A combined monoclonal antibody preparation against rabies virus

Abstract

The present invention discloses a combined monoclonal antibody preparation against rabies virus, which relates to the technical field of biomedicine. The combined monoclonal antibody preparation against rabies virus contains R92 antibody and R71 antibody; the light chain sequence of the R92 antibody is as shown in SEQ ID NO:1, the heavy chain sequence of the R92 antibody is as shown in SEQ ID NO:2, the light chain sequence of the R71 antibody is as shown in SEQ ID NO:3, and the heavy chain sequence of the R71 antibody is as shown in SEQ ID NO:4. It is also disclosed that the ratio of the R92 antibody to the R71 antibody in the combined monoclonal antibody preparation against rabies virus is (1-5):(1-5). The combined monoclonal antibody preparation against rabies virus of the present invention has a high affinity for rabies virus glycoprotein, has good neutralization ability and thermal stability, has good neutralization ability against a variety of street strains, and the two antibodies target different epitopes, providing a wider range of protection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a combined monoclonal antibody preparation against rabies virus. Background Art

[0002] Rabies virus (RABV) is a single-stranded negative-strand RNA virus of the family Rhabdoviridae and the genus Rabies virus. Under transmission electron microscopy, it appears rod-shaped or bullet-shaped, with a diameter of about 75 nm and a length of about 100 - 300 nm. RABV has a tropism for neurons and can invade the central nervous system of the body, leading to fatal nerve infections.

[0003] The RABV genome is about 12 knt in length and encodes five structural proteins in total, namely nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), envelope glycoprotein (G), and large polymerase protein (L). The coding order from the 3' end to the 5' end of the genome is 3'-N-P-M-G-L-5'. The glycoprotein has a size of 65 - 67 kDa and is interconnected with the M protein inside RABV. It is the only exposed protein inserted into the viral lipid envelope and is also the only ligand for the cellular receptor. In the natural state, the G protein forms trimers on the virus surface and is the main surface antigen bound by neutralizing antibodies. The G protein consists of three domains, including an extracellular domain, a transmembrane domain, and an intracellular domain.

[0004] Rabies is a zoonotic global infectious disease caused by rabies virus. For rabies exposure, especially for severely exposed individuals, rabies vaccine and anti-rabies antibody should be injected throughout the process. Currently, the types of rabies vaccines mainly include novel inactivated rabies vaccines, attenuated vaccines, nucleic acid vaccines, subunit vaccines, virus-like particle vaccines, and oral vaccines.

[0005] Currently, international rabies passive immunization preparations can be divided into four types: equine rabies immunoglobulin (ERIG), equine-derived purified F(ab')2 fragment products, human rabies immunoglobulin (HRIG), and recombinant monoclonal antibodies. The first two are customarily referred to as "ERA" in China. The products approved for marketing in China are equine-derived purified F(ab')2 fragment products, HRIG, and recombinant monoclonal antibodies. Currently, there are two recombinant anti-rabies virus monoclonal antibodies on the global market, namely SII RMab of the Serum Institute of India and ormutivimab injection of North China Pharmaceutical Group in China. Since each monoclonal antibody has single specificity, the WHO recommends using a cocktail containing at least two monoclonal antibodies that can bind to non-overlapping antigenic epitopes to limit the risk of failure due to lack of coverage of prevalent RABV strains or sudden viral escape.

[0006] In the prior art, a monoclonal antibody against rabies virus, a humanized antibody derived from mice, has been disclosed. However, compared with other antibodies, its dosage is relatively high, being 0.3 mg / kg (Chao, T.Y., et al., SYN023, a novel humanized monoclonal antibody cocktail, for post-exposure prophylaxis of rabies. PLoS Negl Trop Dis, 2017. 11(12): p.e0006133.).

[0007] Therefore, there is an urgent need to develop a human monoclonal antibody against rabies virus for therapeutic use, which has a lower dosage, high affinity, and strong neutralizing activity. SUMMARY OF THE INVENTION

[0008] The object of the present invention is to provide a combined monoclonal antibody preparation against rabies virus, which has a lower dosage, is a fully human antibody, has high affinity for rabies virus glycoprotein, and strong neutralizing activity.

[0009] To achieve the above object of the invention, the technical solution of the present invention is as follows:

[0010] On the one hand, the present invention provides a combined monoclonal antibody preparation against rabies virus, which contains R92 antibody and R71 antibody; the light chain sequence of the R92 antibody is as shown in SEQ ID NO: 1, the heavy chain sequence of the R92 antibody is as shown in SEQ ID NO: 2, the light chain sequence of the R71 antibody is as shown in SEQ ID NO: 3, and the heavy chain sequence of the R71 antibody is as shown in SEQ ID NO: 4.

[0011] Preferably, the ratio of R92 antibody to R71 antibody in the combined monoclonal antibody preparation against rabies virus is (1 - 5):(1 - 5).

[0012] More preferably, the ratio of R92 antibody to R71 antibody in the combined monoclonal antibody preparation against rabies virus is 1:1, 1:5 or 5:1.

[0013] Even more preferably, the ratio of R92 antibody to R71 antibody in the combined monoclonal antibody preparation against rabies virus is 1:1.

[0014] Preferably, the combined monoclonal antibody preparation against rabies virus further includes one or more of a surfactant, a stabilizer, an amino acid, and a chelating agent.

[0015] Furthermore, the surfactant includes non-ionic surfactants and ionic surfactants.

[0016] Preferably, the non-ionic surfactant is selected from one or more of polysorbate-80, polysorbate-20, polyethylene glycol octyl phenyl ether, nonylphenol polyoxyethylene ether, and fatty alcohol polyoxyethylene ether.

[0017] Preferably, the ionic surfactant is selected from one or more of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, and sodium lauroyl sarcosinate.

[0018] Furthermore, the surfactant is a non-ionic surfactant.

[0019] Still further, the non-ionic surfactant is polysorbate-80.

[0020] Furthermore, the stabilizer is selected from one or more of trehalose, m-cresol, zinc stearate, sodium octanoate, and β-cyclodextrin.

[0021] Still further, the stabilizer is trehalose.

[0022] Furthermore, the amino acid is selected from one or more of histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, and tryptophan.

[0023] Still further, the amino acid is histidine.

[0024] Furthermore, the chelating agent is selected from one or more of EDTA, EDTA-2Na, EDTA-Ca, and EDTA-2K.

[0025] Still further, the chelating agent is EDTA.

[0026] According to some embodiments of the present invention, the anti-rabies virus combined monoclonal antibody preparation may further include one or more of trehalose, histidine, polysorbate-80, and EDTA.

[0027] Preferably, the anti-rabies virus combined monoclonal antibody preparation further includes a buffer solution.

[0028] Furthermore, the buffer solution is selected from one or more of phosphate buffer solution, Tris buffer solution, MES buffer solution, HEPES buffer solution, citric acid-sodium citrate buffer solution, and acetic acid-sodium acetate buffer solution.

[0029] Still further, the buffer solution is phosphate buffer solution.

[0030] Preferably, the pH of the anti-rabies virus combined monoclonal antibody preparation is 5-8.

[0031] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of R92 antibody is 0.1-5 mg / ml, and the concentration of R71 antibody is 0.1-5 mg / ml.

[0032] Further, the concentration of R92 antibody is 2.5 mg / ml, and the concentration of R71 antibody is 2.5 mg / ml.

[0033] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of the buffer solution is 5-15 mmol / L; further, the concentration of the buffer solution is 10 mmol / L.

[0034] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of trehalose is 0-150 mg / ml.

[0035] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of histidine is 0-10 mg / ml.

[0036] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of polysorbate-80 is 0-1 mg / ml.

[0037] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of EDTA is 0-0.02 mg / ml.

[0038] The present invention also provides a preparation method of the above-mentioned anti-rabies virus combined monoclonal antibody preparation, comprising the following steps:

[0039] The anti-rabies virus combined monoclonal antibody preparation consists of two active ingredients, namely anti-rabies virus monoclonal antibody R92 antibody and anti-rabies virus monoclonal antibody R71 antibody. The preparation process of the preparation is to mix the R92 antibody stock solution and the R71 antibody stock solution in a certain proportion. Specifically as follows:

[0040] 1. Dilute the R92 antibody stock solution with the stock solution diluent to a protein concentration of 5 mg / ml;

[0041] 2. Dilute the R71 antibody stock solution with the stock solution diluent to a protein concentration of 5 mg / ml;

[0042] 3. Dilute the R92 antibody stock solution with the preparation diluent to a protein concentration of 1 mg / ml;

[0043] 4. Dilute the R71 antibody stock solution with the preparation diluent to a protein concentration of 1 mg / ml;

[0044] 5. Mix the R92 antibody with a target protein concentration of 1 mg / ml and the R71 antibody with a target protein concentration of 1 mg / ml according to the mass ratio and mix well.

[0045] According to some embodiments of the present invention, the anti-rabies virus combined monoclonal antibody preparation can be prepared by the following steps: calculate the amounts of the two stock solutions and the dilution doses required according to the final concentration and the protein concentrations of the R92 antibody and the R71 antibody, take them out and mix well.

[0046] On the other hand, the present invention also provides the application of the above-mentioned anti-rabies virus combined monoclonal antibody preparation in the preparation of products for neutralizing rabies mutant strains.

[0047] On the other hand, the present invention also provides the application of the above-mentioned anti-rabies virus combined monoclonal antibody preparation in the preparation of drugs for treating and / or preventing rabies virus.

[0048] Preferably, the drug may further include a pharmaceutically acceptable carrier.

[0049] More preferably, the pharmaceutically acceptable carrier is selected from one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, and preservatives.

[0050] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, and mannitol.

[0051] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0052] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglyceryl-3 polyricinoleate.

[0053] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitin ester.

[0054] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0055] Specifically, the binder is selected from at least one of ethanol, starch paste, syrup, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinylpyrrolidone.

[0056] Specifically, the preservative is selected from at least one of methyl paraben, propyl paraben, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethyl paraben.

[0057] Preferably, the dosage form of the drug is selected from any one of injections, tablets, capsules, oral liquid dosage forms, granules, ointments, suspensions, powders, emulsions, solutions, dripping pills, suppositories, and aerosols.

[0058] On the other hand, the present invention also provides the application of the above-mentioned anti-rabies virus combined monoclonal antibody preparation in the preparation of rabies virus detection reagents.

[0059] The beneficial effects of the present invention are as follows:

[0060] The anti-rabies virus combined monoclonal antibody preparation of the present invention is composed of two fully human monoclonal antibodies covering non-overlapping antigenic epitopes, showing good neutralization effects against different street strains in non-clinical trials, having a high affinity for rabies virus glycoprotein, which is 4.799E-10 M, and having good thermal stability. Compared with clinically developed products, the activity is 1500 IU / mg, and the expected dosage is 0.013 mg / kg. Description of the Drawings

[0061] Figure 1 It is a detection chromatogram of the purity of the antibody by SEC-HPLC.

[0062] Figure 2 It is a determination chromatogram of the affinity between the LZR antibody and the recombinant rabies virus glycoprotein.

[0063] Figure 3 It is the serum neutralizing antibody level when the recombinant human anti-rabies virus monoclonal antibody is used alone with the vaccine.

[0064] Figure 4 It is the epitope analysis of the recombinant human anti-rabies virus monoclonal antibody.

[0065] Figure 5 It is the epitope analysis of the recombinant human anti-rabies virus monoclonal antibody.

[0066] Figure 6 It is the neutralization effect of the R71 antibody on the R92E1-E4 escape virus.

[0067] Figure 7 It is the neutralization effect of the R71 antibody on the R92E5-E8 escape virus.

[0068] Figure 8 It is the neutralization effect of the C4 antibody, C7 antibody, and C8 antibody on the C4 antibody escape strain. Detailed implementation manners

[0069] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further clarified below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention, not all of them. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all fall within the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operation methods used are all conventional operation methods, and the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0070] The LZR antibodies described in the present invention include R92 antibody and R71 antibody.

[0071] Example 1

[0072] 1. Antibody

[0073] The light chain sequence of R92 antibody is shown in SEQ ID NO:1.

[0074] The heavy chain sequence of R92 antibody is shown in SEQ ID NO:2.

[0075] The light chain sequence of R71 antibody is shown in SEQ ID NO:3.

[0076] The heavy chain sequence of R71 antibody is shown in SEQ ID NO:4.

[0077] SEQ ID NO:1

[0078] DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYGASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQVNSYPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0079] SEQ ID NO:2

[0080] QVQLQQSGGGVVQPGGSLRLSCAASGFIFSNFGMHWVRQAPGKGLEWVAVISYEGRIPDYSDSVKGRFTISRDNSRNTLYMQLNRLTPEDTAMYYCAKIMTDGSGFDLWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0081] SEQ ID NO:3

[0082] QSVLTQPPSVSEAPRQRVTISCSGSSSNIGENAVNWYQQVPGKAPRLLIYSDDQLSSGISDRFSGSKSGTSASLAISGLLSEDEADYFCAAWDDSLDGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS。

[0083] SEQ ID NO:4

[0084] QVTLKESGPRVVKPSETLSLTCSVSGASTNSYYWHWIRQPPGKGLEWVGRMNYRGTPLYNPSLKSRVTISVDTSKNQLSLKVRSATAADTAMYYCARVDNWNFDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0085] 2. Mix the antibodies in the ratios of 1:5, 1:1, and 5:1 for R92 antibody and R71 antibody respectively, and detect the protein content and neutralizing activity.

[0086] The preparation process of the anti-rabies virus combined monoclonal antibody preparation is to mix the stock solutions of R92 antibody and R71 antibody in a certain ratio. Specifically as follows:

[0087] 1. Dilute the stock solution of R92 antibody with the stock solution diluent to a protein concentration of 5 mg / ml;

[0088] 2. Dilute the stock solution of R71 antibody with the stock solution diluent to a protein concentration of 5 mg / ml;

[0089] 3. Dilute the stock solution of R92 antibody with the preparation diluent to a protein concentration of 1 mg / ml;

[0090] 4. Dilute the stock solution of R71 antibody with the preparation diluent to a protein concentration of 1 mg / ml;

[0091] 5. Mix the R92 antibody with a target protein concentration of 1 mg / ml and the R71 antibody with a target protein concentration of 1 mg / ml by mass ratio and mix well.

[0092] The immunofluorescent focus inhibition assay (RFFIT) was used to determine the anti-rabies virus neutralizing activity.

[0093] 2.1 Determination of virus dilution for neutralization

[0094] (1) The rabies virus CVS-11 to be detected (from the Institute of Virology, Chinese Center for Disease Control and Prevention) was serially diluted 3-fold with DMEM medium containing 10% calf serum. 50 μL of the diluted virus was added to a 96-well plate, with 2 wells for each dilution as duplicates, and 50 μL of a BSR cell suspension at 1.0×10 6 cells / mL was added.

[0095] (2) 50 μL of medium was supplemented to each well for detection, and cultured at 37 °C with 5% CO2 for 24 h. The supernatant was discarded. The cells were washed once with PBS, and then fixed with 200 μL of pre-cooled 80% acetone for 10 min; the acetone was discarded, and the cells were left standing at room temperature for 15 min.

[0096] (3) The FITC-labeled anti-rabies virus nucleoprotein antibody (Beijing Consiltech Medical Research Center, catalog number: CAR100) was diluted 200-fold with PBS, and 100 μL was added to each well. Incubate at 37 °C for 1 h, discard the liquid, and wash 3 times with PBS.

[0097] (4) Observe the cell infection ratio at different dilutions under a fluorescence microscope. The virus dilution at which 80%-95% of the cells are infected by the virus is the virus dilution for neutralization.

[0098] 2.2 Determination of in vitro neutralization activity by RFFIT

[0099] The samples to be analyzed and the national standard of rabies immunoglobulin for human use (purchased from the National Institutes for Food and Drug Control) were serially diluted 1:3 starting from 10-fold, with a total of 8 dilutions; DMEM medium containing 10% newborn calf serum was used as the negative control.

[0100] (1) 50 μL of each of the samples to be detected, the standard product, and the negative control were added to a 96-well cell culture plate. After neutralizing with 50 μL of the virus for neutralization at 37 °C for 1 h, 50 μL of a BSR cell suspension at 1.0×10 6 cells / mL was added, and cultured at 37 °C with 5% CO2 for 24 h.

[0101] (2) The supernatant was discarded, the cells were washed once with PBS, and then fixed with 200 μL of pre-cooled 80% acetone at -30 °C for 10 min; the acetone was discarded, and the cells were left standing at room temperature for 15 min.

[0102] (3) The FITC-labeled anti-rabies virus nucleoprotein antibody was diluted 200-fold with PBS, and 100 μL was added to each well. Incubate at 37 °C for 1 h, discard the liquid, and wash 3 times with PBS.

[0103] (4) Observe and record the infection ratio of each well under a fluorescence microscope. Calculate the antibody activity according to the formula in the method for determining the titer of rabies immunoglobulin in the Pharmacopoeia of the People's Republic of China (Volume III).

[0104] The experimental results are shown in Table 1 below.

[0105] Table 1.

[0106]

[0107]

[0108] 3. Preparation components

[0109] Using DOE design, including a central design with a total of 19 combinations, differential scanning fluorimetry was used in the initial screening to investigate the melting temperature (TM) of R92 antibody and R71 antibody in different formulations respectively. The experimental design and test results are shown in the following table.

[0110] Table 2 Preliminary selection of solvent components of R92 and R71 antibody stock solutions

[0111] Component Range Trehalose 0 - 150 mg / ml Histidine 0 - 10 mg / ml Polysorbate - 80 0 - 1.0 mg / ml Calcium Disodium Edetate 0 - 0.02 mg / ml pH 5.0-8.0 Buffer 10 mmol / L Phosphate Buffer Concentration of R92 or R71 Antibody 2.5 mg / ml

[0112] Table 3 Experimental design and results of preliminary selection of solvent components of R92 and R71 antibody stock solutions

[0113]

[0114]

[0115] Screen the parameters that have a significant impact on the melting temperature and determine the preliminary parameter range. The experimental result model prediction shows that Tm is related to pH and trehalose concentration, but the Tm values of the products within the screening range are relatively high, indicating good product stability.

[0116] Example 2

[0117] Mix R92 antibody and R71 antibody in a ratio of 1:1 and proceed with the following detections.

[0118] 1. Antibody purity analysis

[0119] The main experimental instruments include a high-performance liquid chromatograph (manufacturer: Agilent, model: 1260), and the chromatographic column is a NanoChrom SEC analytical chromatographic column.

[0120] Prepare mobile phase A solution (51.3 mmol / L Na2HPO4, 48.7 mmol / L NaH2PO4, 150 mmol / L NaCl) for the experiment; dilute the reference substance and the test substance to 1 mg / ml with mobile phase A solution in a sample vial for sample injection; turn on the 1260 online workstation, connect the chromatographic column in the correct direction, and the chromatographic parameters are: injection volume 50 μl, detection wavelength 280 nm, column temperature 30 °C, flow rate 0.7 ml / min, upper pressure limit 120 bar, lower pressure limit 0 bar, analysis time 20 minutes, and isocratic elution is used; after the chromatographic column is balanced, run the sample injection sequence.

[0121] Data analysis: After the test is completed, enter the analysis software, open the chromatogram, call the analysis method, and automatically integrate to obtain the area of each peak.

[0122] The detection chromatogram is as Figure 1 shown. SEC-HPLC analysis shows that the monomer content of the antibody immunoglobulin is higher than 95%.

[0123] 2. Affinity determination

[0124] The equilibrium dissociation constant KD of the LZR antibody and rabies virus glycoprotein was detected by the capture method. The capture molecule Anti-His Antibody (manufacturer: Cytiva, product number: 28995056) was coated on the CM5 chip, the glycoprotein was captured as a ligand, and then the antibody to be tested was used as an analyte for determination.

[0125] (1) Dilute Anti-His Antibody to 1 - 10 μg / ml with sodium acetate and mix evenly.

[0126] (2) Program operation: Select the immobilization program, first activate the chip surface with a mixture of NHS / EDC, then couple Anti-His Antibody in the selected channel at a flow rate of 10 μl / min for 420 sec, and finally block the blank channel with ethanolamine.

[0127] (3) Dilute HBS-EP+ Buffer 10 times as the test substance diluent and running buffer, run the singlecycle knetics program. When capturing the ligand, first dilute the glycoprotein 1200 times with the running buffer at a flow rate of 10 μl / min for a capture time of 100 sec.

[0128] (4) Then, the test sample was serially diluted (5 concentration points), with the running buffer as the blank control. The analyte binding time was 120 sec, the dissociation time was 600 sec, the flow rate was 30 μl / min, the regeneration solution was glycine, the binding time was 30 sec, and the flow rate was 30 μl / min.

[0129] (5) Open the analysis software, perform equation fitting using the 1:1 Binding model, and automatically obtain the KD value.

[0130] The affinity map determined by Biacore is shown in Figure 2 As shown. After calculation, the affinity of the LZR antibody for the recombinant rabies virus glycoprotein was 4.799E-10 M.

[0131] 3. Protection against street strains

[0132] According to the above-mentioned rabies virus neutralizing activity analysis by the immunofluorescence focus inhibition method (RFFIT) in 1, different street strains isolated at different times and locations were detected.

[0133] The group of human-derived anti-rabies virus monoclonal antibodies showed very good protective effects against all 10 strains included in the study. The neutralizing antibody titers were between 2400 IU / ml - 8500 IU / ml (protein concentration 1.0 mg / ml). The results are shown in Table 4 below.

[0134] Table 4 Neutralizing activities of the LZR antibody against different strains

[0135]

[0136]

[0137] 4. Use in combination with vaccines

[0138] The interaction between the LZR antibody (referred to as "monoclonal antibody") and the rabies vaccine (referred to as "vaccine", produced by Changchun Institute of Biological Products) was evaluated using guinea pigs. The experimental design consisted of 3 parts, namely, using the vaccine alone, using rabies immunoglobulin for human (referred to as "HRIG", produced by Wuhan Institute of Biological Products Co., Ltd., China National Pharmaceutical Corporation) alone, and using the vaccine in combination with HRIG or the monoclonal antibody. The experimental groups are shown in Table 5, and 5 guinea pigs were injected in each group. The guinea pigs were provided by the Laboratory Animal Room of Lanzhou Institute of Biological Products.

[0139] Table 5 Grouping for the study of drug interactions

[0140]

[0141] Immunization, blood collection, and neutralizing antibody analysis:

[0142] The monoclonal antibody and rabies immunoglobulin (HRIG) were administered only once on day 0 by intramuscular injection into the right hind limb of guinea pigs, with the injection volume being 100 μl per animal. The vaccination schedule was on days 0, 3, 7, and 14 by intramuscular injection into the left hind limb of guinea pigs, 100 μl per animal per time; blood was collected on days 1, 3, 7, 14, and 28 after injection, and the rapid immunofluorescence inhibition test (RFFIT method) was used to detect the neutralizing antibody titer against rabies virus in the sera of guinea pigs.

[0143] Experimental results:

[0144] In guinea pigs, when the vaccine was used in combination with the monoclonal antibody or HRIG, rabies virus neutralizing antibody activity was detected on days 1 and 3 when 20 IU / kg, 100 IU / kg, and 500 IU / kg of the monoclonal antibody and 20 IU / kg of HRIG were used in combination with the vaccine; after 7 days of vaccination, all groups produced a relatively high concentration of rabies virus neutralizing antibodies; the vaccine group produced the highest level of neutralizing antibodies on day 7, and the antibody levels in the groups with HRIG or monoclonal antibody combined with the vaccine were slightly lower; on days 14 and 21, both the vaccine group and the groups with the vaccine combined with the monoclonal antibody or HRIG produced a high level of neutralizing antibodies, but the antibody level in the vaccine + 500 IU / kg monoclonal antibody group was lower than that of other groups, indicating that 500 IU / kg of the monoclonal antibody may inhibit the immune response induced by the vaccine. The results are shown in Figure 3 .

[0145] Example 3

[0146] To verify whether the R92 antibody and R71 antibody target different epitopes of the rabies virus glycoprotein, the present invention analyzed them using the surface plasmon resonance (SPR) technology for biomolecular interactions.

[0147] Experimental method:

[0148] First, the rabies virus glycoprotein was conjugated to a CM5 chip, and then the stock solutions of the R92 antibody (batch number: R7DS20211001) and R71 antibody (batch number: R8DS20211101) were appropriately diluted.

[0149] (1) The diluted R92 antibody was continuously injected 10 times until the response value no longer increased significantly, ensuring that the glycoprotein on the chip surface was saturated with the R71 antibody, and then the R71 antibody was injected, and the binding response value of the R71 antibody to the glycoprotein on the chip surface was recorded.

[0150] (2) The chip was regenerated to return the response to the baseline.

[0151] The diluted R71 antibody was continuously injected 10 times until the response value no longer increased significantly, ensuring that the glycoprotein on the chip surface was saturated with the R92 antibody, and then the R92 antibody was injected, and the binding response value of the R92 antibody to the glycoprotein on the chip surface was recorded.

[0152] Experimental results:

[0153] (1) As shown in Figure 4 . The first injection is the R92 antibody. It can be seen that it binds significantly to the glycoprotein on the chip (Binding1, ΔRU = 147.9). Without regenerating the chip, continuously injecting the R92 antibody, it can be observed that the increase in the response value decreases successively. Continuously inject until the glycoprotein on the chip surface is saturated with the R92 antibody, and then inject the R71 antibody. It can be observed that there is a significant binding (Binding11, ΔRU = 277.5).

[0154] (2) As shown in Figure 5 . Change the injection order, that is, the first injection is the R71 antibody. It can be seen that it binds significantly to the glycoprotein on the chip (Binding1, ΔRU = 297.6). Without regenerating the chip, continuously inject the R71 antibody. It can be observed that the response value decreases successively. Continuously inject until the glycoprotein on the chip surface is saturated with the R71 antibody, and then inject the R92 antibody. It can be observed that there is a significant binding (Binding14, ΔRU = 110.3).

[0155] The increase in the response value of the binding of R92 to the glycoprotein after R71 saturation (110.3 RU) is comparable to the response value of the binding of R92 alone to the glycoprotein (147.9 RU); the increase in the response value of the binding of R71 to the glycoprotein after R92 saturation (277.5 RU) is comparable to the response value of the binding of R71 alone to the glycoprotein (297.6 RU).

[0156] The response level of each antibody after the glycoprotein is saturated is slightly lower than the response level of single injection, which may be caused by factors such as steric hindrance formed after the binding of the previous antibody, in line with theoretical judgment. This result indicates that the binding of the two antibodies to the glycoprotein is independent of each other, there is basically no competition, and it also shows that the epitopes of these two monoclonal antibodies do not overlap. The specific data are shown in Table 6.

[0157] Table 6 Summary of epitope analysis data of recombinant human anti-rabies virus monoclonal antibodies

[0158]

[0159] The SRP competitive binding results show that the two antibodies bind to different epitopes of the rabies virus glycoprotein, and they basically do not interfere with each other, which can further reduce the impact of virus mutation on neutralizing activity.

[0160] Example 4

[0161] Eight CVS-11 mutant viruses were obtained with the R92 antibody, and they were respectively named R92 E1 - E8.

[0162] The preparation method of the mutant virus comprises the following steps:

[0163] The CVS-11 strain virus is serially diluted 10-fold, and 50 μl of the virus at a dilution of 10 7 -10 4 FFU / ml is taken and added with 50 μl of anti-rabies virus monoclonal antibody at 4 IU / ml, and incubated at 37 °C for 1 h. 50 μl of BSR cell suspension at 1.0×10 6 cells / ml is added, and cultured at 5% CO2 and 37 °C for 4 h. The culture medium is discarded, 150 μl of the culture medium containing 2 IU / ml monoclonal antibody is added, and the culture is continued for 72 h. The virus culture supernatant is harvested. 50 μl of the culture supernatant is taken and added with 50 μl of BSR cell suspension at 1.0×10 6 cells / ml and 50 μl of the culture medium, and cultured at 5% CO2 and 37 °C for 24 h. After the supernatant is discarded, the cells are washed once with PBS, and fixed with 200 μl of precooled 80% acetone for 15 min; the acetone is discarded and air-dried at room temperature for 30 min. The FITC-labeled anti-rabies virus nucleoprotein antibody is diluted 200-fold with PBS, 100 μl is added to each well, incubated at 37 °C for 1 h, the liquid is discarded, and washed 3 times with PBS. The infection ratio of cells at different dilutions is observed under a fluorescence microscope. The wells with an infection dose between 10% and 50% are selected for subsequent experiments.

[0164] The virus culture supernatant is taken, neutralized with the antibody at 4 IU / ml at a volume ratio of 1:1, and used to infect BSR cells. The cells are cultured at 5% CO2 and 37 °C for 4 h. The culture medium is discarded, and the culture medium containing 2 IU / ml monoclonal antibody is added, and the culture is continued for 72 h. This step is repeated 3 times to obtain the P4 generation escape virus.

[0165] 50 μl of the P4 generation escape virus is taken and serially diluted 2-fold up to 128-fold. 50 μl of BSR cell suspension at 1.0×10 6 cells / ml is added, and cultured with 50 μl of the culture medium at 5% CO2 and 37 °C for 24 h. After the supernatant is discarded, the cells are washed once with PBS, and fixed with 200 μl of precooled 80% acetone for 10 min; the acetone is discarded and left standing at room temperature for 15 min. The FITC-labeled anti-rabies virus nucleoprotein antibody is diluted 200-fold with PBS, 100 μl is added to each well, incubated at 37 °C for 1 h, the liquid is discarded, and washed 3 times with PBS. The infection ratio of cells at different dilutions is observed under a fluorescence microscope.

[0166] The amino acid sequence of R92 E1 is shown in SEQ ID NO:5; the amino acid sequence of R92 E2 is shown in SEQ ID NO:6; the amino acid sequence of R92 E3 is shown in SEQ ID NO:7; the amino acid sequence of R92 E4 is shown in SEQ ID NO:8; the amino acid sequence of R92 E5 is shown in SEQ ID NO:9; the amino acid sequence of R92 E6 is shown in SEQ ID NO:10; the amino acid sequence of R92 E7 is shown in SEQ ID NO:11; the amino acid sequence of R92 E8 is shown in SEQ ID NO:12.

[0167] Calibrate R92 E1-E8. Mix the mutant viruses with 80%-100% cell infection dose with culture medium, 2 IU / ml R92 antibody and 2 IU / ml R71 antibody respectively. After neutralization for 1 h, add BSR cells, and maintain the types and concentrations of antibodies in the culture system the same as those in the neutralization process. After culturing for 24 h, fix with acetone and detect the cell infection situation using fluorescein-labeled anti-rabies virus nucleoprotein antibody.

[0168] The results are shown in Figure 6 - Figure 7 , in the presence of 2 IU / ml R92 antibody at the final concentration, the replication of R92 E1-E8 viruses is not affected, and their infection levels are comparable to the virus control without antibody. R92 antibody cannot neutralize these mutant viruses. In the presence of 2 IU / ml R71 antibody at the final concentration, no cells were observed to be infected after culturing for 24 h. R71 antibody completely neutralizes these mutant viruses. This result indicates that R71 antibody can effectively neutralize the mutant viruses that escape R92 antibody.

[0169] Since a stable mutant virus could not be obtained for the R71 antibody, the following method was selected to conduct cross-neutralization research. During the early candidate antibody screening process, a cross-neutralization test was performed using the mutant virus of the C4 antibody with the same heavy-chain variable region as the R71 antibody. Three antibodies were transiently expressed in the test and named C4, C7, and C8 (the amino acid sequence of C4 is shown in SEQ ID NO:13, the amino acid sequence of C7 is shown in SEQ ID NO:14, and the amino acid sequence of C8 is shown in SEQ ID NO:15). Among them, the variable region sequences of the C7 and C8 antibodies are the same as those of the R92 antibody and the R71 antibody, respectively. The heavy-chain variable region sequences of the C4 and C8 antibodies are the same and form a group. The mutant virus of the C4 antibody at 80%-100% cell infection dose (the virus can be stably amplified in the culture system in the presence of 4 IU / ml C4 antibody) was added to C4, C7, and C8 antibodies at a final concentration of 2 IU / ml. After neutralization for 1 h, BSR cells were added, and the antibody types and concentrations in the culture system were maintained the same as those during the neutralization process. After culturing for 24 h, the cells were fixed with acetone, and the cell infection was detected using a fluorescein-labeled anti-rabies virus nucleoprotein antibody. The results are shown in Figure 8 . The C4 antibody could not neutralize the mutant escape virus at all, and the C8 antibody with the same heavy chain as the C4 antibody could not effectively neutralize the mutant escape virus of the C4 antibody either, but the C8 antibody had a certain inhibitory effect on the mutant escape virus of the C4 antibody. The C7 antibody completely neutralized the mutant escape virus of the C4 antibody. The results showed that the C7 antibody (with the same variable region as the R92 antibody) could completely neutralize the mutant escape virus that the C8 antibody (with the same variable region as the R71 antibody) could not effectively neutralize.

[0170] In summary, the R92 antibody and the R71 antibody can effectively neutralize the mutant viruses that the other cannot neutralize, and they have a clear cross-protective effect.

[0171] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A combined monoclonal antibody preparation against rabies virus, characterized in that, The anti-rabies virus combined monoclonal antibody preparation contains R92 antibody and R71 antibody; the light chain sequence of the R92 antibody is as shown in SEQ ID NO:1, the heavy chain sequence of the R92 antibody is as shown in SEQ ID NO:2, the light chain sequence of the R71 antibody is as shown in SEQ ID NO:3, the heavy chain sequence of the R71 antibody is as shown in SEQ ID NO:4, and the ratio of R92 antibody to R71 antibody in the anti-rabies virus combined monoclonal antibody preparation is (1-5):(1-5), and the concentration of R92 antibody is 0.1-5 mg / ml, and the concentration of R71 antibody is 0.1-5 mg / ml.

2. The anti-rabies virus combined monoclonal antibody preparation according to claim 1, characterized in that, The ratio of R92 antibody to R71 antibody in the anti-rabies virus combined monoclonal antibody preparation is 1:1, 1:5 or 5:

1.

3. The anti-rabies virus combined monoclonal antibody preparation according to claim 2, characterized in that, The ratio of R92 antibody to R71 antibody in the anti-rabies virus combined monoclonal antibody preparation is 1:

1.

4. The anti-rabies virus combined monoclonal antibody preparation according to claim 1, characterized in that, The concentration of R92 antibody is 2.5 mg / ml, and the concentration of R71 antibody is 2.5 mg / ml.

5. The anti-rabies virus combined monoclonal antibody preparation according to claim 1, characterized in that The anti-rabies virus combined monoclonal antibody preparation further comprises one or more of a surfactant, a stabilizer, an amino acid, and a chelating agent.

6. The anti-rabies virus combined monoclonal antibody preparation according to claim 5, characterized in that, The surfactant is a non-ionic surfactant, and the non-ionic surfactant is selected from one or more of polysorbate-80, polysorbate-20, polyoxyethylene octylphenyl ether, nonylphenol polyoxyethylene ether, and fatty alcohol polyoxyethylene ether.

7. The anti-rabies virus combined monoclonal antibody preparation according to claim 5, characterized in that, The stabilizer is selected from one or more of trehalose, m-cresol, zinc stearate, sodium octanoate, and β-cyclodextrin.

8. The anti-rabies virus combined monoclonal antibody preparation according to claim 5, characterized in that The amino acid is selected from one or more of histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, and tryptophan.

9. The anti-rabies virus combined monoclonal antibody preparation according to claim 5, characterized in that The surfactant is polysorbate-80, the stabilizer is trehalose, the amino acid is histidine, and the chelating agent is EDTA.

10. The anti-rabies virus combined monoclonal antibody preparation according to any one of claims 1-9, characterized in that, The pH of the anti-rabies virus combined monoclonal antibody preparation is 5-8.

11. Use of the anti-rabies virus combined monoclonal antibody preparation according to any one of claims 1-10 in the preparation of a product for neutralizing rabies mutant strains.

12. Use of the anti-rabies virus combined monoclonal antibody preparation according to any one of claims 1-10 in the preparation of a drug for treating rabies virus.

13. Use of the anti-rabies virus combined monoclonal antibody preparation according to any one of claims 1-10 in the preparation of a rabies virus detection reagent.

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