Anti-rabies virus combined monoclonal antibody preparation

Abstract

Provided is an anti-rabies virus combined monoclonal antibody preparation. The present invention relates to the technical field of biomedicine. The anti-rabies virus combined monoclonal antibody preparation comprises an R92 antibody and an R71 antibody. A light chain sequence of the R92 antibody is set forth in SEQ ID NO: 1, and a heavy chain sequence of the R92 antibody is set forth in SEQ ID NO: 2. A light chain sequence of the R71 antibody is set forth in SEQ ID NO: 3, and a heavy chain sequence of the R71 antibody is set forth in SEQ ID NO: 4. The ratio of the R92 antibody to the R71 antibody in the anti-rabies virus combined monoclonal antibody preparation is (1-5):(1-5). The anti-rabies virus combined monoclonal antibody preparation has a high affinity for the rabies virus glycoprotein, has relatively good neutralizing capacity and thermal stability, and has very good neutralizing capacity against a variety of street strains. Since the two antibodies target different epitopes, the preparation can provide broader protection.

Description

A combined monoclonal antibody preparation against rabies virus Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an anti-rabies virus combined monoclonal antibody preparation. Background Art

[0002] Rabies virus (RABV) is a single-stranded negative-sense RNA virus of the Rhabdoviridae family and the Lyssavirus genus. It is rod-shaped or bullet-shaped under a transmission electron microscope, with a diameter of about 75nm and a length of about 100-300nm. RABV is neurotropic and invades the central nervous system of the body, causing fatal neuroinfection.

[0003] The RABV genome is approximately 12 knt long and encodes five structural proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), envelope glycoprotein (G), and large polymerase protein (L). The coding sequence from the 3' to 5' end of the genome is 3'-NPMGL-5'. The glycoprotein is 65-67 kDa in size and is linked to the M protein within the RABV genome. It is the only exposed protein inserted into the viral lipid envelope and the sole ligand for cellular receptors. In its natural state, the G protein forms a trimer on the viral surface and is the primary surface antigen bound by neutralizing antibodies. The G protein is composed of three domains: the extramembrane domain, the transmembrane domain, and the intramembrane domain.

[0004] Rabies is a global zoonotic disease caused by the rabies virus. Those exposed to rabies, especially those with severe exposure, should be fully vaccinated with rabies vaccine and anti-rabies antibodies. Currently, rabies vaccines include new inactivated rabies vaccines, attenuated rabies vaccines, nucleic acid vaccines, subunit vaccines, virus-like particle vaccines, and oral vaccines.

[0005] Currently, passive rabies immunization agents available internationally are categorized into four types: equine immune globulin (ERIG), equine purified F(ab')2 fragments, human rabies immunoglobulin (HRIG), and recombinant monoclonal antibodies. The first two are customarily referred to in China as "ERA." Approved for marketing in my country are equine purified F(ab')2 fragments, HRIG, and recombinant monoclonal antibodies. Currently, two recombinant anti-rabies virus monoclonal antibodies are marketed globally: SII RMab from the Serum Institute of India and ormutivimab injection from North my country Pharmaceutical Group. Because each monoclonal antibody has a single specificity, the WHO recommends using a cocktail containing at least two monoclonal antibodies that bind to non-overlapping epitopes to limit the risk of failure due to lack of coverage against circulating RABV strains or unexpected viral escape.

[0006] The prior art discloses an anti-rabies virus monoclonal antibody, which is a humanized antibody derived from mice. However, compared with other antibodies, its dosage is higher, at 0.3 mg / kg (Chao, TY, 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 therapeutic human anti-rabies virus monoclonal antibodies that have a low dosage, high affinity, and strong neutralizing activity.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide an anti-rabies virus combined monoclonal antibody preparation, which has a low dosage, is a fully human antibody, has a high affinity for rabies virus glycoprotein, and has a strong neutralizing activity.

[0010] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0011] On the one hand, the present invention provides an anti-rabies virus combination monoclonal antibody preparation, which contains R92 antibody and R71 antibody; the light chain sequence of the R92 antibody is shown in SEQ ID NO: 1, the heavy chain sequence of the R92 antibody is shown in SEQ ID NO: 2, the light chain sequence of the R71 antibody is shown in SEQ ID NO: 3, and the heavy chain sequence of the R71 antibody is shown in SEQ ID NO: 4.

[0012] Preferably, the ratio of R92 antibody to R71 antibody in the anti-rabies virus combined monoclonal antibody preparation is (1-5): (1-5).

[0013] Further preferably, the ratio of R92 antibody to R71 antibody in the anti-rabies virus combination monoclonal antibody preparation is 1:1, 1:5 or 5:1.

[0014] Further preferably, the ratio of R92 antibody to R71 antibody in the anti-rabies virus combined monoclonal antibody preparation is 1:1.

[0015] Preferably, the anti-rabies virus combination monoclonal antibody preparation further comprises one or more of a surfactant, a stabilizer, an amino acid, and a chelating agent.

[0016] Furthermore, the surfactant includes a nonionic surfactant and an ionic surfactant.

[0017] Preferably, the nonionic surfactant is selected from one or more of polysorbate 80, polysorbate 20, polyethylene glycol octylphenyl ether, nonylphenol polyoxyethylene ether, and fatty alcohol polyoxyethylene ether.

[0018] Preferably, the ionic surfactant is selected from one or more of sodium lauryl sulfate, sodium dodecylbenzenesulfonate, and sodium lauryl sarcosinate.

[0019] Furthermore, the surfactant is a nonionic surfactant.

[0020] Furthermore, the nonionic surfactant is polysorbate 80.

[0021] Furthermore, the stabilizer is selected from one or more of trehalose, m-cresol, zinc stearate, sodium octanoate, and β-cyclodextrin.

[0022] Furthermore, the stabilizer is trehalose.

[0023] Furthermore, the amino acid is selected from one or more of histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, and tryptophan.

[0024] Furthermore, the amino acid is histidine.

[0025] Furthermore, the chelating agent is selected from one or more of EDTA, EDTA-2Na, EDTA-Ca, and EDTA-2K.

[0026] Furthermore, the chelating agent is EDTA.

[0027] According to some embodiments of the present invention, the anti-rabies virus combination monoclonal antibody preparation may further include one or more of trehalose, histidine, polysorbate 80, and EDTA.

[0028] Preferably, the anti-rabies virus combined monoclonal antibody preparation further comprises a buffer.

[0029] Furthermore, the buffer is selected from one or more of phosphate buffer, Tris buffer, MES buffer, HEPES buffer, citric acid-sodium citrate buffer, and acetic acid-sodium acetate buffer.

[0030] Furthermore, the buffer solution is a phosphate buffer solution.

[0031] Preferably, the pH of the anti-rabies virus combined monoclonal antibody preparation is 5-8.

[0032] 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.

[0033] Furthermore, the concentration of the R92 antibody is 2.5 mg / ml, and the concentration of the R71 antibody is 2.5 mg / ml.

[0034] 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.

[0035] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of trehalose is 0-150 mg / ml.

[0036] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of histidine is 0-10 mg / ml.

[0037] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of polysorbate 80 is 0-1 mg / ml.

[0038] Preferably, in the anti-rabies virus combined monoclonal antibody preparation, the concentration of EDTA is 0-0.02 mg / ml.

[0039] The present invention also provides a method for preparing the above-mentioned anti-rabies virus combined monoclonal antibody preparation, comprising the following steps:

[0040] The anti-rabies virus combined monoclonal antibody preparation consists of two active ingredients, namely the anti-rabies virus monoclonal antibody R92 antibody and the anti-rabies virus monoclonal antibody R71 antibody. The preparation process is to mix the R92 antibody stock solution and the R71 antibody stock solution in a certain proportion. The details are as follows:

[0041] 1. Dilute the R92 antibody stock solution with stock solution diluent to a protein concentration of 5 mg / ml;

[0042] 2. Dilute the R71 antibody stock solution with stock solution diluent to a protein concentration of 5 mg / ml;

[0043] 3. Dilute the R92 antibody stock solution with formulation diluent to a protein concentration of 1 mg / ml;

[0044] 4. Dilute the R71 antibody stock solution with formulation diluent to a protein concentration of 1 mg / ml;

[0045] 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.

[0046] According to some embodiments of the present invention, the anti-rabies virus combination monoclonal antibody preparation can be prepared by the following steps: calculating the required amount and dilution of the two stock solutions according to the final concentration and protein concentration of R92 antibody and R71 antibody, taking out and mixing.

[0047] In another aspect, the present invention also provides the use of the above-mentioned anti-rabies virus combined monoclonal antibody preparation in the preparation of a product for neutralizing rabies mutant strains.

[0048] In yet another aspect, the present invention further provides use of the above-mentioned anti-rabies virus combination monoclonal antibody preparation in the preparation of a drug for treating and / or preventing rabies virus.

[0049] Preferably, the drug may further include a pharmaceutically acceptable carrier.

[0050] Further preferably, the pharmaceutically acceptable carrier is selected from one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, and preservatives.

[0051] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, and mannitol.

[0052] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0053] 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.

[0054] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan.

[0055] 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.

[0056] Specifically, the binder is selected from at least one of ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinyl pyrrolidone.

[0057] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric cyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0058] Preferably, the dosage form of the drug is selected from any one of injection, tablet, capsule, oral liquid dosage form, granule, ointment, suspension, powder, emulsion, solution, pill, suppository, and aerosol.

[0059] In yet another aspect, the present invention further provides use of the above-mentioned anti-rabies virus combined monoclonal antibody preparation in the preparation of a rabies virus detection reagent.

[0060] The beneficial effects of the present invention are:

[0061] The anti-rabies virus combination monoclonal antibody preparation of the present invention is composed of two fully human monoclonal antibodies covering non-overlapping antigenic epitopes. It has shown good neutralization effect against different street virus strains in non-clinical trials, has a high affinity for rabies virus glycoprotein of 4.799E-10M, and has good thermal stability. Compared with the product in clinical development, it has an activity of 1500IU / mg and an estimated dosage of 0.013mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is a graph showing the purity of the antibody detected by SEC-HPLC.

[0063] Figure 2 is a graph showing the affinity determination between LZR antibody and recombinant rabies virus glycoprotein.

[0064] Figure 3 shows the serum neutralizing antibody levels when the recombinant human anti-rabies virus monoclonal antibody and the vaccine are used alone.

[0065] Figure 4 shows the epitope analysis of the recombinant human anti-rabies virus monoclonal antibody.

[0066] Figure 5 shows the epitope analysis of the recombinant human anti-rabies virus monoclonal antibody.

[0067] Figure 6 shows the neutralizing effect of R71 antibody on R92E1-E4 escape virus.

[0068] Figure 7 shows the neutralizing effect of R71 antibody on R92E5-E8 escape virus.

[0069] FIG8 shows the neutralizing effects of C4 antibody, C7 antibody, and C8 antibody on C4 antibody escape strains. DETAILED DESCRIPTION

[0070] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0071] The LZR antibodies described in the present invention include R92 antibodies and R71 antibodies.

[0072] Example 1

[0073] 1. Antibodies

[0074] The light chain sequence of the R92 antibody is shown in SEQ ID NO: 1.

[0075] The heavy chain sequence of the R92 antibody is shown in SEQ ID NO: 2.

[0076] The light chain sequence of the R71 antibody is shown in SEQ ID NO: 3.

[0077] The heavy chain sequence of the R71 antibody is shown in SEQ ID NO: 4.

[0078] SEQ ID NO: 1

[0079] SEQ ID NO:2

[0080] SEQ ID NO:3

[0081] SEQ ID NO:4

[0082] 2. R92 antibody and R71 antibody were mixed at a ratio of 1:5, 1:1, and 5:1, respectively, and the protein content and neutralizing activity were tested.

[0083] The preparation process of the anti-rabies virus combined monoclonal antibody preparation is to mix the R92 antibody stock solution and the R71 antibody stock solution in a certain proportion. The details are as follows:

[0084] 1. Dilute the R92 antibody stock solution with stock solution diluent to a protein concentration of 5 mg / ml;

[0085] 2. Dilute the R71 antibody stock solution with stock solution diluent to a protein concentration of 5 mg / ml;

[0086] 3. Dilute the R92 antibody stock solution with formulation diluent to a protein concentration of 1 mg / ml;

[0087] 4. Dilute the R71 antibody stock solution with formulation diluent to a protein concentration of 1 mg / ml;

[0088] 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.

[0089] The anti-rabies virus neutralization activity was determined using the fluorescent focus inhibition immunoassay (RFFIT).

[0090] 2.1 Neutralization Virus Dilution Determination

[0091] (1) The rabies virus CVS-11 to be tested (from the Institute of Virology, Chinese Center for Disease Control) was diluted three-fold using DMEM medium containing 10% calf serum. 50 μL of the diluted virus was added to a 96-well plate, with two wells of each dilution as parallels. 1.0×10 6 50 μL of BSR cell suspension with a concentration of 1 cell / mL.

[0092] (2) Add 50 μL of culture medium to each well and incubate at 37°C for 24 h in a 5% CO2 atmosphere. Discard the supernatant. Wash the cells once with PBS and fix them with 200 μL of pre-chilled 80% acetone for 10 min. Discard the acetone and let them stand at room temperature for 15 min.

[0093] (3) FITC-labeled anti-rabies virus nucleoprotein antibody (Beijing Kangsiertai Medical Research Center, catalog number: CAR100) was diluted 200 times with PBS, 100 μL was added to each well, incubated at 37°C for 1 h, the liquid was discarded, and the cells were washed three times with PBS.

[0094] (4) Observe the infection ratio of cells at different dilutions under a fluorescence microscope. The virus dilution at which 80%-95% of cells are infected is the virus dilution used for neutralization.

[0095] 2.2 RFFIT determination of in vitro neutralization activity

[0096] The samples to be analyzed and the national standard of rabies immunoglobulin (purchased from the China Food and Drug Inspection Institute) were diluted 1:3 starting from 10 times, for a total of 8 dilutions; DMEM medium containing 10% newborn calf serum was used as a negative control.

[0097] (1) Take 50 μL of each of the sample to be tested, standard and negative control and add them to a 96-well cell culture plate. Add 50 μL of neutralizing virus to each well and neutralize at 37°C for 1 hour. Then add 1.0×10 6 50 μL of BSR cell suspension with a concentration of 100 cells / mL was cultured in 5% CO2 at 37°C for 24 h.

[0098] (2) Discard the supernatant, wash the cells once with PBS, add 200 μL of pre-cooled 80% acetone at -30°C and fix for 10 min; discard the acetone and let it stand at room temperature for 15 min.

[0099] (3) FITC-labeled anti-rabies virus nucleoprotein antibody was diluted 200-fold with PBS, 100 μL was added to each well, incubated at 37°C for 1 h, the liquid was discarded, and the wells were washed three times with PBS.

[0100] (4) Observe and record the infection rate of each well under a fluorescence microscope. Calculate the antibody activity according to the formula for the rabies immunoglobulin titer assay in the Pharmacopoeia of the People's Republic of China (Volume III).

[0101] The experimental results are shown in Table 1 below.

[0102] Table 1.

[0103] 3. Preparation ingredients

[0104] A DOE design, including a central design, totaling 19 combinations was used. Initial screening used differential scanning fluorimetry to investigate the melting temperatures (TM) of the R92 and R71 antibodies in different formulations. The experimental design and test results are shown in the following table.

[0105] Table 2 Preliminary selection of solvent components for R92 and R71 antibody stock solutions

[0106] Table 3 Preliminary experimental design and results for the selection of solvent components for R92 and R71 antibody stock solutions

[0107] Parameters with a significant impact on melting temperature were screened and a preliminary parameter range was determined. The experimental results showed that the Tm was related to pH and trehalose concentration, but the Tm values ​​of the products within the screening range were relatively high, indicating good product stability.

[0108] Example 2

[0109] Mix the R92 antibody and the R71 antibody at a ratio of 1:1 and perform the following test.

[0110] 1. Antibody purity analysis

[0111] The main instruments used in the experiment are high performance liquid chromatography (manufacturer: Agilent, model: 1260), and the chromatographic column is a nano-SEC analytical column.

[0112] Prepare the mobile phase A solution (51.3mmol / L Na2HPO4, 48.7mmol / L NaH2PO4, 150mmol / L NaCl) used in the experiment; dilute the reference sample and the test sample to 1mg / ml with mobile phase A solution and prepare them in a sample vial for loading; turn on the 1260 online workstation and connect the chromatographic column in the correct direction. The chromatographic parameters are: injection volume 50μl, detection wavelength 280nm, column temperature 30℃, flow rate 0.7ml / min, upper pressure limit 120bar, lower pressure limit 0bar, analysis time 20 minutes, and isocratic elution; after the chromatographic column is equilibrated, run the loading sequence.

[0113] 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.

[0114] The detection spectrum is shown in FIG1 . SEC-HPLC analysis shows that the content of antibody immunoglobulin monomers is higher than 95%.

[0115] 2. Affinity determination

[0116] The equilibrium dissociation constant (KD) between LZR antibody and rabies virus glycoprotein was determined using a capture assay. Anti-His Antibody (Cytiva, Cat. No. 28995056) was coated on a CM5 chip, capturing the glycoprotein as a ligand and measuring the antibody as the analyte.

[0117] (1) Dilute the Anti-His Antibody to 1-10 μg / ml with sodium acetate and mix well.

[0118] (2) Program operation: Select the immobilization program, first activate the chip surface with a mixture of NHS / EDC, then couple the Anti-His Antibody to the selected channel at a flow rate of 10 μl / min for 420 sec, and finally block the blank channel with ethanolamine.

[0119] (3) HBS-EP+Buffer was diluted 10-fold as the test sample diluent and running buffer, and the single cycle knetics program was run. When capturing the ligand, the glycoprotein was first diluted 1200-fold with running buffer, the flow rate was 10 μl / min, and the capture time was 100 sec.

[0120] (4) The test sample was then diluted in a gradient (5 concentration points), with running buffer as a blank control. The analyte binding time was 120 sec, the dissociation time was 600 sec, and the flow rate was 30 μl / min. The regeneration solution was glycine, with a binding time of 30 sec and a flow rate of 30 μl / min.

[0121] (5) Open the analysis software, use the 1:1 binding model to fit the equation, and automatically obtain the KD value.

[0122] The affinity map determined by Biacore is shown in Figure 2. After calculation, the affinity of LZR antibody to recombinant rabies virus glycoprotein is 4.799E-10M.

[0123] 3. Protection against street strains

[0124] According to the above-mentioned 1st step, the anti-rabies virus neutralization activity analysis was performed on street virus strains isolated at different times and spaces.

[0125] The human anti-rabies virus monoclonal antibodies produced very good protection against all 10 strains included in the study, with neutralizing antibody titers ranging from 2400 IU / ml to 8500 IU / ml (protein concentration 1.0 mg / ml). The results are shown in Table 4 below.

[0126] Table 4 Neutralizing activity of LZR antibodies against different strains

[0127] 4. Used in combination with vaccines

[0128] The interaction between LZR antibody (abbreviated as "mAb") and rabies vaccine (abbreviated as "vaccine," produced by the Changchun Institute of Biological Products) was evaluated in guinea pigs. The study design consisted of three parts: vaccine alone, rabies human immunoglobulin (abbreviated as "HRIG," produced by Sinopharm Wuhan Institute of Biological Products), and vaccine combined with HRIG or mAb. The experimental groups are shown in Table 5, with five guinea pigs injected into each group. Guinea pigs were provided by the Laboratory Animal Laboratory of the Lanzhou Institute of Biological Products.

[0129] Table 5 Drug interaction study groups

[0130] Immunization, blood collection, and neutralizing antibody analysis:

[0131] Monoclonal antibodies and rabies human immune globulin (HRIG) were administered only once on day 0, intramuscularly into the right hind limb of guinea pigs, with an injection volume of 100 μl per animal. Guinea pigs were vaccinated on days 0, 3, 7, and 14, intramuscularly into the left hind limb at a dose of 100 μl per animal. Blood was collected on days 1, 3, 7, 14, and 28 after injection, and anti-rabies virus neutralizing antibody titers in guinea pig serum were measured using the rapid fluorescence inhibition immunoassay (RFFIT).

[0132] Experimental results:

[0133] In guinea pigs, when the vaccine was combined with a monoclonal antibody or HRIG, rabies virus-neutralizing antibody activity was detected on days 1 and 3 at 20 IU / kg, 100 IU / kg, and 500 IU / kg of monoclonal antibody, as well as at 20 IU / kg of HIRG combined with the vaccine. Seven days after vaccination, all groups produced high concentrations of rabies virus-neutralizing antibodies. The vaccine group produced the highest level of neutralizing antibodies on day 7, while the antibody levels in the HRIG or monoclonal antibody combined with the vaccine group were slightly lower. On days 14 and 21, the vaccine group and the vaccine combined with the monoclonal antibody or HRIG group all produced high levels of neutralizing antibodies, but the antibody levels in the vaccine + 500 IU / kg monoclonal antibody group were lower than those in the other groups, suggesting that the 500 IU / kg monoclonal antibody may have inhibited the vaccine-induced immune response. The results are shown in Figure 3.

[0134] Example 3

[0135] In order to verify whether the R92 antibody and the R71 antibody are directed against different epitopes of the rabies virus glycoprotein, the present invention used the biological macromolecular interaction (SPR) technology to analyze them.

[0136] Experimental methods:

[0137] First, the rabies virus glycoprotein was coupled to the CM5 chip, and then the R92 antibody stock solution (batch number: R7DS20211001) and the R71 antibody stock solution (batch number: R8DS20211101) were appropriately diluted.

[0138] (1) Inject the diluted R92 antibody 10 times continuously until the response value no longer increases significantly, ensuring that the glycoprotein on the chip surface is saturated with the R71 antibody. Then inject the R71 antibody and record the response value of the R71 antibody binding to the glycoprotein on the chip surface.

[0139] (2) Regenerate the chip to return the response to baseline.

[0140] The diluted R71 antibody was injected continuously for 10 times until the response value no longer increased significantly to ensure that the glycoprotein on the chip surface was saturated with the R92 antibody. Then the R71 antibody was injected and the response value of the R92 antibody binding to the glycoprotein on the chip surface was recorded.

[0141] Experimental results:

[0142] (1) As shown in Figure 4. The first injection was the R92 antibody, which was found to bind significantly to the glycoprotein on the chip (Binding 1, ΔRU = 147.9). Continuous injection of the R92 antibody without chip regeneration showed an increase in the response value followed by a decrease. Continuous injection was performed until the glycoprotein on the chip surface was saturated with the R92 antibody. Then, the R71 antibody was injected, and significant binding was observed (Binding 11, ΔRU = 277.5).

[0143] (2) As shown in Figure 5. Changing the injection order, that is, the first injection is R71 antibody, it can be seen that it binds significantly to the glycoprotein on the chip (Binding 1, ΔRU = 297.6). Without chip regeneration, R71 antibody is continuously injected, and the response values ​​can be observed to decrease successively. The injection is continued until the glycoprotein on the chip surface is saturated with R71 antibody, and then R92 antibody is injected, and significant binding can be observed (Binding 14, ΔRU = 110.3).

[0144] After R71 saturation, the increase in the response value of R92 binding to glycoprotein (110.3RU) was equivalent to the response value of R92 binding to glycoprotein alone (147.9RU); after R92 saturation, the increase in the response value of R71 binding to glycoprotein (277.5RU) was equivalent to the response value of R71 binding to glycoprotein alone (297.6RU).

[0145] After saturation of the glycoprotein, the response levels of each antibody were slightly lower than those observed when injected alone. This is likely due to factors such as steric hindrance after the binding of the preceding antibody, which is consistent with theoretical assumptions. This result indicates that the binding of the two antibodies to the glycoprotein is independent, with little competition. It also suggests that the epitopes of the two monoclonal antibodies do not overlap. Specific data are shown in Table 6.

[0146] Table 6 Summary of epitope analysis data of recombinant human anti-rabies virus monoclonal antibodies

[0147] The SRP competitive binding results showed that the two antibodies bound to different epitopes of the rabies virus glycoprotein and basically did not interfere with each other, which could further reduce the impact of viral mutations on neutralizing activity.

[0148] Example 4

[0149] The R92 antibody obtained 8 CVS-11 mutant viruses, named R92E1-E8.

[0150] The method for preparing the mutant virus comprises the following steps:

[0151] The CVS-11 virus was diluted 10-fold serially and 10 7 -10 4 Add 50 μl of virus diluted to FFU / ml, add 50 μl of anti-rabies virus monoclonal antibody at 4 IU / ml, and incubate at 37°C for 1 hour. 650 μl of BSR cell suspension (100 μg / ml) was added, incubated at 37°C for 4 h with 5% CO2. The culture medium was discarded and 150 μl of culture medium containing 2 IU / ml monoclonal antibody was added. The culture was continued for 72 h and the virus culture supernatant was harvested. 50 μl of culture supernatant was taken and 1.0×10 6 Add 50 μl of a BSR cell suspension (100 μg / ml) to 50 μl of culture medium in 5% CO2 at 37°C for 24 hours. Discard the supernatant, wash the cells once with PBS, and fix them with 200 μl of pre-chilled 80% acetone for 15 minutes. Discard the acetone, and let the cells dry at room temperature for 30 minutes. Add 100 μl of FITC-labeled anti-rabies virus nucleoprotein antibody diluted 200-fold with PBS to each well. Incubate at 37°C for 1 hour, discard the liquid, and wash three times with PBS. Observe the infection rate of cells at different dilutions under a fluorescence microscope. Select wells with an infection dose between 10% and 50% for subsequent experiments.

[0152] Take the viral culture supernatant and neutralize it with 4 IU / ml of antibody at a 1:1 volume ratio. Infect BSR cells and incubate at 37°C with 5% CO2 for 4 hours. Discard the culture medium and add medium containing 2 IU / ml of monoclonal antibody. Continue incubation for 72 hours. Repeat this step three times to obtain P4 generation escape viruses.

[0153] Take 50 μl of the P4 generation escape virus and dilute it 2-fold to 128-fold. Add 1.0×10 6 50 μl of a BSR cell suspension (100 μg / ml) was added to 50 μl of culture medium and incubated at 37°C in 5% CO2 for 24 hours. After discarding the supernatant, the cells were washed once with PBS and fixed with 200 μl of pre-chilled 80% acetone for 10 minutes. The acetone was discarded and the cells were allowed to stand at room temperature for 15 minutes. 100 μl of FITC-labeled anti-rabies virus nucleoprotein antibody was diluted 200-fold with PBS and added to each well. The cells were incubated at 37°C for 1 hour, the solution was discarded, and the cells were washed three times with PBS. The percentage of cells infected at different dilutions was observed under a fluorescence microscope.

[0154] The amino acid sequence of R92E1 is shown in SEQ ID NO:5; the amino acid sequence of R92E2 is shown in SEQ ID NO:6; the amino acid sequence of R92E3 is shown in SEQ ID NO:7; the amino acid sequence of R92E4 is shown in SEQ ID NO:8; the amino acid sequence of R92E5 is shown in SEQ ID NO:9; the amino acid sequence of R92E6 is shown in SEQ ID NO:10; the amino acid sequence of R92E7 is shown in SEQ ID NO:11; and the amino acid sequence of R92E8 is shown in SEQ ID NO:12.

[0155] For calibration of R92E1-E8, 80%-100% cell-infective doses of the mutant virus were mixed with culture medium, 2 IU / ml of R92 antibody, and 2 IU / ml of R71 antibody, respectively. After neutralization for 1 hour, cells were added to BSR cells, maintaining the same antibody species and concentrations as during the neutralization process. After 24 hours of incubation, cells were fixed with acetone and infected with fluorescein-labeled anti-rabies virus nucleoprotein antibodies.

[0156] The results are shown in Figures 6 and 7. In the presence of R92 antibody at a final concentration of 2 IU / ml, the replication of the R92E1-E8 viruses was unaffected, with infection levels comparable to those of the virus control without antibody. R92 antibody failed to neutralize these mutant viruses. In the presence of R71 antibody at a final concentration of 2 IU / ml, no cells were infected after 24 hours of culture, indicating that R71 antibody completely neutralized these mutant viruses. This result demonstrates that R71 antibody can effectively neutralize mutant viruses that have escaped R92 antibody.

[0157] Since it was not possible to obtain a stable mutant virus for the R71 antibody, a cross-neutralization study was conducted in the following manner. During the early candidate antibody screening process, a cross-neutralization test was performed using a 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, 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). 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. 80%-100% cell infection dose of the mutant virus of the C4 antibody (the virus can stably amplify in the presence of 4 IU / ml C4 antibody in the culture system) was added to the C4, C7, and C8 antibodies at a final concentration of 2 IU / ml, and after 1 hour of neutralization, BSR cells were added to maintain the same antibody type and concentration in the culture system as during the neutralization process. After 24 hours of culture, the cells were fixed with acetone and infected with fluorescein-labeled anti-rabies virus nucleoprotein antibodies. The results are shown in Figure 8. The C4 antibody was completely unable to neutralize the mutant escape virus. The C8 antibody, which has the same heavy chain as the C4 antibody, was also unable to effectively neutralize the mutant escape virus of the C4 antibody, but the C8 antibody had a certain inhibitory effect on the C4 antibody mutant escape virus. The C7 antibody completely neutralized the C4 antibody mutant escape virus. The results showed that the C7 antibody (which is exactly the same as the R92 antibody variable region) can completely neutralize the mutant escape virus that the C8 antibody (which is exactly the same as the R71 antibody variable region) cannot effectively neutralize.

[0158] In summary, R92 antibody and R71 antibody can effectively neutralize mutant viruses that cannot neutralize each other, and the two have a clear cross-protection effect.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-rabies virus combined monoclonal antibody preparation, characterized in that: The anti-rabies virus combined monoclonal antibody preparation comprises R92 antibody and R71 antibody; the light chain sequence of the R92 antibody is shown in SEQ ID NO: 1, the heavy chain sequence of the R92 antibody is shown in SEQ ID NO: 2, the light chain sequence of the R71 antibody is shown in SEQ ID NO: 3, and the heavy chain sequence of the R71 antibody is shown in SEQ ID NO:

4.

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 (1-5): (1-5).

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, 1:5 or 5:

1.

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

1.

5. The anti-rabies virus combined monoclonal antibody preparation according to claim 1, characterized in that: The concentration of R92 antibody was 0.1-5 mg / ml, and the concentration of R71 antibody was 0.1-5 mg / ml.

6. The anti-rabies virus combined monoclonal antibody preparation according to claim 5, characterized in that: The concentration of R92 antibody was 2.5 mg / ml, and the concentration of R71 antibody was 2.5 mg / ml.

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

8. The anti-rabies virus combined monoclonal antibody preparation according to claim 7, characterized in that: The surfactant is a nonionic surfactant, and the nonionic surfactant is selected from one or more of polysorbate-80, polysorbate-20, polyethylene glycol octylphenyl ether, nonylphenol polyoxyethylene ether, and fatty alcohol polyoxyethylene ether.

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

10. The anti-rabies virus combined monoclonal antibody preparation according to claim 7, characterized in that: The amino acids are selected from one or more of histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine and tryptophan.

11. The anti-rabies virus combined monoclonal antibody preparation according to claim 7, characterized in that: It also includes one or more of polysorbate-80, trehalose, histidine, and EDTA.

12. The anti-rabies virus combined monoclonal antibody preparation according to any one of claims 1 to 11, characterized in that: The pH of the anti-rabies virus combined monoclonal antibody preparation is 5-8.

13. Use of the anti-rabies virus combined monoclonal antibody preparation according to any one of claims 1 to 12 in the preparation of a product for neutralizing rabies mutant strains.

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

15. Use of the anti-rabies virus combined monoclonal antibody preparation according to any one of claims 1 to 12 in the preparation of a rabies virus detection reagent.

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