein expression in mammalian cells. The aforementioned properties or characteristics may be observed, measured, or evaluated using techniques known in the art, including but not limited to enzyme-linked immunosorbent assay (ELISA), flow cytometry sorting (FACS) or BIACORE plasma resonance analysis, any in vitro or in vivo neutralization assay, receptor binding assay, cytokine or growth factor production and / or secretion assay, signal transduction assay, and immunohistochemical analysis of tissue sections from various sources (including human, primate, or any other source).

[0106] The term "epitope" refers to the antigenic region to which an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein.

[0107] The term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair (such as antigen and antibody). Affinity can typically be expressed by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art, such as those using the ForteBio biological molecular interaction workstation.

[0108] The terms “antigen-binding fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment of the antibody of the present invention that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single-chain antibodies, Fab, Fab', F(ab')2, Fv, or scFv. Further, as used herein, the term “antibody” includes both the antibody and its antigen-binding fragment.

[0109] The term "nucleic acid" or "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term "nucleic acid" or "nucleic acid molecule" also includes nucleic acids containing analogs of known natural nucleotides, having similar binding properties to reference nucleic acids, and being metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules with uridine replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as explicitly stated sequences.

[0110] The term "vector" includes shuttle vectors and expression vectors. Typically, plasmid structures also include an origin of replication (e.g., ColE1 origin of replication) and selectable markers (e.g., ampicillin or tetracycline resistance) for plasmid replication and selection in bacteria, respectively. The term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene upon transformation, transfection, or transduction into host cells. Expression vectors typically contain one or more phenotypic selection markers and origins of replication for maintaining the vector and, if necessary, amplifying it within the host.

[0111] The term "detectable label" is any type of label that makes an antibody detectable when linked to an antibody of this disclosure. Detectable labels can also be cytotoxic or cellular, such as therapeutic agents or cytotoxic agents. Typically, detectable labels can include luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescence quenchers, colored molecules, radioisotopes, radionuclides, metal atoms, biotin, avidin, streptavidin, antibodies or fragments thereof, Grb2, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donor / acceptor, acridinium esters, and colorimetric substrates. Those skilled in the art will readily recognize other useful labels not mentioned above that can be used in the operation of this disclosure.

[0112] The term "therapeutic agent" is any compound known in the art for the detection, diagnosis, or treatment of a symptom or disease. Such compounds can be naturally occurring, modified, or synthetic. Non-limiting examples of therapeutic agents may include drugs, therapeutic compounds, toxins, genetic material, metals (such as radioisotopes), proteins, peptides, carbohydrates, lipids, steroids, nucleic acid-based substances, or derivatives, analogs, or combinations thereof in their natural form or derivatized with hydrophobic or charged portions to enhance incorporation or adsorption into cells. Such therapeutic agents can be water-soluble or hydrophobic.

[0113] The term "antibody fragment" includes a portion of a complete antibody, preferably the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0114] The term "Fv" refers to the smallest antibody fragment containing both a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain and a light chain variable region domain. The folding of these two domains produces six hypervariable rings (three rings each in the H chain and L chain), which provide the amino acid residues for antigen binding and confer the antibody's antigen-binding specificity. However, even a single variable region (or half of an Fv containing only the three CDRs specific to the antigen) has the ability to recognize and bind antigens, although its affinity is lower than that of the entire binding site.

[0115] The term "single-chain Fv," also abbreviated as "sFv" or "scFv," refers to an antibody fragment comprising VH and VL antibody domains linked together to form a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker located between the VH and VL domains, enabling the sFv to form the desired antigen-binding structure.

[0116] The term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell damage.

[0117] The term "natural sequence" polynucleotide refers to a polynucleotide that has the same nucleotide sequence as a naturally derived polynucleotide. "Natural sequence" polypeptide refers to a polypeptide that has the same amino acid sequence as a polypeptide derived from nature (such as an antibody). Such natural sequence polynucleotides and polypeptides can be isolated from nature or produced through recombinant or synthetic methods.

[0118] The term "hypervariable region" refers to the amino acid residues in an antibody responsible for antigen binding. Hypervariable regions typically contain amino acid residues from the "complementarity-determining region" or "CDR".

[0119] The term "humanized antibody" is generally considered to be a human antibody having one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "input" residues, which are typically derived from the "input" variable region. Humanization is traditionally performed by replacing the corresponding sequence of a human antibody with an introduced hypervariable region sequence. Therefore, such "humanized" antibodies are chimeric antibodies in which essentially less than the complete human variable region has been replaced by a corresponding sequence from a non-human species.

[0120] The term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzyme labeling, may catalyze a chemical change in the detectable substrate compound or composition.

[0121] When the term "competition" is used in the context of competing antigen-binding proteins for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., N protein or a fragment thereof).

[0122] The term "polynucleotide variant" is a polynucleotide that typically differs from the polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or synthetically produced, for example, by modifying one or more polynucleotide sequences of the present invention and evaluating one or more biological activities of the polypeptides encoded herein and / or using any of a variety of techniques, as is well known in the art. A polypeptide "variant" is a polypeptide that typically differs from the polypeptide specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or synthetically produced, for example, by modifying one or more of the polypeptide sequences described above of the present invention and evaluating one or more biological activities of the polypeptides described herein and / or using any of a variety of techniques, as is well known in the art. In specific embodiments, the polynucleotide variants and polypeptide variants have at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% homology to the said polynucleotide or polypeptide.

[0123] Example 1: Plasma Cell Screening

[0124] 1. Isolation of PBMCs (peripheral blood mononuclear cells)

[0125] Healthy volunteers were recruited, immunized, and their peripheral blood samples were collected for plasma cell isolation.

[0126] Plasma and PBMC cells were separated using density centrifugation: 15 mL of lymphocyte separation medium was added to a centrifuge tube, followed by 10 mL of whole blood from a volunteer diluted with an equal volume of PBS buffer. Centrifuge at 1250 rpm, 22°C for 35 min. After centrifugation, the upper clear plasma layer was aspirated to obtain the plasma sample, which was then aliquoted and stored at -80°C. The intermediate PBMC cells were aspirated and brought to a final volume of 35 mL with washing buffer; centrifuged at 1800 rpm, 22°C for 8 min. After centrifugation, the cell pellet was collected, and the volume was adjusted to 5-10 mL. Centrifuged at 1500 rpm, 4°C for 5 min, the supernatant was discarded, and cell cryopreservation buffer (FBS:DMSO = 9:1) was added to dilute to approximately 1 × 10⁻⁶ cells / mL. 7 / mL. After aliquoting and labeling, cool to -80°C using a programmed freezer, and then store in a liquid nitrogen tank.

[0127] 2. Sample screening

[0128] Using purchased rabies G protein (PaddyChang), individuals with high titers of G protein antibodies in their plasma were screened using the ELISA detection method.

[0129] The specific method is as follows: Rabies G protein was diluted to 2 μg / mL with pH 9.6 phosphate coating buffer (1.59g Na2CO3, 2.93g NaHCO3, pH adjusted to 9.6, dissolved and then brought to a final volume of 1000mL with ultrapure water; the pH 9.6 phosphate coating buffer described below is the same formula). 0.1mL of the solution was added to each well of an ELISA 96-well plate. The plate was coated overnight at 4°C. Blocking buffer (50g skim milk powder, 150mL sheep serum, 5mL Tween 20, 50mL 20x concentration PBS stock solution, dissolved and then brought to a final volume of 1000mL with ultrapure water; the blocking buffer described below is the same formula) was then applied at 37°C for 2 hours. Each plasma sample obtained during the isolation of peripheral blood mononuclear cells in step (1) was initially diluted 1:50 (3 μL of plasma and 150 μL of blocking buffer), and then serially diluted 3-fold. 100 μL of primary antibody was added to each well, and the samples were incubated at 37°C for 1 h. Then, 100 μL / well of horseradish peroxidase (HRP)-labeled goat anti-human IgG (1:10000 dilution) secondary antibody was added, and the samples were incubated at 37°C for 1 h. Next, 100 μL / well of substrate chromogenic buffer (TMB) was added, and the samples were incubated at 37°C in the dark for 5 min. The reaction was then stopped with 2M sulfuric acid, and the OD450-OD630 values ​​were read. The results showed that the plasma from volunteer No. 11 had a good binding curve and dose-response relationship, indicating that plasma sample No. 11 contained specific anti-rabies antibodies. Sample No. 11 was selected for flow cytometry sorting.

[0130] 3. Flow cytometry for sorting individual plasma cells

[0131] Based on the serological results of step 2, flow cytometry was used to sort PBMCs of sample 11 to obtain individual plasma cells. As the terminal stage of B cell differentiation, plasma cells can directly secrete antibodies. Therefore, for samples in the rapid proliferation phase of plasma cells, antibody gene amplification using single plasma cell sorting can yield a large number of specific antibodies. The sorting scheme was CD3- / CD14- / CD16- / CD235a- / CD19+ / IgD- / CD27-BV421+ / CD38-APC+. The flow cytometry sorting results are shown in Figure 1. In the Q2-1 gate, the selected double-positive specific plasma cells were identified. A total of 688 plasma cells were sorted in the first round, 800 plasma cells were obtained in the second round, and 825 plasma cells were obtained in the third round. Subsequent single-cell PCR experiments were then conducted to isolate antibody genes.

[0132] Example 2: Isolation of antibody variable region gene from plasma cells by single-cell RT-PCR

[0133] First-strand cDNA synthesis via reverse transcription: The double-positive single plasma cells obtained in Example 1 were added to a 96-well plate containing 20 μL of single-cell lysis buffer (Sigma, catalog number I8896-100 mL; Invitrogen, catalog number 10777019) and the cells were lysed. Primers for the constant regions of each heavy and light chain subtype were added at 0.5 μM, along with Superscript IV reverse transcriptase (Invitrogen, catalog number 18090200), and the cells were incubated at 55°C for 1 h to synthesize the first-strand cDNA via reverse transcription. The genes for the VH variable region of the heavy chain or the Vκ / λ variable region of the light chain were then obtained through two rounds of PCR amplification. Primer information for the two rounds of PCR amplification can be found in patent document CN201711013251.3. The obtained VH and Vκ / λ gene products were identified by 2% agarose gel electrophoresis and sequenced.

[0134] Thus, the first round of screening yielded 586 antibodies, the second round yielded 704 antibodies, and the third round yielded 650 antibodies derived from human plasma cell VH and Vκ / λ pairing (also known as fully human antibodies). Among them, the fully human antibodies that specifically target rabies G protein include TRN1040, TRN1041, TRN1042, TRN1043, and TRN1044.

[0135] Example 3 Antibody Construction

[0136] Antibody screening involved constructing linear DNA fragments containing promoters, antibody constant regions, and terminators for the VH and Vκ / λ gene products using overlapping PCR. The construction steps for the linear DNA fragments of the complete antibody expression systems TRN1040, TRN1041, TRN1042, TRN1043, and TRN1044 were as follows: The VH gene products of TRN1040, TRN1041, TRN1042, TRN1043, and TRN1044 were respectively combined with DNA fragments containing CMV promoters and DNA fragments containing the heavy chain constant region of human IgG1 (including the hinge region, CH1, CH2). 2 and CH3) were ligated by PCR; the VK gene products of TRN1040, TRN1041, TRN1042 and TRN1044 were ligated by PCR with DNA fragments containing CMV promoters and constant region DNA fragments containing human Kappa (including hinge region and C-DOMAIN); the VL gene product of TRN1043 was ligated by PCR with DNA fragments containing CMV promoters and constant region DNA fragments containing human Lambda (including hinge region and C-DOMAIN) to obtain linear DNA fragments encoding antibody expression systems containing heavy chain or light chain nucleic acids.

[0137] Example 4: Construction of an expression vector for recombinant antibodies

[0138] The linear DNA fragments of the antibody expression systems TRN1040, TRN1041, TRN1042, TRN1043, and TRN1044 obtained in Example 3 were ligated into the pcDNA3.3 vector using homologous recombination cloning to construct heavy and light chain expression vectors for fully human anti-rabies virus antibodies. These expression vectors were then transformed into DH5α competent bacteria and cultured overnight at 37°C on ampicillin-containing plates. Single colonies were picked and amplified by PCR using specific primers under the following conditions: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 100 s, 28 cycles; 72°C extension for 5 min. 5 μL of the PCR product was detected by 1% agarose gel electrophoresis. Transformants containing antibody heavy and light chain genes were sequenced for verification, and the antibody amino acid sequences were obtained, as shown in Table 1.

[0139] Table 1. Sequences of the 5 antibodies

[0140] Example 5 Antibody Expression and Purification

[0141] The plasmid that was positive for sequencing verification in Example 4 was transformed into DH5α for large-scale amplification. After rapid extraction of the recombinant plasmid, HEK293I cells were co-transfected with heavy and light chain expression vectors of different antibodies using PEI as the transfection reagent. The culture medium was replaced with fresh medium 6-8 hours after transfection, and the cells were cultured at 37°C in an 8% CO2 incubator for 96 hours. The cell supernatant was collected for detection. The transfection supernatant was collected, centrifuged at 4000 rpm for 1 hour, and purified using protein A magnetic beads. The expression and purification status of the purified antibody were examined using SDS-PAGE and Western Blot (WB). The antibody used for Western Blot detection was an alkaline phosphatase (AP)-labeled anti-human IgG antibody (brand: Promega, catalog number: w4031). The difference between non-reducing SDS-PAGE and reducing SDS-PAGE gel electrophoresis lies in the addition of β-mercaptoethanol loading buffer to the antibody sample for reducing electrophoresis, followed by boiling in a water bath for 5 minutes, and then adding PBS to a final volume of 10-15 μL before loading. The SDS-PAGE and Western Blot (WB) results are shown in Figure 2. The left figure shows the results for non-reduced samples, and the right figure shows the results for reduced samples. Figure 2 shows that after antibody reduction, there are two bands: the heavy chain and the light chain. The molecular weights of the reduced antibody light and heavy chains are consistent with the typical antibody size. The non-reduced antibody has a size of 180 kDa, consistent with the size of a typical intact antibody, indicating correct antibody assembly and structure.

[0142] Example 6: ELISA detection of antibody binding activity

[0143] Rabies G protein was used as the antigen for ELISA detection. The corresponding antigen was diluted to 2 μg / mL, and 100 μL was added to each well of a 96-well plate. The plate was incubated overnight at 4°C and blocked with blocking buffer at 37°C for 2 h. Each obtained rabies antibody was serially diluted, starting at 10 μg / mL, with 12 3-fold dilutions, and a minimum concentration of 0.056 ng / mL. The antibodies from each dilution were then added to 96-well plates and incubated at 37°C for 1 h. After washing with PBST buffer, 100 μL of Goat-Anti-IgG-Fab-HRP (secondary antibody) diluted 1:10000 with blocking buffer (purchased from Sigma, catalog number A02931ML) was added to each well and incubated at 37°C for 1 h. After washing with PBST buffer and protecting from light, 100 μL of TMB chromogenic buffer was added to each well and incubated at 37°C for 5 min. The incubation was immediately stopped with 50 μL of 2M H2SO4. OD values ​​were detected at dual wavelengths from 450-630 nm. The results are shown in Table 2. The fully human anti-rabies virus monoclonal antibodies TRN1040, TRN1041, TRN1042, TRN1043 and TRN1044 can all bind to the rabies G protein. Even after the antibodies were diluted 3-fold in 12 serial dilutions (the antibody concentration was about 0.056 ng / mL), they could still bind to the antigen.

[0144] Table 2 Wavelength detection results

[0145] Example 7: Determination of antibody-antigen affinity using surface plasmon resonance (SPR) technique.

[0146] The SPR method was employed, using 1×HBS-EP+Buffer (pH 7.4) as the experimental buffer. Antibodies such as TRN1040 and TRN1041 (3 nM loading concentration) were captured on a Protein A chip at a flow rate of 10 μL / min, with a binding time of 90 s. Channel 2 was injected, and channel 1 served as a blank reference channel. The analyte was rabies virus G protein, initially diluted 2-fold at 96 nM in seven serial dilutions. Channels 1 and 2 were injected at a flow rate of 30 μL / min, with a binding time of 90 s and a dissociation time of 900 s. The regeneration solution was 10 mM glycine (pH 1.5), injected at a flow rate of 30 μL / min into channels 1 and 2, with a binding time of 90 s. Data were analyzed using Biacore 8K analysis software (Biacore Insight Evaluation), with channel 1 serving as a blank reference channel. Background signal was subtracted before analyzing the binding of each sample. The analysis model used was 1:1 binding. The results are shown in Table 3. The fully human anti-rabies virus monoclonal antibodies TRN1040, TRN1041, TRN1042, TRN1043 and TRN1044 of the present invention have a strong affinity for rabies G protein antigen.

[0147] Table 3 Summary of Affinity of Anti-Rabies Virus Monoclonal Antibodies

[0148] Example 8: Determination of neutralizing titer of anti-rabies virus antibody

[0149] According to the standard Pharmacopoeia of the People's Republic of China 2020 Edition (Part IV), General Chapter 3512, the samples were tested for rabies virus neutralizing antibodies. The human control antibody was purchased free of charge from the China National Institutes for Food and Drug Control (batch number: 250011-201306). TRN073 is referenced in patent CN106432486B, and TRN006 is referenced in patent CN103910796B. The specific procedure is as follows: Add 50 μL of neutralizing virus to each well of the diluted standard and test sample. Simultaneously, set up normal cell control wells (add only 100 μL of DMEM to each well) and neutralizing virus control wells (containing 100 μL of DMEM with 5% inactivated newborn calf serum, plus 50 μL of neutralizing virus). After mixing, incubate at 37℃ for 1 h for neutralization. Add 1×10⁻⁶ oz.5 μL of neutralizing virus to each well. 6 50 μL of BSR cell suspension per cell / mL was cultured at 37℃ and 5% CO2 for 24 h. After culture, the culture medium was aspirated, and 100 μL of PBS was added to each well for washing and aspiration. Then, 50 μL of 80% acetone pre-cooled to 4℃ was added to each well, and the cells were fixed at 4℃ for 30 min or -30℃ for 10 min. The acetone was discarded, and after evaporation and drying, 50 μL of fluorescently labeled rabies virus nucleoprotein antibody at the working concentration was added to each well. The cells were incubated at 37℃ for 30 min, and the liquid was discarded. The cells were washed 2-3 times with PBS, dried, and 50 μL of 80% glycerol was added to each well. The cells were observed under a fluorescence microscope. The fluorescence staining was recorded, and the results were calculated. The results are shown in Table 4. The fully human anti-rabies virus monoclonal antibodies TRN1040, TRN1041, TRN1042, TRN1043, and TRN1044 of the present invention have high rabies virus neutralizing activity.

[0150] Table 4 shows the neutralizing titer of the rabies virus CVS-11 cell strain.

[0151] Example 9: Broad-spectrum neutralization experiment with pseudoviruses

[0152] The G protein sequences of 31 representative RABV strains from domestic and foreign markets, as well as other rabies virus strains, were collected and analyzed (including complete background information and full-length 524 amino acids). A rabies virus pseudovirus library was constructed, and neutralization experiments were conducted, following the procedures outlined in Example 8. The operational steps are shown in Figure 3. The types and results of domestic, foreign, and other rabies virus strains are shown in Tables 5-9. The experimental results show that the antibody of this invention can neutralize all 16 domestic and 6 foreign prevalent rabies viruses detected, and can neutralize 9 other rabies viruses, demonstrating excellent broad-spectrum neutralizing ability.

[0153] Table 5. Representative rabies strains found in domestic street viruses.

[0154] Table 6. Representative Rabies Strains from Abroad

[0155] Table 7 Neutralizing titers of different domestic and international street virus strains

[0156] Table 8. Neutralizing activity of rabies monoclonal antibodies against other rabies virus genera.

[0157] Table 9 Summary of the neutralizing activity of rabies monoclonal antibodies against rabies viruses from domestic and international sources.

[0158] Example 10: Effect of the dual antibody

[0159] The surface plasmon resonance (SPR) method was used to determine the binding activity of natural fully human anti-rabies virus monoclonal antibodies against the rabies virus G protein, and the correlation between antibody and antigen binding sites was analyzed.

[0160] A His capture kit was used to prepare a chip for capturing His-tag proteins: channel 1 was used as the reference channel, and channel 2 as the sample channel. Anti-histidine antibody was immobilized on channels 1 and 2 of the CM5 sensor chip surface using an amino-coupled method. Capture conditions: G protein concentration was 400 μg / mL, binding time was 180 s, and flow rate was 5 μL / min. TRN1040, TRN1041, TRN1042, TRN1043, and TRN1044 antibody concentrations were 50 μg / mL, binding time was 180 s, flow rate was 10 μL / min, regeneration solution was Glycine 1.5, and regeneration time was 30 s.

[0161] The results of antigen competition analysis are shown in Figure 4. The analysis revealed that TRN1044 can block the binding of TRN1040 to the antigen protein, and vice versa (see Figures 4-1 and 4-2). TRN1044 can block the binding of TRN1043 to the antigen protein, but not vice versa (see Figures 4-3 and 4-4). Furthermore, TRN1044 cannot block the binding of TRN1041 (see Figures 4-5 and 4-6) and TRN1042 (see Figures 4-7 and 4-8) to the antigen protein, and vice versa. These results indicate that TRN1044 has epitopes that are close to or identical to those of TRN1040, partially overlaps with the epitope of TRN1043, and represents different epitopes from TRN1041 and TRN1042. Similarly, it can be seen that TRN1042 has different epitopes from the other four antibodies (see Figures 4-7, 4-8, 4-9, 4-10, 4-13, 4-14, 4-15, 4-16); TRN1041 and TRN1043 have partially overlapping epitopes (see Figures 4-17, 4-18), and have different epitopes from the other three antibodies (see Figures 4-5, 4-6, 4-11, 4-12, 4-13, 3-14); the epitope situation of TRN1040 is the same as that of TRN1044 (see Figures 4-1, 4-2); TRN1043 and TRN1042 have different epitopes (see Figures 4-15, 4-16), and have partially overlapping epitopes from the other three antibodies (see Figures 4-3, 4-4, 4-17, 4-18, 4-19, 4-20).

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A fully human neutralizing antibody against rabies virus, characterized in that, complementarity determining regions of a heavy chain variable region and complementarity determining regions of a light chain variable region comprising any one of: complementarity determining regions of a heavy chain variable region and complementarity determining regions of a light chain variable region comprising any one of: complementarity determining regions (CDRs) of a heavy chain variable region include any one of: (1) H1 CDR1 : SEQ ID NO: 11; H1 CDR2: SEQ ID NO: 12; H1 CDR3: SEQ ID NO: 13; (2) H2 CDR1 : SEQ ID NO: 14; H2 CDR2: SEQ ID NO: 15; H2 CDR3: SEQ ID NO: 16; (3) H3 CDR1 : SEQ ID NO: 17; H3 CDR2: SEQ ID NO: 18; H3 CDR3: SEQ ID NO: 19; (4) H4 CDR1 : SEQ ID NO: 20; H4 CDR2: SEQ ID NO: 21; H4 CDR3: SEQ ID NO: 22; (5) H5 CDR1 : SEQ ID NO: 23; H5 CDR2: SEQ ID NO: 24; H5 CDR3: SEQ ID NO: 25; or an amino acid sequence that is at least 70% identical to a complementarity determining region of a heavy chain variable region; complementarity determining regions (CDRs) of a light chain variable region include any one of: (1') L1 CDR1 : SEQ ID NO: 26; L1 CDR2: SEQ ID NO: 27; L1 CDR3: SEQ ID NO: 28; (2') L2 CDR1 : SEQ ID NO: 29; L2 CDR2: SEQ ID NO: 30; L2 CDR3: SEQ ID NO: 31 ; (3') L3 CDR1 : SEQ ID NO: 32; L3 CDR2: SEQ ID NO: 33; L3 CDR3: SEQ ID NO: 34; (4') L4 CDR1 : SEQ ID NO: 35; L4 CDR2: SEQ ID NO: 36; L4 CDR3: SEQ ID NO: 37; (5') L5 CDR1 : SEQ ID NO: 38; L5 CDR2: SEQ ID NO: 39; L5 CDR3: SEQ ID NO: 40; or an amino acid sequence that is at least 70% identical to a complementarity determining region of a light chain variable region; Preferably, the neutralizing antibody comprises complementarity determining regions of a heavy chain variable region and complementarity determining regions of a light chain variable region comprising any one of: complementarity determining regions (CDRs) of a heavy chain variable region include any one of: H1 H1 CDR1 : SEQ ID NO: 11; H1 CDR2: SEQ ID NO: 12; H1 CDR3: SEQ ID NO: 13; complementarity determining regions (CDRs) of a light chain variable region include any one of: L1 CDR1 : SEQ ID NO: 26; L1 CDR2: SEQ ID NO: 27; L1 CDR3: SEQ ID NO: 28; or the complementarity determining region (CDR) sequences of the heavy chain variable region include: H2 CDR1 : SEQ ID NO: 14; H2 CDR2: SEQ ID NO: 15; H2 CDR3: SEQ ID NO: 16; and the complementarity determining region (CDR) sequences of the light chain variable region include: L2 CDR1 : SEQ ID NO: 29; L2 CDR2: SEQ ID NO: 30; L2 CDR3: SEQ ID NO: 31 ; or the complementarity determining region (CDR) sequences of the heavy chain variable region include: H3 CDR1 : SEQ ID NO: 17; H3 CDR2: SEQ ID NO: 18; H3 CDR3: SEQ ID NO: 19; and the complementarity determining region (CDR) sequences of the light chain variable region include: L3 CDR1 : SEQ ID NO: 32; L3 CDR2: SEQ ID NO: 33; L3 CDR3: SEQ ID NO: 34; or the complementarity determining region (CDR) sequences of the heavy chain variable region include: H4 CDR1 : SEQ ID NO: 20; H4 CDR2: SEQ ID NO: 21 ; H4 CDR3: SEQ ID NO: 22; and the complementarity determining region (CDR) sequences of the light chain variable region include: L4 CDR1 : SEQ ID NO: 35; L4 CDR2: SEQ ID NO: 36; L4 CDR3: SEQ ID NO: 37; or the complementarity determining region (CDR) sequences of the heavy chain variable region include: H5 CDR1 : SEQ ID NO: 23; H5 CDR2: SEQ ID NO: 24; H5 CDR3: SEQ ID NO: 25; and the complementarity determining region (CDR) sequences of the light chain variable region include: L5 CDR1 : SEQ ID NO: 38; L5 CDR2: SEQ ID NO: 39; L5 CDR3: SEQ ID NO: 40; or an amino acid sequence having at least 70% identity to the complementarity determining region of the heavy chain variable region and the complementarity determining region of the light chain variable region. Preferably, the neutralizing antibody includes any one of the following heavy chain variable region (VH) and light chain variable region (VL): the heavy chain variable region includes: a sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, a sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2, a sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3, a sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4, a sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5; The light chain variable region comprises: a sequence as set forth in SEQ ID NO: 6 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6; a sequence as set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7; a sequence as set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8; a sequence as set forth in SEQ ID NO: 9 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9; a sequence as set forth in SEQ ID NO: 10 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10; Preferably, the neutralizing antibody comprises any one of 1) - 5) as follows: 1) a heavy chain variable region as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, a light chain variable region as set forth in SEQ ID NO: 6 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 6; 2) a heavy chain variable region as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2, a light chain variable region as set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7; 3) a heavy chain variable region as set forth in SEQ ID NO: 3 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3, a light chain variable region as set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8; 4) a heavy chain variable region as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4, a light chain variable region as set forth in SEQ ID NO: 9 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9; 5) a heavy chain variable region as set forth in SEQ ID NO: 5 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 5; a light chain variable region as set forth in SEQ ID NO: 10 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

10.

2. The fully human neutralizing antibody against rabies virus of claim 1, wherein, The neutralizing antibody is isolated from human donors' plasma cells infected with rabies virus; Preferably, the neutralizing antibody is a fully human neutralizing antibody; Preferably, the neutralizing antibody can broadly and effectively neutralize multiple strains of rabies virus; Preferably, the neutralizing antibody comprises a full-length antibody, an antibody fragment, a single-chain antibody, a bispecific antibody, a domain antibody, an antibody fusion, a ScFv, a Fab, a Fab', a F(ab')2, or a Fv; Preferably, the neutralizing antibody comprises an IgG, IgA, IgD, IgE, or IgM antibody; Preferably, the constant region of the neutralizing antibody includes any one of the constant regions of IgG, IgA, IgD, IgE or IgM, more preferably the IgG constant region; Preferably, the neutralizing antibody comprises a modified or conjugated neutralizing antibody; Preferably, the modification includes disulfide bond formation, glycosylation, esterification, acetylation, methylation, and phosphorylation; Preferably, the coupling includes a detectable marker connection; Preferably, the detectable markers include radioactive substances, enzymes, coenzymes, fluorescent agents, chemiluminescent agents, bioluminescent agents, chromogenic agents, enzyme substrates or cofactors, enzyme inhibitors, complexes, and dyes.

3. A neutralizing antibody composition, characterized in that, Includes at least two of the fully human anti-rabies virus neutralizing antibodies as described in claim 1 or 2; Preferably, it includes combinations of 3) and 4), 3) and 2), 3) and 1), and 3) and 5), and more preferably, combinations of 3) and 4) and 3) and 2).

4. A polynucleotide encoding a fully human anti-rabies virus neutralizing antibody as described in claim 1 or 2; Preferably, the polynucleotide comprises a nucleotide sequence encoding part or all of the heavy chain variable region and / or a nucleotide sequence encoding part or all of the light chain variable region; Preferably, the nucleic acid molecule comprises a nucleic acid molecule having a conserved nucleotide sequence variant of the nucleotide sequence.

5. The polynucleotide of claim 4 or the expression vector expressing the neutralizing antibody of claim 1 or 2.

6. A host cell comprising the polynucleotide of claim 4 or the expression vector of claim 5.

7. A pharmaceutical composition, characterized by, Includes the neutralizing antibody of claim 1 or 2, the neutralizing antibody composition of claim 3, the polynucleotide of claim 4, the expression vector of claim 5, and / or the host cell of claim 6; Preferably, the pharmaceutical composition further includes one or more other therapeutic agents; Preferably, the other therapeutic agents are used to treat a specific indication, more preferably to treat infection or prevent side effects; Preferably, the other therapeutic agents are administered in combination with the anti-rabies virus neutralizing antibody; Preferably, the anti-rabies virus neutralizing antibody is administered in combination with other antibodies targeting other antigens of the rabies virus.

8. A rabies virus detection product, characterized by, It includes the neutralizing antibody of claim 1 or 2, the neutralizing antibody composition of claim 3, the polynucleotide of claim 4, the expression vector of claim 5, and / or the host cell of claim 6.

9. A formulation characterized in that, Includes the neutralizing antibody of claim 1 or 2, the neutralizing antibody composition of claim 3, the polynucleotide of claim 4, the expression vector of claim 5, and / or the host cell of claim 6; Preferably, the formulation further includes a pharmaceutically acceptable carrier; Preferably, the formulation includes a lyophilized formulation, a solution, or a suspension; Preferably, the formulation is administered via intravenous, intramuscular, intraperitoneal, cerebrospinal, subcutaneous, intramedullary, intrathecal, oral, local, or inhalation routes.

10. A method for detecting whether a biological sample contains a rabies virus for non-diagnostic and therapeutic purposes, characterized by, contacting a biological sample with the neutralizing antibody of claim 1 or 2, the neutralizing antibody composition of claim 3, the polynucleotide of claim 4, the expression vector of claim 5, the host cell of claim 6 and / or the detection product of claim 8, and qualitatively or quantitatively detecting.

11. A method for producing the neutralizing antibody of claim 1 or 2, characterized by, comprising culturing the host cell of claim 6 under suitable conditions, and isolating the antibody molecule; preferably, further comprising a purification step.

12. Use of the neutralizing antibody of claim 1 or 2, the neutralizing antibody composition of claim 3, the polynucleotide of claim 4, the expression vector of claim 5, the host cell of claim 6 and / or the detection product of claim 8 in the manufacture of a medicament or reagent for any of the following uses: 1) inhibiting rabies virus; 2) preventing a disease caused by infection with rabies virus; 3) detecting rabies virus.

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