Anti-rabies virus fully human antibody and composition and application thereof

Abstract

The application provides two kinds of anti-rabies virus full human monoclonal antibodies or a combination thereof, a heavy chain variable region of the antibodies comprises three CDR regions, sequences of the three CDR regions are respectively shown as SEQ ID NO:1-3 and SEQ ID NO:9-11, and a light chain variable region of the antibodies comprises three CDR regions, sequences of the three CDR regions are respectively shown as SEQ ID NO:4-6 and SEQ ID NO:12-14. The application also provides application of the antibodies to anti-rabies virus. The antibodies are high in affinity to different antigen epitopes of a rabies virus G protein, and are high in potency in neutralizing rabies virus strains derived from different genetic subtypes, and the combination of the antibodies does not affect antigen binding and virus neutralization activities of the antibodies.

Description

[0001] The present application claims priority to the Chinese patent application No. 202311206762.2, filed on September 19, 2023, entitled "Anti-rabies virus fully human antibody and composition and application thereof". TECHNICAL FIELD

[0002] The present application belongs to the field of biomedical technology, and relates to an antibody against rabies virus, in particular to an anti-rabies virus fully human antibody and composition and application thereof. BACKGROUND

[0003] Rabies is a fatal, acute and progressive encephalomyelitis caused by Lyssavirus, and all warm-blooded animals are susceptible to rabies virus, and once the disease occurs, the mortality rate is almost 100%.

[0004] WHO recommends that rabies vaccine and rabies immune globulin (RIG) should be used in combination after rabies III exposure. The currently used main products include equine rabies immune globulin (ERIG) and human rabies immune globulin (HRIG), which are two types of polyclonal antibodies. HRIG is derived from the plasma of healthy volunteers after vaccination with rabies vaccine, and the yield is extremely low and the price is expensive, which is difficult to meet the demand for clinical prophylactic drugs, and the quality of such products is difficult to control, and there is a risk of potential viral reinfection. ERIG is derived from the serum of immunized horses, which is a heterologous serum, and the incidence of adverse reactions is high (12.3%-46.2%), mostly serious serum sickness or anaphylactic shock, which seriously affects the application in clinical immunoprophylaxis. Due to these reasons, it has become an inevitable trend to develop human therapeutic monoclonal antibodies against rabies virus to replace the currently widely used blood-derived antibodies for the prevention and treatment of clinical rabies exposure.

[0005] The G protein of rabies virus is arranged in the trimeric spike structure on the surface of the viral envelope, and is the only antigen exposed on the surface of the virus, which is also the main target of neutralizing antibodies. The main antigenic sites on the G protein are I, II, III, IV, G1 and G5. Due to the natural variability of rabies virus, especially the variability of the neutralizing epitopes in monoclonal antibodies, WHO recommends using a cocktail composition containing at least two monoclonal antibodies against different non-overlapping antigenic sites on the G protein of rabies virus to replace traditional serum products to ensure the effectiveness of the monoclonal antibody combination preparation against different virus strains or different genotypes of viruses. However, the use of multiple monoclonal antibodies may produce inhibitory effects due to mutual antagonism or competition, which cannot achieve the desired ideal effect.

[0006] In fact, there is only one product of anti-rabies monoclonal antibody drug currently listed in China, that is, the recombinant human anti-rabies virus monoclonal antibody injection (Xunke, also known as Ormutivimab Injection) listed by North China Pharmaceutical in February 2022. The corresponding Chinese patent document is CN101100663A, which adopts a complete chemical synthesis method to synthesize DNA sequences encoding the variable regions of the heavy chain and light chain of NM57, respectively, and links them with the vector pCAdhfr to obtain a recombinant vector that can be used to express the complete NM57 antibody. The vector is used to transfect mammalian cells to construct a high-efficiency engineering cell, and the obtained monoclonal antibody is renamed as NM57. This method has simple operation process, high product expression level (more than 100 milligrams per liter), uniform and stable antibody quality, and good stability, and has industrialization value.

[0007] This product, although a human monoclonal antibody, can only target specific rabies virus gene subtypes and cannot recognize and neutralize some gene subtypes of rabies virus, and has certain defects in recognition and neutralization of broad spectrum.

[0008] In addition, Chinese patent document CN113185608A discloses a full human monoclonal antibody with broad spectrum binding to rabies virus. By constructing a prokaryotic expression vector of rabies virus specific antigen protein, and using an Escherichia coli prokaryotic system for expression and purification, five specific full human monoclonal antibody sequences against rabies virus GP fragments are successfully screened.

[0009] However, this document only provides preliminary binding data and does not disclose evidence of broad spectrum binding, let alone broad spectrum neutralization activity and in vivo and in vitro effect data, and of course does not provide a cocktail composition of full human monoclonal antibodies without antagonistic effect. SUMMARY

[0010] The present application provides an anti-rabies virus full human monoclonal antibody, named 5-7G, which is composed of a heavy chain and a light chain. The variable region of the heavy chain includes three CDR regions, the sequences of which are shown in SEQ ID NO: 1-3, respectively. The variable region of the light chain includes three CDR regions, the sequences of which are shown in SEQ ID NO: 4-6, respectively.

[0011] Preferably, the full-length sequence of the variable region of the heavy chain of the 5-7G antibody is shown in SEQ ID NO: 7, and the full-length sequence of the variable region of the light chain is shown in SEQ ID NO: 8.

[0012] The present invention also provides another fully human monoclonal antibody against rabies virus, named 26-12G, which is composed of a heavy chain and a light chain, wherein the variable region of the heavy chain includes three CDR regions, the sequences of which are shown in SEQ ID NO:9-11 respectively; and the variable region of the light chain includes three CDR regions, the sequences of which are shown in SEQ ID NO:12-14 respectively.

[0013] Preferably, the full-length sequence of the variable region of the 26-12G antibody heavy chain is shown in SEQ ID NO:15, and the full-length sequence of the variable region of the light chain is shown in SEQ ID NO:16.

[0014] The present invention also provides a nucleic acid molecule encoding the above-mentioned antibody.

[0015] The present invention also provides expression cassettes, recombinant vectors or recombinant microorganisms containing the above-mentioned nucleic acid molecules.

[0016] The present invention also provides pharmaceutical compositions comprising the above-described antibody and pharmaceutically acceptable excipients, diluents or carriers.

[0017] Preferably, the pharmaceutical composition is a dual antibody "cocktail" composition, wherein the first antibody is a 5-7G antibody and the second antibody is a 26-12G antibody.

[0018] The present invention also provides the use of the antibodies, nucleic acid molecules, expression cassettes, recombinant vectors or recombinant microorganisms, or pharmaceutical compositions described herein in the preparation of reagents or drugs for the diagnosis, prevention and / or treatment of rabies virus infection.

[0019] The present invention has the following beneficial effects:

[0020] The recombinant monoclonal antibody against rabies virus prepared by this invention is derived from hybridoma cell screening, followed by recombinant expression after sequence analysis. The entire recombinant expression sequence is a fully human sequence, with no safety risks such as allergies or viral reinfection. The purity of the recombinant protein after purification is as high as 90% or more, making it suitable for large-scale production of high-purity fully human recombinant monoclonal antibodies against rabies virus.

[0021] Western blotting analysis, ELISA antigen epitope analysis, and ECG analysis were performed. 50 Detection methods, including ForteBio Octet affinity analysis, have demonstrated that the two fully human recombinant monoclonal antibodies against rabies virus of this invention bind to different antigenic epitopes of the rabies virus G protein with high affinity. The two fully human recombinant monoclonal antibodies against rabies virus of this invention can be used alone or in combination without affecting their binding to the antigenic epitopes.

[0022] In vitro and in vivo virus neutralization analyses showed that the two fully human recombinant monoclonal antibodies against rabies virus of this invention exhibited high titers for neutralizing rabies virus strains derived from different genotypes. The neutralizing titers for the vast majority were higher than those of existing human anti-rabies virus antibodies, demonstrating broad-spectrum neutralizing activity. These two fully human recombinant monoclonal antibodies against rabies virus of this invention can be used alone or in combination without affecting each other's virus neutralizing activity. Furthermore, when used in combination, they exhibit broad-spectrum neutralizing activity against all genotypes.

[0023] Instruction manual illustrations

[0024] Figure 1 The SDS-PAGE results of the anti-rabies virus monoclonal antibody of the present invention are shown. Lane M is the marker, lane 1 is the 5-7G antibody, and lane 2 is the 26-12G antibody.

[0025] Figure 2 The Western blot results of the anti-rabies virus monoclonal antibody of the present invention are shown. The left panel (A) shows the results for 5-7G antibodies, and the right panel (B) shows the results for 26-12G antibodies. Lane M is the marker, lane 1 is the rabies G protein treated with non-reducing buffer, and lane 2 is the rabies G protein treated with reducing buffer.

[0026] Figure 3 EC showing the anti-rabies virus monoclonal antibody of the present invention 50 Test results. The left image shows the results for 5-7G antibodies, and the right image shows the results for 26-12G antibodies.

[0027] Figure 4 The results of affinity testing for the anti-rabies virus monoclonal antibody of this invention are shown. The left figure shows the results for 5-7G antibodies, and the right figure shows the results for 26-12G antibodies.

[0028] Figure 5The results of the mouse challenge protection test of the anti-rabies virus monoclonal antibody of the present invention are shown. The horizontal axis of the graph represents the number of days, and the vertical axis represents the survival percentage. In the legend: solid circles represent the PBS control group; hollow circles represent the vaccine-only group; solid triangles represent the vaccine + 20 IU / kg HRIG group; hollow inverted triangles represent the vaccine + 0.03 mg / kg 5-7G / 26-12G group; solid inverted triangles represent the vaccine + 0.06 mg / kg 5-7G / 26-12G group; solid rhombuses represent the vaccine + 0.12 mg / kg 5-7G / 26-12G group; hollow hexagons represent the 20 IU / kg HRIG-only group; hollow squares represent the 0.03 mg / kg 5-7G / 26-12G group; hollow rhombuses represent the 0.06 mg / kg 5-7G / 26-12G group; hollow triangles represent the 0.12 mg / kg 5-7G / 26-12G group; solid hexagons represent the group with no neutralizing antibodies. Detailed Implementation

[0029] The following detailed description provides further details through specific embodiments. However, it should be noted that the embodiments described below are merely for illustrating the content of the invention and do not represent that the invention is limited to the described embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention still fall within the protection scope of the invention, and the scope of protection of the appended claims shall prevail.

[0030] As is known to those skilled in the art, the rabies virus described in this invention is the pathogen that causes rabies. Biologically, this virus belongs to the genus *Rabiesvirus* within the family Rhabdoviridae. The virus particle consists of a capsid and a core. The capsid is a lipoprotein double envelope, containing glycoproteins on the outer side and matrix membrane proteins on the inner side. The nucleocapsid of the core is composed of single-stranded RNA and proteins. Rabies virus contains five proteins: glycoprotein (G), nucleoprotein (N), dimeric enzyme (L), phosphoprotein (NS), and matrix (M). Glycoprotein G can lead to the formation of neutralizing antibodies in the body, which can combat viral attack and serve as the main antigen of rabies virus. The other antigen is nucleoprotein N. Rabies virus can persist in wild animals for extended periods and is widespread among wild animals worldwide. Host animals include carnivores and bats. Foxes, wolves, jackals, badgers, raccoon dogs, skunks, raccoons, meerkats, and bats are all natural reservoir hosts of rabies and can be infected with the rabies virus, becoming sources of infection and subsequently infecting livestock such as pigs, cattle, sheep, and horses.

[0031] Currently, rabies prevention and treatment mainly rely on vaccination and antibody administration. Antibodies can be derived from the serum of immunized animals or humans, or immunoglobulins can be isolated and prepared from serum. In my country, HRIG (human rabies immunoglobulin) is widely used. However, due to the limited supply, high price, and potential risks of serum sickness-like allergic reactions and blood-borne diseases, research has begun on humanized monoclonal antibodies against rabies virus. However, as commercially available products, they still have many drawbacks. For example, monoclonal antibodies target only a single epitope, which may not be as broad-spectrum or comprehensive as polyclonal antibodies in serum. Different monoclonal antibodies may be needed for different rabies viruses. Furthermore, the expression efficiency is affected after full humanization of the sequence, and the high price remains a problem.

[0032] As is known to those skilled in the art, the polyclonal antibodies described in this invention are mixtures of antibodies against the same antigen produced by different B cells in the body, and they can recognize and bind to different antigenic epitopes on that single antigen. Monoclonal antibodies, on the other hand, are produced by the same B cells and can only recognize a specific single antigenic epitope.

[0033] As is known to those skilled in the art, the chain structure and regions of the antibody molecules described in this invention have the following meanings: Antibody molecules are generally composed of two heavy chains (i.e., H chains) and two light chains (i.e., L chains), each chain including a variable region (i.e., V region), a constant region (i.e., C region), and a hinge region. The composition and arrangement of residues near the amino terminus of the heavy and light chains vary greatly; this region is called the variable region, while the composition and arrangement of residues near the carboxyl terminus are relatively stable, hence it is called the constant region. Within the variable regions of the heavy and light chains, there are three specific segments in each chain with highly variable amino acid composition and arrangement, called hypervariable regions (i.e., HVR). These regions together constitute the antigen-binding site of the antibody molecule. This site can bind complementaryly to the corresponding antigenic epitope; therefore, the hypervariable region is also called the complementarity-determining region (i.e., CDR), denoted as CDR1, CDR2, and CDR3, respectively. The different amino acid compositions and sequences of the CDRs of different antibodies determine the specificity of the antibody's binding to the corresponding antigenic epitope, responsible for recognizing and binding the antigen, thereby exerting an immune effect. The amino acid composition and sequence of the variable regions of an antibody molecule, excluding the hypervariable region or CDR region, are relatively small and are called the backbone region (i.e., FR region).

[0034] As we know from the above common knowledge, the basic immune function of an antibody depends on three CDR regions on both the heavy and light chains. These regions are all located in the variable regions of the antibody heavy and light chains. The other amino acid sequences on the antibody molecule are generally conserved, and even if there are slight changes, they usually do not affect the basic immune function of the antibody molecule. Therefore, a sequence expressing all CDR regions can express an antibody or evaluate whether they belong to the same antibody.

[0035] Other antibody-related technologies, instruments, and materials are well known to those skilled in the art. Examples include immunizing animals (e.g., mice) with antigens (e.g., viruses or their immunogenic proteins), performing hybridoma fusion and screening after immunization, antibody expression and purification, and antibody structure and performance analysis (e.g., fluorescent antibody-virus neutralization assay (FAVN), affinity, neutralization performance, in vitro and in vivo protective effects). Unless otherwise specified in this invention, any other technologies, instruments, and materials known to those skilled in the art that can achieve the same purpose may be used. Even if specific technologies, instruments, and materials are specified in this invention, it does not mean that this invention can only use these technologies, instruments, and materials; it merely represents preferred embodiments of the invention. Those skilled in the art can still use any other technologies, instruments, and materials known to them that can achieve the same purpose.

[0036] Example 1: Preparation of Hybridoma Cells

[0037] 1.1 Immunization of Animals

[0038] CAMouse was immunized with rabies G protein (rabies virus glycoprotein, from nearshore organisms) and SRV9 virus (attenuated rabies virus strain). HG Mice (Chongqing Jinmaibo Biotechnology Co., Ltd.) were initially immunized with rabies G protein, 50 μL / mouse, intramuscularly, followed by immunization once a week. After three protein immunizations, two booster immunizations were performed using SRV9 virus (100 μL / mouse, 10 doses). 7.5 / mL TCID 50 (Half-maximum tissue culture infection dose) immunize once a week, collect serum on day 7 for fluorescent antibody virus neutralization test (FAVN) detection, select mice with high virus neutralization titer for booster immunization with rabies G protein, 50 μL / mouse, intraperitoneal injection, and perform hybridoma fusion experiment 4 days later.

[0039] 1.2 Hybridoma Fusion and Culture

[0040] Splenic lymphocytes from immunized mice obtained in step 1.1 were separated and mixed with SP2 / 0 cells (mouse myeloma cells) at a ratio of 3:1. The mixture was centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 10 mL of PBS. The mixture was centrifuged again at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 10 mL of electrofusion buffer. The mixture was centrifuged again at 1200 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in electrofusion buffer. The cell suspension was added to the electrofusion tank, the voltage parameters were set, and the fusion button was clicked to perform electrofusion. The resulting hybridoma cell mixture was transferred to a semi-solid medium containing HAT (a selective medium containing hypoxanthine, aminopterin, and thymidine deoxyribonucleoside) and cultured in a 37°C, 5% CO2 incubator. After 7-10 days, single clones were picked and transferred to a 96-well plate containing HT medium (a selective medium containing hypoxanthine and thymidine deoxyribonucleoside) and cultured for another 2-3 days before detection.

[0041] 1.3 Virus Binding Screening

[0042] BHK-21 hamster kidney cells were divided into groups of 1×10⁻⁶ cells. 5 Prepare 96-well plates at a density of 100 μL cells / mL. Add 100 μL of basal medium (MEM) containing 5% FBS and 1% penicillin and streptomycin to each well and incubate at 37°C and 5% CO2 for 12 h until the cells are fully adhered. Dilute the cytotoxic virus in the deep-well plates according to the titer of the rabies virus strain CVS-11. Discard the 96-well plate culture medium, add 100 μL of virus dilution to each well, and seed the cells according to the multiplicity of infection (MOI = 0.1). Set up uninoculated cells as a control. BHK-21 cells, both inoculated and uninoculated with the virus, were cultured at 37°C in a 5% CO2 incubator for 72 hours. After 72 hours, the culture medium was discarded, and each well was fixed with 80% cold acetone at -20°C for 2 hours. The acetone was then discarded, and the cells were washed three times with PBST (250 μL of Tween-20 was added to 500 mL of PBS). 50 μL of the hybridoma cell supernatant obtained in step 1.2 was added to each well and incubated for 1 hour. HT medium was used as a negative control, and immunized mouse serum was used as a positive control. The culture medium was discarded, and the cells were washed three times with PBST. 1:200 diluted anti-human IgG-FITC (abcam) secondary antibody was added and incubated for 1 hour. The secondary antibody solution was discarded, and the cells were washed three times with PBST. Under a fluorescence microscope, cells inoculated with the virus showed green fluorescence, while uninoculated cells did not show fluorescence. These were hybridoma cell clones that had bound to the CVS-11 virus.

[0043] 1.4 Virus Neutralization Screening

[0044] Serum samples were serially diluted 3-fold in a 96-well plate, with each sample tested in a 4×6-well area. A 96-well plate was used as a control plate, and standard positive serum (0.5 IU / mL) and standard negative serum (non-immunized canine serum) were diluted as described above. A pre-determined titer of eGFP-SRV9 virus (a rabies virus SRV9 strain carrying the fluorescent protein eGFP) was diluted with DMEM to 100 TCID. 50 / 50μL, add 50μL to each well except for the cells and culture medium control wells, and neutralize in an incubator at 37℃ for 60min; resuspend the hybridoma cells that can bind to the virus obtained in step 1.3 to 4×10 5 Cells / mL were added to each well, except for the culture medium control, at a rate of 50 μL. The cells were then cultured for 48 h using standard methods. Observation under a fluorescence microscope was performed; fluorescence in the wells indicated infection, while the absence of fluorescence indicated complete neutralization. The supernatant from hybridoma cells capable of completely neutralizing the eGFP-SRV9 virus was selected as the hybridoma cell supernatant capable of producing the monoclonal antibody of this invention for analysis and detection.

[0045] Example 2: Detection of virus neutralization titer in hybridoma cell supernatant (FAVN assay)

[0046] Antibody samples were serially diluted 3-fold in 96-well plates, with each antibody tested in a 4×6 well region. A 96-well plate was used as a control plate, and standard positive serum (0.5 IU / mL) and standard negative serum (non-immunized dog serum) were diluted as described above. A pre-determined titer of CVS-11 strain was diluted with DMEM to 100 TCID⁻¹. 50 Add 50 μL of the medium to each well, except for the control wells containing cells and culture medium, and neutralize in an incubator at 37°C for 60 min; resuspend the BHK-21 cells to 4 × 10⁻⁵. 5 Add 50 μL of antibody per well (except for the culture medium control) and incubate for 72 h using standard methods. Discard the culture medium, add 80% cold acetone, and fix at -20℃ for 1 h. Discard the fixative, wash 3 times with PBST, add 1:1000 diluted FITC Anti-Rabies Monoclonal Globulin (FUJIREBIO) to each well, and incubate at 37℃ for 1 h. Discard the antibody, wash 3 times with PBST, add 50 μL / well of 90% glycerol buffer, and observe the fluorescence signal in the wells under a fluorescence microscope. Wells showing specific green fluorescence are considered positive, and the virus neutralization titer of the sample is calculated according to the Spearman-Karber formula.

[0047] Example 3: Expression and purification of recombinant antibodies against rabies virus

[0048] The hybridoma cell supernatant obtained in Example 2 was sequenced using the 5' RACE method to obtain the heavy and light chain variable region sequences of two monoclonal antibodies (named 5-7G and 26-12G antibodies, respectively). Following the conventional recombinant antibody expression method, an IgG1 recombinant antibody was constructed using the pcDNA3.4 vector (Invitrogen). Logarithmically growing 293F cells were passaged until they reached a cell size of 1.5 × 10⁻⁶. 6 Once the cells were at a density of 100 cells / mL and the cell viability was greater than 95%, the cells were diluted to 1×10⁻⁶ cells / mL. 6 Transfection was performed after achieving a cell / mL concentration. Two sterile 2mL EP tubes were used. One tube contained 200μL of Opti-MEM culture medium and 20μg of recombinant plasmid DNA, while the other contained 200μL of Opti-MEM culture medium and 60μL of polyethyleneimine (PEI). Both tubes were mixed thoroughly and incubated at room temperature for 5 min. The PEI solution was then rapidly added to the recombinant plasmid DNA mixture, and the mixture was gently pipetted and incubated at room temperature for 15 min. The mixture was then added dropwise to the prepared 293F cells while gently shaking the cells. The transfected cells were cultured in a shaking incubator. Cell viability was calculated daily using trypan blue staining. When the viable cell count dropped to approximately 60%, the cells were centrifuged for 20 min and the cell supernatant was collected.

[0049] Protein purification from the supernatant was performed using a HITRAP PROTEIN AHP protein purification column. The column was first washed with 5 column volumes of ultrapure water, then equilibrated with 5 column volumes of binding buffer (20 mmol / L PB buffer, 0.2 mol / L NaCl, pH 7.2). The collected cell supernatant was filtered through a 0.45 μm filter membrane and mixed with an equal volume of binding buffer, then loaded at a flow rate of 1 mL / min, and the flow-through was collected. The column was washed with 10 column volumes of binding buffer until UV and conductivity were at baseline. Elution buffer was then added to neutralization buffer at a flow rate of 1 mL / min. Immediately after elution, the column was reequilibrated with 10 column volumes of binding buffer. The purified protein was then dialyzed against PBS overnight, concentrated using an ultrafiltration tube, and adjusted to a concentration of 1 mg / mL. The purity of the recombinant monoclonal antibody was verified by SDS-PAGE.

[0050] After treating the two recombinant monoclonal antibodies with a reducing buffer, the results were analyzed by SDS-PAGE. Figure 1 The results showed that the heavy chain bands of both antibodies were 45-60 kDa in size, while the light chain bands were about 25 kDa in size, which was in line with the expected size, and the purity of both was above 90%.

[0051] Example 4: Analysis of the antigen-binding performance of monoclonal antibodies

[0052] 4.1 Western Blot Binding Analysis

[0053] After treating rabies G protein with non-reducing and reducing buffers, the two monoclonal antibodies 5-7G and 26-12G of this invention were reacted with rabies G protein as primary antibodies, respectively. Then, Rabbit anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, DyLight, was used. TM 680 (Invitrogen) is used for color development, such as Figure 2 As shown, both 5-7G and 26-12G recognize conformational epitopes of the rabies G protein.

[0054] 4.2 ELISA Epitope Analysis

[0055] The antigenic epitopes of the recombinant antibody of this invention were detected using a competitive ELISA method, as follows: Rabies G protein was coated onto an ELISA plate at a concentration of 0.5 μg / mL (100 μL per well) and incubated overnight at 4°C; the plate was washed three times with PBST, and 200 μL of 5% BSAPBS was added to each well for blocking at room temperature for 1 h; the plate was washed three times with PBST, and 25 μg / mL of recombinant antibody was added to each well (50 μL per well), and incubated at 37°C for 1 h; simultaneously, a conjugated antibody control and a human rabies immunoglobulin control were set up, with 2 μg / mL of horseradish peroxidase (HRP) conjugated recombinant antibody added to each well (50 μL per well), and incubated at 37°C for 1 h; the plate was washed three times with PBST, and tetramethylbenzidine (TMB) was added for color development for 10 min; 2.5 M sulfuric acid solution was added to stop the reaction, and the OD values ​​were read using an ELISA reader. 450nm value.

[0056] According to literature reports, existing monoclonal antibodies NM57 recognize rabies G protein epitope I, and CR4098 recognize rabies G protein epitope III. As shown in Table 1, the 26-12G antibody of this invention can completely block the NM57 antibody, therefore it recognizes rabies G protein epitope I; the 5-7G antibody of this invention can be completely blocked by the CR4098 antibody, therefore it recognizes rabies G protein epitope III.

[0057] Table 1. Results of antigen epitope competition ELISA detection of the recombinant antibody of this invention.

[0058]

[0059] 4.3 Antibody binding EC 50 Detection

[0060] The purified 5-7G and 26-12G recombinant monoclonal antibodies of this invention were subjected to ECMO. 50The detection method is as follows: Rabies G protein was diluted to 2 μg / mL with coating buffer, 100 μL / well, and coated overnight at 4℃. The coated ELISA plate was then removed, washed three times with PBST, and blocked at 37℃ for 1 h with 5% BSAPBS (5 g BSA added to 100 mL PBS). The blocked ELISA plate was then removed, washed three times with PBST, and the culture supernatant was added. An 8-fold serial dilution was performed according to the measured concentration (concentration range set at 100,000 ng / mL - 0.1 ng / mL, diluted with 5% BSAPBS). 5% BSAPBS was used as a blank control. The plate was incubated at 37℃ for 2 h. The incubated ELISA plate was then removed, washed three times with PBST, and Mouse Anti-Human IgG(Fab)-HRP (GenScript, 1:5000, diluted with 5% BSAPBS) was added. The plate was incubated at 37℃ for 2 h. Remove the sealed ELISA plate, wash three times with PBST, add TMB for 10 min of color development, and terminate the reaction with stop solution (2.5M sulfuric acid). Read the OD value using a microplate reader. 450nm Numerical value. Calculate EC 50 Value: OD 450nm Input the values ​​and corresponding concentration gradients into Graphpad Prism software to calculate EC. 50 value.

[0061] like Figure 3 As shown in the figure, the test results indicate that the 5-7G antibody of this invention binds to the rabies G protein in EC2. 50 The value was 0.0332 μg / mL, indicating that the 26-12G antibody of this invention binds to rabies G protein at an EC50 value. 50 The value was 0.005363 μg / mL, demonstrating excellent binding performance.

[0062] 4.4 Antibody Affinity Analysis

[0063] Using the ForteBio Octet molecular interaction technology platform, the 5-7G / 26-12G antibody of this invention was immobilized onto an Anti Human IgG Fc sensor. The sensor was equilibrated with 1% BSAPBS, and then different concentrations of rabies G protein were added to the sensor to ensure sufficient binding with the 5-7G / 26-12G antibody. The sensor was then equilibrated in PBS buffer, and parameters were measured by changes in molecular concentration at the sensor-media interface. Data analysis was performed using ForteBio Data analysis v11.0 and Dataanalysis HT v11.0 software (ForteBio).

[0064] As attached Figure 4As shown in Table 2, the affinity of the 5-7G antibody of this invention to rabies G protein is 1.62E-9, and the affinity of the 26-12G antibody of this invention to rabies G protein is 1.96E-12, both of which have good affinity.

[0065] Table 2. Results of Affinity Detection of Recombinant Monoclonal Antibodies

[0066]

[0067] Example 5: Virus neutralization performance analysis of monoclonal antibodies

[0068] 5.1 Detection of Broad Spectrum Neutralizing Activity of Recombinant Monoclonal Antibodies

[0069] The recombinant antibody was tested for viral neutralization activity using a method similar to the FAVN assay. The antibody samples were serially diluted 3-fold in a 96-well plate. Antibodies and human rabies immunoglobulin were tested in 4×6-well areas. A 96-well plate was used as a control plate, and standard positive serum (0.5 IU / mL) and standard negative serum (non-immunized dog serum) were diluted as described above. Rabies virus strains with pre-determined titers, including SHJDD14 (OR122986.1), ZJZSD1601 (MG383893.1), NeiMeng927A (EU284095.2), NMALSF01 (MW411451.1), and GDMM, were used. D16 (MW411450.1), HuND02 (KT221115.1), HuNPB01 (GU186386.1), IRKV-THChina12 (JX442979.1), and CVS-11 (EU126641.1) were diluted with DMEM to 100 TCID50 / 50 μL. 50 μL was added to each well except for the control wells containing cells and culture medium. The cells were neutralized at 37°C for 60 min. BHK-21 cells were then resuspended at 4 × 10⁻⁶ cells / well. 5 Add 50 μL of antibody per well (except for the culture medium control) and incubate for 72 h using standard methods. Discard the culture medium, add 80% cold acetone, and fix at -20℃ for 1 h. Discard the fixative, wash 3 times with PBST, add 1:1000 diluted FITC-anti-rabies virus immunoglobulin to each well, and incubate at 37℃ for 1 h. Discard the antibody, wash 3 times with PBST, add 50 μL / well of 90% glycerol buffer, and observe the fluorescence signal in the wells under a fluorescence microscope. Wells showing specific green fluorescence are considered positive, and the virus neutralization titer of the sample is calculated according to the Spearman-Karber formula.

[0070] Seven rabies virus street strains of prevalent genotypes in my country (Asian type I: SHJDD14, ZJZSD1601, and HuND02; Asian type II: HuNPB01 and GDMM D16; Arctic-associated group: NeiMeng927A; and World group grassland type: NMALSF01) and one rabies virus strain isolated from bats, IRKV-THChina12, were tested. CVS-11 was used as a standard control strain. As shown in Table 3, the 5-7G and 26-12G antibodies of this invention exhibited good neutralizing activity against all genotypes of rabies virus street strains prevalent in my country. The neutralizing titers of most of these antibodies were significantly higher than those of the most commonly used human rabies immunoglobulin in my country, which served as the control. Furthermore, the 26-12G antibody (which recognizes rabies G protein epitope I) showed extremely high neutralizing activity against IRKV-THChina12, reaching an astonishing 1439.1 IU / mL.

[0071] Table 3 Broad-spectrum neutralizing titer of the recombinant antibody of this invention

[0072]

[0073]

[0074] 5.2 Detection of Synergistic Combination Broad Spectrum Neutralizing Activity of Recombinant Monoclonal Antibodies

[0075] The two recombinant antibodies of the present invention were combined in a 1:1 ratio for virus neutralization activity detection, using the same method as in 5.1.

[0076] As shown in Table 4, after mixing the two antibodies 5-7G and 26-12G of the present invention in a 1:1 ratio, the 5-7G and 26-12G combination showed the same good neutralizing activity against the above-mentioned seven street rabies virus genotypes circulating in my country. At the same time, the combination also showed the same neutralizing activity against IRKV-THChina12 bat virus as the 26-12G antibody alone. Furthermore, the combined use of the two antibodies did not show any antagonistic or competitive inhibitory effect on the neutralization of the virus.

[0077] Table 4 Broad-spectrum neutralizing titer of recombinant antibody combinations

[0078]

[0079] Note: "+" indicates <50 IU / mL, "++" indicates 50-100 IU / mL, "+++" indicates >100 IU / mL, and "-" indicates neutralizing activity <0.5 IU / mL.

[0080] Example 6 Animal challenge protection experiment

[0081] 110 female Kunming white mice aged 6-8 weeks were selected and divided into groups 1-11, with 10 mice in each group. All mice in the 11 groups were injected intramuscularly with 50 μL of the standard challenge virus strain CVS-11 rabies virus in the left hind leg (10 μL / L). 7.5 TCID 50 The vaccine was administered intramuscularly in the left hind leg at 50 μL / mL, with Group 1 receiving PBS as a blank control. Groups 2-5 received 50 μL of human rabies vaccine (Liaoning Chengda) intramuscularly at the CVS-11 virus injection site at a dose of 20 IU / kg. Group 2 received 50 μL of human rabies immunoglobulin (Taibang Biotechnology) intramuscularly at the CVS-11 virus injection site. Groups 3-5 received a 1:1 mixture of the recombinant antibody composition of this invention (0.03 mg / kg, 0.06 mL) intramuscularly at the CVS-11 virus injection site. Group 6 received an intramuscular injection of 50 μL of human rabies immunoglobulin at a dose of 20 IU / kg at the CVS-11 virus injection site. Groups 7-10 received an intramuscular injection of a 1:1 mixture of the recombinant antibody composition of this invention (0.03 mg / kg, 0.06 mg / kg, and 0.12 mg / kg) at the CVS-11 virus injection site. Group 11 received an intramuscular injection of 0.12 mg / kg of antibody without virus-neutralizing activity at the CVS-11 virus injection site. Mice were observed twice daily, and mortality was recorded.

[0082] like Figure 5 As shown in Table 5, the 5-7G / 26-12G antibody combination of the present invention at doses of 0.03 mg / kg, 0.06 mg / kg, and 0.12 mg / kg are equivalent to 10 IU / kg, 20 IU / kg, and 40 IU / kg, respectively. According to the experimental results, under the premise of simultaneous vaccination, the 5-7G / 26-12G antibody combination of the present invention at a dose of 0.06 mg / kg can have a better protective effect than the existing human rabies immunoglobulin (HRIG) (20 IU / kg), with the protection rate increasing from 40% (4 / 10) to 60% (6 / 10), and reaching 70% (7 / 10) protection rate at a dose of 0.12 mg / kg.

[0083] Table 5 Results of mouse challenge protection test

[0084]

[0085] The above descriptions are merely embodiments of the present invention. Commonly known technical knowledge in the solutions is not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the filing date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical well-known technologies should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A fully human monoclonal antibody against rabies virus, comprising a heavy chain and a light chain, wherein the full-length sequence of the variable region of the heavy chain is shown in SEQ ID NO:7, and the full-length sequence of the variable region of the light chain is shown in SEQ ID NO:

8.

2. A fully human monoclonal antibody against rabies virus, comprising a heavy chain and a light chain, wherein the full-length sequence of the variable region of the heavy chain is shown in SEQ ID NO:15, and the full-length sequence of the variable region of the light chain is shown in SEQ ID NO:

16.

3. A nucleic acid molecule encoding the antibody of any one of claims 1-2.

4. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule of claim 3.

5. A pharmaceutical composition comprising the antibody of claim 1 or the antibody of claim 2, and a pharmaceutically acceptable excipient, diluent, or carrier.

6. The pharmaceutical composition of claim 5, wherein it is a dual antibody "cocktail" composition, wherein the first antibody is the antibody of claim 1 and the second antibody is the antibody of claim 2.

7. The use of the antibody of any one of claims 1-2, or the nucleic acid molecule of claim 3, or the expression cassette, recombinant vector or recombinant microorganism of claim 4, or the pharmaceutical composition of claim 5 or 6 in the preparation of a reagent or medicament for the diagnosis, prevention and / or treatment of rabies virus infection.

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