Anti-RABV antibody with neutralizing binding site at non-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 a non-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, allowed mutation of 12% or below), CDR2 (SEQ ID NO 3, allowed mutation of 12% or below) and CDR3 (SEQ ID NO 4, allowed mutation of 12% or below), and a light chain variable region of the anti-RABV antibody comprises CDR1 (SEQ ID NO 6, allowed mutation of 12% or below), CDR2 (SEQ ID NO 7, allowed mutation of 12% or below) and CDR3 (SEQ ID NO 8, allowed mutation of 12% or below). 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 having a neutralizing binding site at a non-antigen epitope III and its application. Background Art

[0002] Rabies virus (RABV) is a zoonotic pathogen with both wild and domesticated animal reservoirs. Human infection with RABV causes an acute, fatal illness accompanied by rapidly progressive encephalitis. Approximately 61,000 human deaths from rabies occur annually worldwide, the majority of whom are children, making human rabies a major public health problem. In Asia and Africa, dog bites account for nearly all causes of human rabies infection. However, in the United States, most human rabies cases result from exposure to insectivorous bats and terrestrial animals, including skunks and raccoons. Deaths from rabies can be prevented through timely post-exposure prophylaxis (PEP), which consists of wound care and combined passive and active immunization with rabies immune globulin (RIG) and rabies vaccine, respectively. With proper administration, PEP is effective in preventing disease, but treatment of human rabies following PEP administration often deviates from the treatment protocol used in the setting of severe exposure.

[0003] An estimated 36,000 courses of rabies PEP are performed annually in the United States. The cost of rabies PEP in the United States and worldwide varies widely, with the majority of the cost of RIG derived from serum from either RABV-immunized human donors (hRIG) or hyperimmune horses (eRIG). Due to the expense of producing large quantities of fractionated blood products, a global shortage of RIG exists. Administration of RIG from human or equine donors also poses a potential safety threat due to adventitious factors.

[0004] Human monoclonal antibodies (HuMAbs) targeting RABV glycoprotein (G) have been proposed as alternatives to RIG, and several candidate antibodies have entered the marketing and clinical stages. Marketed products include: SII RMAb (17C7, Rabishield) from the Serum Institute of India; RabiMabs (M777-16-3 and 62-71-3) from Zydus Cadila, India; SYN023 (HU1A9 and CIB012) from Xingmeng Biopharmaceutical Co., Ltd.; MN57 from North China Pharmaceutical Co., Ltd.; GR1801 from Zhixiang Jintai Biopharmaceutical Co., Ltd. has been approved for marketing; and CBB1 from Changchun Biotech Co., Ltd. has entered Phase II clinical trials. Summary of the Invention

[0005] The present application provides an anti-RABV antibody with a neutralizing binding site at non-epitope III and its application. The present application obtains a non-epitope III epitope antibody through research, which has good neutralizing activity and animal protection level.

[0006] The anti-RABV antibodies CDD12 and CDE8 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 CDD12 and CDE8 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.

[0007] In the first aspect, the present application provides an anti-RABV antibody having a neutralizing binding site in non-epitope III, using the following technical solution:

[0008] An anti-RABV antibody having a neutralizing binding site at non-epitope III, the anti-RABV antibody comprising a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region includes CDR1, CDR2, and CDR3,

[0010] 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 12% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 2;

[0011] 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 12% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 3;

[0012] 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 12% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 4;

[0013] The light chain variable region includes CDR1, CDR2, and CDR3,

[0014] 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 12% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 6;

[0015] 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 12% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 7;

[0016] 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 12% or less of the sites are allowed to be mutated based on the amino acid sequence shown in SEQ ID NO 8.

[0017] 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 5% 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In a third aspect, the present application provides a vector comprising the aforementioned nucleic acid molecule.

[0022] 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.

[0023] 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.

[0024] In a sixth aspect, the present application provides a pharmaceutical composition, which adopts the following technical solution:

[0025] A pharmaceutical composition comprising the anti-RABV antibody, the nucleic acid molecule, the vector, the cell, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0026] In summary, this application includes at least one of the following beneficial technical effects:

[0027] The anti-RABV antibodies CDD12 and CDE8 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 CDD12 and CDE8 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. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The present application is further described in detail below in conjunction with the examples and test results.

[0035] Example

[0036] Example 1

[0037] This example provides a process for preparing anti-RABV antibodies.

[0038] 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).

[0039] 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.

[0040] 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.

[0041] 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.

[0042] After screening, two anti-RABV antibodies were obtained, named CDD12 and CDE8. The specific sequence information of these antibodies is shown in Table 1.

[0043] Table 1 Sequence information of CDD12 and CDE8 and comparison results with CDD12

[0044]

[0045]

[0046] Example 2

[0047] In this example, affinity testing was performed on the anti-RABV antibodies screened and purified in Example 1.

[0048] 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).

[0049] The specific process is as follows:

[0050] 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.

[0051] 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.

[0052] Kinetic association rates (kon) and dissociation rates (koff) were determined using Biacore TM8K 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.

[0053] 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 17, and the light chain variable region sequence is represented by the amino acid sequence set forth in SEQ ID NO 18.

[0054] The test results are shown in Table 2.

[0055] Table 2 Affinity test results for CDD12, CDE8 and Hu2G11

[0056] Serial number Antibody type kon(1 / Ms) koff(1 / s) KD(M) 1 <![CDATA[Hu2G11 ana log]]> 1.64E+05 3.13E-04 1.91E-09 2 CDD12 1.84E+05 2.41E-04 1.31E-09 3 CDE8 1.81E+05 2.55E-04 1.41E-09

[0057] As shown in Table 2, anti-RABV antibodies CDD12 and CDE8 have good binding affinity to rabies virus CVS-11G protein.

[0058] Example 3

[0059] In this example, the binding properties of the anti-RABV antibodies obtained by screening and purification in Example 1 were detected.

[0060] The binding characteristics of anti-RABV antibodies to rabies virus CVS-11G protein (ACRO Biosystems) were detected by ELISA. The specific steps include:

[0061] 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.

[0062] After washing twice with 300 μL 1×PBST, serial dilution samples of RABV antibodies (maximum concentration 5 μg / mL, 3-fold dilution, 12 steps) were added and incubated at 37°C for 1 h.

[0063] 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.

[0064] 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 -OD 570 .

[0065] The measured data were processed using GraphPad Prism 7.00 analysis software. Transform analysis was first performed with the X-axis plotted as Log(antibody concentration) and the chemiluminescence intensity plotted as the ordinate. The EC50 value was calculated using a four-parameter fitting method.

[0066] At the same time, affinity testing was performed on Hu2G11 as a control antibody.

[0067] The results are shown in Table 3.

[0068] Table 3 Binding characteristics test results for CDD12, CDE8 and Hu2G11

[0069]

[0070]

[0071] As shown in Table 3 , the anti-RABV antibodies CDD12 and CDE8 have good binding activity to rabies virus CVS-11G protein and are better than the control antibody Hu2G11 analog.

[0072] Example 4

[0073] In this example, the neutralizing activity of the anti-RABV antibodies screened and purified in Example 1 was tested.

[0074] Detecting the neutralizing activity of anti-RABV antibodies against rabies virus CVS-11 series pseudovirus strains. Specifically, the following steps are included:

[0075] (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.

[0076] (2) Neutralization of virus and sample: The concentration of CVS-11 wild-type (WT) and a series of CVS-11 mutant pseudovirus solutions was adjusted to 3.2 × 104 TCID 50 / mL. A series of CVS-11 mutants include CVS-11(T36A), CVS-11(K226M), CVS-11(R264Q), CVS-11(W251R), CVS-11(K342R), CVS-11(I338T), CVS-11(K330Q), CVS-11(R333H), and CVS-11(N336G). 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.

[0077] Information about 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.

[0078] 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.

[0079] (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 sample and virus neutralizer, and the cells were cultured in a cell culture incubator at 37° C. and 5% CO 2 for 24 h.

[0080] (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.

[0081] (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).

[0082] 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%.

[0083] According to the neutralization inhibition rate results, the EC50 value of the antibody was calculated using the Reed-Muench method.

[0084] At the same time, the neutralization activity of Hu2G11 was detected as a control antibody.

[0085] The test results are shown in Table 4.

[0086] Table 4 Binding characteristics test results for CDD12, CDE8 and Hu2G11

[0087]

[0088] As shown in Table 4, the anti-RABV antibodies CDD12 and CDE8 have good neutralizing activity against rabies virus CVS-11 (WT) and related mutants.

[0089] Example 5

[0090] In this example, the binding epitopes of the anti-RABV antibodies screened and purified in Example 1 were analyzed.

[0091] 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:

[0092] 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 described 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. Test antibodies (analyte 1 and analyte 2 premixed at a 1:1 ratio, with a final concentration of 200 nM for both antibodies in the premix) were injected at 1000 rpm to bind to the ligand and determine whether the binding of different antibodies interfered with each other. The antibody binding time was 300 s.

[0093] 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).

[0094] The test results are shown in Table 5.

[0095] At the same time, the binding epitope of Hu2G11 antibody was analyzed.

[0096] Table 5 Binding epitope analysis results for CDD12, CDE8 and Hu2G11

[0097]

[0098] As shown in Table 5, CDD12, CDE8, and Hu2G11 analog bind to similar epitopes. Combined with the results of the pseudovirus neutralization experiments with the wild-type (WT) and CVS-11(I338T) rabies virus strains, it can be determined that CDD12 and CDE8 bind to the non-epitope III site of the rabies virus G protein.

[0099] Example 6

[0100] In this example, the in vitro neutralizing activity of the anti-RABV antibodies screened and purified in Example 1 was tested.

[0101] The specific steps include:

[0102] Pipette DMEM culture medium containing 10% newborn calf serum into a 96-well cell culture plate, 100 μL per well.

[0103] 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.

[0104] 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.

[0105] Neutralization: Incubate at 37°C for 1 hour to neutralize the antibodies and virus.

[0106] Inoculation of cells: BSR cells in the logarithmic growth phase were taken, 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.

[0107] 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.

[0108] 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.

[0109] Seal the plate: discard the fluorescent antibody, add 200-300 μL / well of 0.01M PBS, wash the plate once, and add 80% glycerol, 1 drop / well.

[0110] Observe and count, and count the corresponding wells with fluorescent foci more or less than 50% of the infection amount under a fluorescence microscope.

[0111] The test results are shown in Table 6.

[0112] Table 6 Results of in vitro neutralization activity test on CDD12 and CDE8

[0113] Serial number Antibody type RFFIT (IU / mg) 1 CDE8 1615.1 2 CDD12 2281.4

[0114] As shown in Table 6, the anti-RABV antibodies CDD12 and CDE8 have good in vitro neutralizing activity against rabies virus CVS-11.

[0115] Example 7

[0116] In this example, the in vivo neutralizing activity of the anti-RABV antibodies screened and purified in Example 1 was tested.

[0117] The specific steps include:

[0118] Anti-RABV antibodies were diluted 10-fold in a 10-fold serial dilution series at 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 antibody activity in mice was calculated based on the survival rate of mice in different dose groups.

[0119] The test results are shown in Table 7.

[0120] Table 7 Results of in vivo neutralization activity test on CDD12 and CDE8

[0121] Serial number Antibody type Activity in mice (IU / mg) 1 CDE8 1676.42 2 CDD12 1413.77

[0122] As shown in Table 7, the anti-RABV antibodies CDD12 and CDE8 have good in vivo neutralizing activity against rabies virus CVS-11.

[0123] Example 8

[0124] This example conducted an interference test on vaccination using the anti-RABV antibodies obtained by screening and purification in Example 1.

[0125] To determine the effect of the antibody cocktail on vaccine efficacy, in vivo animal experiments were performed in the absence of rabies virus challenge.

[0126] Experimental groups were set up: Balb / C female mice were administered 50 μg / kg of anti-RABV antibody via intramuscular injection into the gastrocnemius muscle of the left hind leg, and simultaneously received rabies vaccine (Liaoning Chengda Biological Co., Ltd., injection volume 1 / 25 of the human dose) ectopically intramuscularly, resulting in two experimental groups; or Balb / C female mice were administered 20 IU / kg of human rabies immune globulin (HRIG) via intramuscular injection into the gastrocnemius muscle of the left hind leg, and simultaneously received rabies vaccine (Liaoning Chengda Biological Co., Ltd., injection volume 1 / 25 of the human dose) ectopically intramuscularly, resulting in one experimental group. Control mice were administered only with rabies vaccine. Rabies vaccine was administered on days 0 and 7.

[0127] 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).

[0128] The test results are shown in Table 8.

[0129] Table 8 Results of the interference test of CDD12 and CDE8 on vaccination

[0130]

[0131] As shown in Table 8, compared with human rabies immunoglobulin (HRIG), the combined use of anti-RABV antibodies CDD12 and CDE8 with the vaccine does not affect the production of vaccine antibodies.

[0132] Example 9

[0133] This example studies the street virus protection of the anti-RABV antibodies obtained by screening and purification in Example 1.

[0134] To test whether anti-RABV antibodies exhibit neutralizing activity against lethal rabies virus infection in vivo, this example conducted studies in mice.

[0135] 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.

[0136] The test results are shown in Table 9.

[0137] Table 9 Results of the study on the protective effect of CDD12 and CDE8 antibodies against street viruses

[0138]

[0139]

[0140] As shown in Table 9, the anti-RABV antibodies CDD12 and CDE8 had a good protective effect at a concentration of 10 μg / ml.

[0141] 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.

[0142] 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 in non-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 12% 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 12% 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 12% 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 12% 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 12% 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 12% or less of the sites are allowed to mutate 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 5% 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 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.

4. The anti-RABV antibody according to claim 1, wherein 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; 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.

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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