Anti-RABV antibody with neutralizing binding site at epitope III and application of anti-RABV antibody

Abstract

The invention relates to the technical field of rabies antibodies, in particular to an anti-RABV antibody with a neutralizing binding site at an epitope III and application of the anti-RABV antibody. A heavy chain variable region of the anti-RABV antibody comprises CDR1 (SEQ ID NO 2, allowing 20% or less of mutation), CDR2 (SEQ ID NO 3, allowing 25% or less of mutation) and CDR3 (SEQ ID NO 4, allowing 40% or less of mutation), and a light chain variable region of the anti-RABV antibody comprises CDR1 (SEQ ID NO 6, allowing 10% or less of mutation), CDR2 (SEQ ID NO 7, allowing 15% or less of mutation) and CDR3 (SEQ ID NO 8, allowing 15% or less of mutation). The anti-RABV antibody provided by the invention has good neutralizing activity on the rabies virus CVS-11 and related mutant strains.

Description

Technical Field

[0001] The present application relates to the technical field of rabies antibodies, and in particular to an anti-RABV antibody with a neutralizing binding site at antigen epitope III and its application. Background Art

[0002] Rabies virus (RABV) belongs to the genus Lyssavirus of the family Rhabdoviridae. It is a single-stranded negative-sense RNA virus with an extremely high mortality rate (close to 100%). Its genome encodes five structural proteins (N, P, M, G, L), of which G protein (glycoprotein) is the main antigen, which determines the virus invasion and immunogenicity. The main transmission route is through bites from infected animals (dogs, bats, etc.) or contact of saliva with mucous membranes. The virus migrates along the peripheral nerves to the central nervous system (CNS), causing acute encephalitis, and the incubation period is usually 1-3 months (can be as short as a few days). The main hosts of rabies virus are dogs (accounting for 99% of human cases) and bats (important hosts in the Americas and Europe), and high-incidence areas are parts of Asia and Africa (due to low dog vaccination rates).

[0003] Rabies is a fatal zoonotic disease caused by the lyssavirus. Clinically, it presents with hydrophobia, aerophobia, pharyngeal muscle spasms, and progressive paralysis. It is one of the most dangerous infectious diseases worldwide. According to the World Health Organization (WHO), approximately 59,000 people die from rabies annually worldwide. Over 95% of these cases occur in Asia and Africa, where medical resources are scarce. Children account for a significant 40% of these deaths, highlighting the severe threat the disease poses to vulnerable groups.

[0004] Currently, post-exposure prophylaxis (PEP) for rabies mainly relies on passive immunotherapy with a rabies vaccine combined with anti-rabies immunoglobulin (RIG). However, existing RIG therapy has many limitations: First, human RIG (HRIG) relies on plasma from blood donors, and its production capacity is severely limited, making it difficult to meet global demand; second, although equine RIG (ERIG) has a higher production volume, it is prone to causing severe allergic reactions such as serum sickness and has poor safety; in addition, RIG is expensive and unaffordable for low-income people, resulting in many patients being unable to receive timely and effective treatment. More critically, traditional single monoclonal antibody therapy is prone to neutralization escape due to mutations in the viral glycoprotein (G protein) epitope, and the protection rate against some prevalent strains (such as the bat-derived GT1 variant) is less than 60%, seriously affecting the treatment effect.

[0005] To address the above issues, cocktail combination antibody therapy has emerged. This therapy targets multiple key epitopes of the viral G protein (such as antigenic sites II, III, IV and linear epitope CR57) and uses the synergistic effect of different antibodies to comprehensively block the binding of the virus to neuronal receptors, thereby significantly reducing the risk of neutralization escape (escape rate <0.1%). International studies have shown that a cocktail consisting of 2-3 antibodies can achieve a neutralization coverage rate of more than 98% for the world's seven rabies virus genotypes (GT1-GT7), demonstrating excellent broad spectrum and therapeutic potential. Summary of the Invention

[0006] The present application provides an anti-RABV antibody having a neutralizing binding site at antigen epitope III and its application.

[0007] The anti-RABV antibodies CDA8, CDB4, and CDC4 provided herein are a cocktail combination antibody preparation that has good binding affinity and activity to the rabies virus CVS-11 G protein and exhibits good neutralizing activity against rabies virus CVS-11 and related mutants, effectively neutralizing multiple street rabies strains. Furthermore, the anti-RABV antibodies CDA8, CDB4, and CDC4 exhibit good in vitro and in vivo neutralizing activity against rabies virus CVS-11, and the combined use of anti-RABV antibodies with vaccines does not affect the production of vaccine antibodies.

[0008] In a first aspect, the present application provides an anti-RABV antibody having a neutralizing binding site at epitope III, using the following technical solution:

[0009] An anti-RABV antibody having a neutralizing binding site at epitope III, the anti-RABV antibody comprising a heavy chain variable region and a light chain variable region;

[0010] The heavy chain variable region includes CDR1, CDR2, and CDR3,

[0011] Wherein, the CDR1 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO 2, and an amino acid sequence in which 20% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 2;

[0012] The CDR2 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO 3, and an amino acid sequence in which 25% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 3;

[0013] The CDR3 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO 4, and an amino acid sequence in which 40% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 4;

[0014] The light chain variable region includes CDR1, CDR2, and CDR3,

[0015] Wherein, the CDR1 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO 6, and an amino acid sequence in which 10% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 6;

[0016] The CDR1 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO 7, and an amino acid sequence in which 15% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 7;

[0017] The CDR1 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO 8, and an amino acid sequence in which 15% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 8.

[0018] Optionally, the heavy chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 1, and an amino acid sequence in which 15% or less of the sites are allowed to mutate based on the amino acid sequence shown in SEQ ID NO 1; the light chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 5, and an amino acid sequence in which 10% or less of the sites are allowed to mutate based on the amino acid sequence shown in SEQ ID NO 5.

[0019] Optionally, the heavy chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 9; the light chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 13,

[0020] Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 10-12, respectively; and the CDR1, CDR2, and CDR3 of the light chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 14-16, respectively.

[0021] Optionally, the heavy chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 17; the light chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 21.

[0022] Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 18-20, respectively; and the CDR1, CDR2, and CDR3 of the light chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 22-24, respectively.

[0023] In a second aspect, the present application provides a nucleic acid molecule encoding the heavy chain variable region and / or light chain variable region of the anti-RABV antibody.

[0024] In a third aspect, the present application provides a vector comprising the aforementioned nucleic acid molecule.

[0025] In a fourth aspect, the present application provides a cell that expresses the heavy chain variable region and / or light chain variable region of the anti-RABV antibody, or comprises the nucleic acid molecule, or comprises the vector.

[0026] In a fifth aspect, the present application provides the use of the above-mentioned anti-RABV antibodies, the above-mentioned nucleic acid molecules, the above-mentioned vectors, and the above-mentioned cells in preparing a composition for treating rabies.

[0027] In a sixth aspect, the present application provides a pharmaceutical composition, which adopts the following technical solution:

[0028] A pharmaceutical composition comprising the anti-RABV antibody, the nucleic acid molecule, the vector, the cell, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0029] In summary, this application includes at least one of the following beneficial technical effects:

[0030] The anti-RABV antibodies CDA8, CDB4, and CDC4 provided herein have good binding affinity and activity to the rabies virus CVS-11 G protein and exhibit good neutralizing activity against rabies virus CVS-11 and related mutants. Furthermore, the anti-RABV antibodies CDA8, CDB4, and CDC4 exhibit good in vitro and in vivo neutralizing activity against rabies virus CVS-11, and the combined use of the anti-RABV antibodies with the vaccine does not affect the production of vaccine antibodies. DETAILED DESCRIPTION

[0031] Before describing the embodiments of the present application in detail, it should be understood that the terminology used herein is only for the purpose of describing particular embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the term belongs.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0033] The endpoints of the ranges and any values ​​disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0034] In this application, the term "comprise" or "include" is an open expression, that is, it includes the content specified in this application, but does not exclude other aspects of the content.

[0035] To make the purpose, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making any creative work are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be understood as limiting this application.

[0036] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0037] The present application is further described in detail below in conjunction with the examples and test results.

[0038] Example

[0039] Example 1

[0040] This example provides a process for preparing anti-RABV antibodies.

[0041] To produce anti-RABV antibodies (rabies virus neutralizing antibodies), lyophilized human rabies vaccine (PV strain) or veterinary rabies vaccine (Flury strain) or human rabies vaccine (CTN strain) were used to immunize RenMab. TM Mice (Beijing Biocytogen, Inc., mouse antibody variable region genes encoding heavy and kappa light chains were replaced in situ with their human counterparts, retaining intact mouse constant regions and key regulatory elements, while the mouse lambda chain gene was knocked out).

[0042] The specific immunization method is as follows: 14-day intervals between immunizations, using the vaccine as the antigen and adjuvant for four to five immunizations, followed by serum antibody qualitative testing. The surge immunization uses tail vein cell immunization and intraperitoneal injection of the antigen simultaneously.

[0043] The serum titers of the anti-RABV antibodies obtained were simultaneously measured by ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting), and immunized mice with high titers were selected for antibody screening. Once the desired immune response was achieved, antigen-specific immune cells were isolated from the immunized mice to further obtain anti-RABV antibodies and the light and heavy chain variable region sequences of the anti-RABV antibodies.

[0044] For example, using single-cell technology (e.g., Berkeley Light The Optoflidic System was used to screen and identify plasma cells secreting antigen-specific monoclonal antibodies, and reverse transcription and PCR sequencing were used to obtain the antibody variable region sequences. The obtained variable region sequences were cloned into a human IgG1 constant region backbone vector to construct an antibody expression plasmid. CHO-S cells or 293T-S cells were transfected with the antibody plasmid and cultured. The culture supernatant of these cells was collected and purified using Protein A affinity chromatography. The binding of the obtained antibody to the antigen was verified using FACS, and antibodies that specifically bind to rabies virus G protein, i.e., anti-RABV antibodies, were obtained.

[0045] After screening, three anti-RABV antibodies were obtained, named CDA8, CDB4, and CDC4. The detailed sequence information of these antibodies is shown in Table 1.

[0046] Table 1 Sequence information of CDA8, CDB4 and CDC4 and their alignment with CDA8

[0047]

[0048]

[0049] Example 2

[0050] In this example, affinity testing was performed on the anti-RABV antibodies screened and purified in Example 1.

[0051] Using a Biacore sensor chip equipped with pre-immobilized Protein A TM The affinity of anti-RABV antibodies for rabies virus CVS-11 (standard challenge virus) G protein (Glycoprotein, ACRO Biosystems, Catalog No.: RAG-V55H5) was detected by surface plasmon resonance (SPR) using a Biacore 8K biosensor (Biacore, Inc., Piscataway, NJ).

[0052] The specific process is as follows:

[0053] The rabies virus CVS-11G protein antigen was diluted to 2 μg / mL using 10× HBS-EP+ buffer (pH 7.4). The anti-RABV antibodies obtained by screening and purification were diluted to 100 nM and then diluted 2-fold to final concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0.78125 nM, respectively. 0 nM was used as a reference for comparison.

[0054] Antigen Glycoprotein protein (2 μg / mL) was captured using a CM5-AntiHis-FlowCell1-8-Chip chip at a flow rate of 10 μL / min for 50 s to achieve the desired protein density (e.g., approximately 50 response units (RU)). The anti-RABV antibody was then allowed to bind to the antigen at a flow rate of 30 μL / min for 180 s and dissociate for 600 s to obtain a binding curve.

[0055] Kinetic association rates (kon) and dissociation rates (koff) were determined using Biacore TM 8K Evaluation Software 3.0 was used to globally fit the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110). Affinity was calculated based on the quotient of the kinetic rate constants (KD = koff / kon). Those skilled in the art will appreciate that specific parameters (e.g., antibody concentration) can be appropriately adjusted for each test antibody in the above-described assay.

[0056] Affinity testing was also performed on Hu2G11, a humanized anti-rabies monoclonal immunoglobulin IgG1 / κ antibody. The heavy chain variable region sequence of Hu2G11 is represented by the amino acid sequence set forth in SEQ ID NO: 25, and the light chain variable region sequence is represented by the amino acid sequence set forth in SEQ ID NO: 26.

[0057] The test results are shown in Table 2.

[0058] Table 2 Affinity test results for CDA8, CDB4, CDC4 and Hu2G11

[0059] Serial number Antibody type kon(1 / Ms) koff(1 / s) KD(M) 1 Hu2G11analog 1.64E+05 3.13E-04 1.91E-09 2 CDA8 1.71E+06 2.390E-04 1.40E-10 3 CDB4 1.44E+06 1.94E-04 1.35E-10 4 CDC4 1.77E+06 2.43E-04 1.38E-10

[0060] As shown in Table 2, anti-RABV antibodies CDA8, CDB4 and CDC4 have good binding affinity to rabies virus CVS-11G protein.

[0061] Example 3

[0062] In this example, the binding properties of the anti-RABV antibodies obtained by screening and purification in Example 1 were detected.

[0063] The binding characteristics of anti-RABV antibodies to rabies virus CVS-11G protein (ACRO Biosystems) were detected by ELISA. The specific steps include:

[0064] Rabies virus CVS-11G protein antigen was diluted to 5 μg / mL using Elisa coating buffer (pH 9.6 carbonate) and 100 μL was added to each well of a 96-well plate. Coating was allowed to proceed overnight at 4°C. Each well was washed three times with 300 μL of 1× PBST, followed by addition of 200 μL of 1% BSA diluted in 1× PBST to each well and blocking at 37°C for 2 h.

[0065] After washing twice with 300 μL 1× PBST, serial dilution samples of anti-RABV antibodies (maximum concentration 5 μg / mL, 3-fold dilution, 12 steps) were added and incubated at 37°C for 1 h.

[0066] After washing again, 100 μL of HRP Goat x-Human IgG Fc Fragament (secondary antibody) diluted 100,000 times with 1× PBST was added to each well and incubated at 37°C for 1 hour. Then, 100 μL of TMB color development solution was added to each well and color was developed at 37°C in the dark for 15 minutes.

[0067] Then add 50 μL of stop solution to each well and read the OD value on a microplate reader. 450 and OD 570 , standard OD value = OD 450 -OD570 .

[0068] The measured data were processed using GraphPad Prism 7.0 analysis software. Transform analysis was first performed with the x-axis plotted as Log(antibody concentration) and the y-axis plotted as chemiluminescence intensity. The EC50 value was calculated using a four-parameter fitting method.

[0069] At the same time, affinity testing was performed on Hu2G11 as a control antibody.

[0070] The results are shown in Table 3.

[0071] Table 3 Binding characteristics test results of CDA8, CDB4, CDC4 and Hu2G11

[0072] Serial number Antibody type EC50 (μg / mL) 1 Hu2G11 analog 1.331 2 CDA8 0.012 3 CDB4 0.015 4 CDC4 0.009

[0073] As shown in Table 3, the anti-RABV antibodies CDA8, CDB4, and CDC4 have good binding activity to rabies virus CVS-11G protein, and are better than the control antibody Hu2G11 analog.

[0074] Example 4

[0075] In this example, the neutralizing activity of the anti-RABV antibodies screened and purified in Example 1 was tested.

[0076] Detecting the neutralizing activity of anti-RABV antibodies against rabies virus CVS-11 series pseudovirus strains. Specifically, the following steps are included:

[0077] (1) Sample dilution: The initial concentration of the anti-RABV antibody to be tested was adjusted to 10 μg / mL with PBS. Then, the sample was diluted at an initial dilution of 1:30 and serially diluted 3-fold in 7 steps to obtain a series of anti-RABV antibodies.

[0078] (2) Neutralization of virus and sample: The concentration of CVS-11 wild-type (WT) and CVS-11 series mutant pseudovirus solution was adjusted to 3.2×10 4 TCID 50 / mL. The CVS-11 series of mutants includes CVS-11(T36A), CVS-11(K226M), CVS-11(R264Q), CVS-11(W251R), CVS-11(K342R), CVS-11(I338T), CVS-11(K330Q), CVS-11(R333H), CVS-11(N336G), CVS-11(L231P), CVS-11(D262N), CVS-11(I133V), CVS-11(S160L), and CVS-11(K346R). Among them, CVS-11(I338T), CVS-11(K330Q), CVS-11(R333H), and CVS-11(N336G) are all key mutation sites in rabies virus epitope III.

[0079] Information on CVS-11 mutants can be found in the article "Antigenic variants of rabies virus," Wiktor TJ, Oprowski HJ Exp Med. 1980 Jul 1; 152(1): 99-112. The construction of CVS-11 mutant pseudoviruses was performed with reference to the article "Development of in vitro and in vivo rabies virus neutralization assays based on a high-titer pseudovirus system," Nie J et al. Sci Rep. 2017 Feb 20; 7: 427-69.

[0080] Add 50 μL of pseudovirus solution to each well of the antibody sample as the sample group and incubate in a 37°C, 5% CO2 cell culture incubator for 1 hour. A cell control group (CC) and a virus control group (VC) were set up. The virus control group (VC) added 50 μL of pseudovirus, while the cell control group (CC) only received culture medium.

[0081] (3) Cell culture: HEK 293T cells were digested and the concentration was adjusted to 5.0×10 5 100 μL of cell suspension was added to each well of the 96-well cell plate containing the antibody sample and virus neutralizer, and cultured in a cell culture incubator at 37° C. and 5% CO 2 for 24 h.

[0082] (4) Cell lysis: After the culture is completed, 150 μL of supernatant was aspirated and luciferase detection reagent was added to the cell culture plate. The reaction was allowed to proceed at room temperature in the dark for 2 min.

[0083] (5) Fluorescence value detection: Use a multichannel pipette to repeatedly pipette the liquid in the reaction well to fully lyse the cells. Aspirate 150 μL of liquid from each well and transfer it to a 96-well white plate chemiluminescence detection plate. Use a multifunctional imaging microplate reader to read the luminescence value (RLU).

[0084] Calculation results: Neutralization inhibition rate = [1-(mean luminescence intensity of the sample group-mean luminescence intensity of the cell control group (CC)) / (mean luminescence intensity of the virus control group (VC)-mean luminescence intensity of the cell control group (CC))] × 100%.

[0085] According to the neutralization inhibition rate results, the EC50 value of the antibody was calculated using the Reed-Muench method.

[0086] HU1A9 was also tested for neutralizing activity as a control antibody. HU1A9 is a humanized anti-rabies monoclonal immunoglobulin IgG1 / κ antibody. The heavy chain variable region sequence of HU1A9 is represented by the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region sequence is represented by the amino acid sequence set forth in SEQ ID NO: 28.

[0087] The test results are shown in Table 4.

[0088] Table 4 Binding characteristics test results for CDA8, CDB4, CDC4 and HU1A9

[0089]

[0090] As shown in Table 4, the anti-RABV antibodies CDA8, CDB4 and CDC4 all had good neutralizing activity against rabies virus CVS-11 (WT) and related mutants.

[0091] Example 5

[0092] In this example, the binding epitopes of the anti-RABV antibodies screened and purified in Example 1 were analyzed.

[0093] The relative positions of target protein epitopes between a pair of purified anti-RABV antibodies were analyzed by biolayer interferometry (BLI) using the ForteBio Octet system. The following steps were performed:

[0094] Throughout the experiment, 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% polysorbate 20 (P20), pH 7.4) was used as the buffer. The anti-RABV antibodies mentioned above were diluted to 200 nM and 500 nM, and rabies virus CVS-11G protein (ACROBiosystems) was diluted to 5 μg / mL as the ligand. Antigen was captured using a HIS1K probe at 1000 rpm for 200 s. Two anti-RABV antibodies (analyte 1 and analyte 2 premixed at a 1:1 ratio, with a final concentration of 200 nM for both antibodies) were injected at 1000 rpm to bind to the ligand and determine whether the binding of the different antibodies interfered with each other. The antibody binding time was 300 s.

[0095] Binding values ​​for each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of one antibody with another, a binding ratio was calculated to compare each pair of antibodies. The binding ratio was defined as the binding value of the secondary antibody (analyte 2) divided by the binding value of the primary antibody (analyte 1). The threshold for determining blocking or non-blocking was 0.6 (a binding ratio below 0.6 indicates that the antibodies compete for binding to the same epitope).

[0096] The test results are shown in Table 5.

[0097] The binding epitope of HU1A9 antibody was also analyzed.

[0098] Table 5 Results of binding epitope analysis for CDA8, CDB4, CDC4 and HU1A9

[0099]

[0100] As shown in Table 5, the binding ratios of HU1A9 analog, CDA8, CDB4, and CDC4 are all lower than 0.6, indicating that they bind to the same epitope. Since HU1A9 binds to epitope III of the rabies virus G protein (relevant content can be found in the article "Antigenic variants of rabies virus" Wiktor TJ, Koprowski HJ Exp Med. 1980 Jul 1; 152(1): 99-112, which shows that mutations in epitope III lead to significant resistance to HU1A9, it can be inferred that its neutralization binding site is on epitope III), and combined with the results of the pseudovirus neutralization experiments with the rabies virus CVS-11 (WT) and CVS-11 (I338T) mutant strains, it can be determined that CDA8, CDB4, and CDC4 bind to epitope III of the rabies virus G protein.

[0101] Example 6

[0102] In this example, the in vitro neutralizing activity of the anti-RABV antibodies screened and purified in Example 1 was tested.

[0103] The specific steps include:

[0104] Pipette DMEM culture medium containing 10% newborn calf serum into a 96-well cell culture plate, 100 μL per well.

[0105] Add samples: Add 50 μL of the antibody to be tested, standard serum (3-fold dilution), and negative control (internal reference) to the first column of wells on each plate. After thorough mixing (15-20 times), pipette 50 μL into the second column of wells, and so on for 3-fold dilution. Pipet 50 μL from the last well and discard.

[0106] Add virus: Dilute the wild-type rabies virus CVS-11 (WT) to an appropriate multiple using DMEM culture medium containing 10% newborn calf serum, and add 50 μL per well to a 96-well cell plate.

[0107] Neutralization: Incubate at 37°C for 1 hour to neutralize the antibodies and virus.

[0108] Inoculation of cells: BSR cells in the logarithmic growth phase were obtained, digested with trypsin solution, and diluted to 8×10 cells with DMEM culture medium containing 10% newborn calf serum. 5 / mL~1×10 6 After neutralization, 50 μL of diluted BSR cells were added to each well of the neutralized 96-well cell plate and cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 h.

[0109] Fixation and staining, acetone fixation: remove the 96-well cell culture plate, discard the culture medium, wash once with 0.01M PBS, 200-300μL / well, add -20℃ pre-cooled 80% acetone, 50μL / well, and fix at 2-8℃ for 30 minutes.

[0110] Add fluorescent antibody: discard the acetone, absorb the residual liquid with filter paper, add 100-fold diluted fluorescent antibody (with 1% Evans blue staining solution), 50 μL / well, and incubate at 37°C for 2 h.

[0111] Seal the plate: discard the fluorescent antibody, add 0.01M PBS 200-300 μL / well, wash the plate once, and add 80% glycerol, 1 drop / well.

[0112] Observe and count, and count the corresponding wells with fluorescent foci more or less than 50% of the infection amount under a fluorescence microscope.

[0113] The test results are shown in Table 6.

[0114] Table 6 Results of in vitro neutralization activity test on CDA8, CDB4 and CDC4

[0115] Serial number Antibody type RFFIT (IU / mg) 1 CDC4 2129.1 2 CDA8 2619.4 3 CDB4 3007.5

[0116] As shown in Table 6, the anti-RABV antibodies CDA8, CDB4 and CDC4 have good in vitro neutralizing activity against rabies virus CVS-11.

[0117] Example 7

[0118] In this example, the in vivo neutralizing activity of the anti-RABV antibodies screened and purified in Example 1 was tested.

[0119] The specific steps include:

[0120] Anti-RABV antibodies were diluted 10-fold in a starting concentration of 1 mg / mL and mixed with an equal volume of wild-type rabies virus CVS-11 (WT). The mixture was neutralized at 37°C for 1 hour, and 25 μL was injected intracerebrally into 6-8 week-old Balb / C female mice. The mice were observed for 30 days. The activity of the anti-RABV antibodies in mice was calculated based on the survival rate of mice in different dose groups.

[0121] The test results are shown in Table 7.

[0122] Table 7 Results of in vivo neutralization activity test on CDA8, CDB4 and CDC4

[0123] Serial number Antibody type Activity in mice (IU / mg) 1 CDC4 4736.00 2 CDA8 2814.37 3 CDB4 3352.83

[0124] As shown in Table 7, the anti-RABV antibodies CDA8, CDB4 and CDC4 have good in vivo neutralizing activity against rabies virus CVS-11.

[0125] Example 8

[0126] This example conducted an interference test on vaccination using the anti-RABV antibodies obtained by screening and purification in Example 1.

[0127] To determine the effect of the antibody cocktail on vaccine efficacy, in vivo animal experiments were performed in the absence of rabies virus challenge.

[0128] Three experimental groups were established: 50 μg / kg of anti-RABV antibody was administered intramuscularly into the gastrocnemius muscle of the left hind leg to Balb / C female mice, along with an ectopic intramuscular injection of rabies vaccine (Liaoning Chengda Biological Co., Ltd., 1 / 25 of the human dose). Alternatively, 20 IU / kg of human rabies immune globulin (HRIG) was administered intramuscularly into the gastrocnemius muscle of the left hind leg to Balb / C female mice, along with an ectopic intramuscular injection of rabies vaccine (Liaoning Chengda Biological Co., Ltd., 1 / 25 of the human dose). A control group was established: mice in the control group received only the rabies vaccine. Rabies vaccination was administered on days 0 and 7.

[0129] Then, on days 1, 2, 4, 8, 16 and 28, blood was collected from the mouse orbits and the serum was tested for neutralizing activity (neutralization RFFIT test against rabies virus CVS-11).

[0130] The test results are shown in Table 8.

[0131] Table 8 Results of the interference test of CDA8, CDB4 and CDC4 on vaccination

[0132]

[0133] As shown in Table 8, compared with human rabies immunoglobulin (HRIG), the combined use of anti-RABV antibodies CDA8, CDB4 and CDC4 with rabies vaccine does not affect the production of vaccine antibodies.

[0134] Example 9

[0135] This example studies the street virus protection of the anti-RABV antibodies obtained by screening and purification in Example 1.

[0136] To test whether anti-RABV antibodies exhibit neutralizing activity against lethal rabies virus infection in vivo, this example conducted studies in mice.

[0137] Different street viruses (BD06, JX09-17, JX10-67, GN07, ZJ-LA, and DRV) were mixed at a completely lethal dose with 10 μg / mL or 1 μg / mL of anti-RABV antibodies and 20 IU / mL of human immunoglobulin G (HRIG) at a 1:1 ratio. After neutralization at 37°C for 1 hour, 30 μL of the antibody-virus mixture was injected intracerebrally and observed for 30 days. A control group (HRIG) of 20 IU / mL served as a control.

[0138] The test results are shown in Table 9.

[0139] Table 9 Results of the study on the protection of CDA8, CDB4 and CDC4 antibodies against street viruses

[0140]

[0141]

[0142] As shown in Table 9, the anti-RABV antibodies CDA8, CDB4, and CDC4 had a good protective effect at a concentration of 10 μg / mL.

[0143] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An anti-RABV antibody with a neutralizing binding site at epitope III, characterized in that: The anti-RABV antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR1, CDR2, and CDR3, Wherein, the CDR1 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO 2, and an amino acid sequence in which 20% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 2; The CDR2 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO 3, and an amino acid sequence in which 25% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 3; The CDR3 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO 4, and an amino acid sequence in which 40% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 4; The light chain variable region includes CDR1, CDR2, and CDR3, Wherein, the CDR1 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO 6, and an amino acid sequence in which 10% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 6; The CDR1 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO 7, and an amino acid sequence in which 15% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 7; The CDR1 of the light chain variable region includes the amino acid sequence shown in SEQ ID NO 8, and an amino acid sequence in which 15% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 8.

2. The anti-RABV antibody according to claim 1, wherein The heavy chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 1, and an amino acid sequence in which 15% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 1; the light chain variable region of the anti-RABV antibody is the amino acid sequence shown in SEQ ID NO 5, and an amino acid sequence in which 10% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 5.

3. The anti-RABV antibody according to claim 1, wherein The heavy chain variable region of the anti-RABV antibody has the amino acid sequence shown in SEQ ID NO 9; the light chain variable region of the anti-RABV antibody has the amino acid sequence shown in SEQ ID NO 13; Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 10-12, respectively; and the CDR1, CDR2, and CDR3 of the light chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 14-16, respectively.

4. The anti-RABV antibody according to claim 1, wherein The heavy chain variable region of the anti-RABV antibody has the amino acid sequence shown in SEQ ID NO 17; the light chain variable region of the anti-RABV antibody has the amino acid sequence shown in SEQ ID NO 21; Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 18-20, respectively; and the CDR1, CDR2, and CDR3 of the light chain variable region of the anti-RABV antibody are the amino acid sequences shown in SEQ ID NOs 22-24, respectively.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the heavy chain variable region and / or light chain variable region of the anti-RABV antibody according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. A cell, characterized in that The cell expresses the heavy chain variable region and / or light chain variable region of the anti-RABV antibody according to any one of claims 1 to 4, or comprises the nucleic acid molecule according to claim 5, or comprises the vector according to claim 6.

8. Use of the anti-RABV antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, or the cell according to claim 7 in preparing a composition for treating rabies.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-RABV antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the cell according to claim 7, and a pharmaceutically acceptable excipient, diluent or carrier.

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