CN105481979A - Humanized anti-rabies-virus glycoprotein neutralizing antibody RV3A5 and application thereof - Google Patents

Abstract

The invention discloses a human anti-rabies virus glycoprotein neutralizing antibody RV3A5, which is obtained by screening with phage antibody library technology. The antibody of the present invention specifically recognizes the rabies virus particle antigen, and is all directed against the rabies virus glycoprotein G, has obvious immunofluorescence reaction and enzyme-linked immune reaction with the rabies virus, and has a neutralizing activity function against rabies virus infection. The antibody of the present invention can be made into a specific antibody drug for preventing and treating rabies, so that it can be clinically used for preventing and treating rabies caused by rabies virus.

Description

Human anti-rabies virus glycoprotein neutralizing antibody RV3A5 and its application

technical field

The invention relates to genetic engineering antibody technology, in particular to human-sourced anti-rabies virus glycoprotein neutralizing antibody RV3A5 and its application.

Background technique

Rabies is a worldwide zoonotic disease caused by rabies virus, once the onset of 100% death. Rabies has been reported in 87 countries in the world, and more than 50,000 people die from rabies every year (KnobelDL, et al.2005). Rabies post-exposure prophylaxis is the main measure to prevent and control rabies. For severely exposed people, the World Health Organization (World Health Organization, WHO) recommends the use of rabies vaccine injection combined with anti-rabies immune globulin (rabiesimmuneglobulin, RIG) method. The two types of RIG currently in use are human rabies immune globulin (HRIG) and equine anti rabies immune globulin (Equinerabies immune globulin, ERIG). ERIG has severe side effects and inhibits the antibody response to certain vaccines, while HRIG is expensive, limited in supply and has potential pathogenic threats. Therefore, it has become a new research and development goal to prepare highly efficient, inexpensive, and less side effect passive immunization preparations.

Human or animal serum immunoglobulins containing specific antibodies have a long history for the prevention and treatment of infectious diseases. The anti-virus neutralizing activity of monoclonal antibodies in vitro and the protection of the body against virus attacks in vivo have been proved by many experiments. In vivo 100% protection of animals from virus attack. The way to obtain multiple antisera by immunizing animals with antigens has always been a classic method to obtain antibodies, but it lacks specificity and uniformity. The subsequent establishment of B lymphocyte hybridoma technology enables many scientists to prepare various monoclonal antibodies (monoclonal antibody, McAb) in vitro through cell engineering. It has strong specificity, uniform properties, and is easy to produce in large quantities. However, most McAbs are of murine origin, and the heterogeneous reaction of murine McAbs greatly limits the application of McAbs as therapeutic agents in humans. As an antibody component, immunoglobulin (Vacciniaimmuneglobulin, VIG) mainly comes from the immune serum of donors (convalescent patients). It takes a lot of manpower and financial resources to obtain positive serum and pass safety testing, which limits its mass production. At the same time, since it is derived from serum, it is prone to infection of blood-borne diseases. Therefore, using human genetically engineered products instead of blood products can overcome these defects. With the continuous deepening of research on human genetically engineered antibodies, it has brought new hope and broad prospects for the development of biological products in this field. A variety of specific mouse and human antibodies can be obtained through the recombination of the antibody molecular gene level, which has made a breakthrough in the research of monoclonal antibodies and has increasingly shown its significance and practical application prospects. The development of human monoclonal antibody against rabies virus and the generation of phage antibody library technology provide new ideas for solving the problem of passive immunization preparations. A monoclonal antibody cocktail made of rabies monoclonal antibodies CR57 and CR4098 neutralized 26 typical street virus strains. Protective experiments on animals have shown that the use of monoclonal antibody cocktails for treatment is feasible and superior (GoudsmitJ, et al. 2006).

The rise of phage antibody library technology in the late 1980s and early 1990s and the development of the entire field of genetic engineering antibody technology research have made great progress in the development and research of human or genetic engineering antibodies in the world today, and have entered the stage of basic research. Substantive applied research and development phase. The successful research of human-derived antiviral genetically engineered antibodies, especially human-derived full antibodies, has brought new hope to the specific prevention and treatment of various viral infectious diseases, and gradually formed a class of anti-viral infection biopharmaceuticals. New antiviral drugs, the so-called antibody drugs (AntibodyDrug). Just like the transformation from blood-derived vaccines to genetically engineered vaccines, there is an urgent need to replace blood-derived VIG with genetically engineered antibodies, such as through chimeric antibody technology (Boulianne, G.L.etal., 1984; Morrison, S.L.etal., 1984), Humanized antibody technology (Jones, P.T. et al., 1986), transgenic mouse technology carrying human monoclonal antibody (Green, L.L. et al., 1994), heterohybridoma technology (James, K. et al., 1987), phage Surface display technology (Barbas, C.F.etal., 1991) and other humanized antibodies have become a major research direction at home and abroad, and are gradually becoming successful.

Contents of the invention

The purpose of the present invention is to provide a human anti-rabies virus glycoprotein neutralizing antibody RV3A5 or an active fragment thereof.

Another object of the present invention is to provide a gene encoding the neutralizing antibody RV3A5 or its active fragment.

Another object of the present invention is to provide the application of the neutralizing antibody RV3A5 and its active fragments in the preparation of drugs or diagnostic reagents for preventing or treating rabies.

The present invention uses the phage antibody library technology to collect a plurality of peripheral blood lymphocytes of vaccinators with high titers of rabies virus antibodies, constructs a human anti-rabies virus genetic engineering antibody library by means of genetic engineering, and screens to obtain specific anti-rabies virus Genetically engineered antibody Fab fragment. The obtained Fab fragment antibody was named RV3A5.

This recombinant antibody is determined by the hypervariable regions (CDRs) specific gene sequences present in the variable regions of the antibody light chain and heavy chain genes, and is effectively expressed in prokaryotic cells to specifically bind rabies virus Antibody. They specifically recognize rabies virus particle antigens, and are all directed against rabies virus glycoprotein G, have obvious immunofluorescence reaction (IFA) and enzyme-linked immunosorbent immunoassay (ELISA) reactions with rabies virus, and have neutralizing activity against rabies virus infection.

The RV3A5-specific light chain variable region gene is derived from the specific enrichment screening of the human anti-rabies virus antibody gene library. The light chain variable region gene of the antibody was amplified by RT-PCR in blood lymphocytes, and the light chain gene of the monoclonal antibody CR4098 was replaced to construct a light chain replacement library. The heavy chain variable region gene specific for RV3A5 is the same as CR4098. The combination of the three CDR region sequences corresponding to the light chain and heavy chain variable regions and the framework region sequence between the CDR regions constitute the sequence characteristics of each antibody variable region, and RV3A5 belongs to the antibody light chain family VL6. The function of the antibody protein is determined by the specific nucleotide sequence and its complementarity in the determinant complementary regions CDR1, CDR2 and CDR3 of the light chain and heavy chain variable regions of the antibody gene. The six corresponding CDR region amino acid sequences constitute the antibody The specific antigen-binding region of each antibody determines the antigen-binding characteristics and anti-rabies virus functional characteristics of each antibody. The amino acid sequences of the antibody light chain and heavy chain variable regions that determine the function of each neutralizing antibody are shown in Table 1:

Table 1

The amino acid sequence of the variable region of the light chain of the neutralizing antibody RV3A5 is shown in SEQ ID No: 1, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID No: 2.

The gene sequence encoding the variable region of the light chain of the neutralizing antibody RV3A5 is shown in SEQ ID No: 3, and the gene sequence of the variable region of the heavy chain is shown in SEQ ID No: 4.

It should be understood that, without affecting the activity of the scFv antibody, those skilled in the art can perform various substitutions, additions and/or deletions of one or several amino acids to the amino acid sequence shown in SEQ ID No: 1-2 to obtain amino acids with equivalent functions Sequence, for example, in the non-hypervariable region, amino acids with similar properties are replaced, such as replacing Val at position 6 of the light chain VL sequence of RV3A5 with Ala.

The present invention also provides an expression vector containing genes encoding the variable region of the light chain and the variable region of the heavy chain of the neutralizing antibody RV3A5.

The invention also provides engineering bacteria and transgenic cell lines containing genes encoding the variable region of the light chain and the variable region of the heavy chain of the neutralizing antibody RV3A5.

The present invention also provides the use of the neutralizing antibody RV3A5 in the preparation of medicaments or diagnostic reagents for preventing or treating rabies.

The present invention further provides medicines or diagnostic reagents containing the neutralizing antibody RV3A5.

In addition, considering the degeneracy of codons, for example, the gene sequence encoding the above-mentioned scFv fragment antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody. Those skilled in the art can artificially synthesize and modify the gene according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

Further, the present invention recombines the variable region of the light chain and the variable region of the heavy chain of the above-mentioned scFv antibody to obtain other molecular forms such as Fab antibody, which can also specifically recognize the surface antigen of rabies virus and have the effect of intracellular immunity. Single-chain antibodies have strong penetrating power and are easy to enter local tissues to play a role.

The above-mentioned genes encoding Fab antibodies and scFv genes can be cloned into expression vectors, and then transformed into hosts, and Fab antibodies and single-chain antibodies can be obtained by inducing expression.

In addition, the genes encoding the light and heavy chains of the above scFv antibodies can be cloned into a full-antibody expression vector and introduced into host cells to obtain full-anti-immunoglobulins expressing anti-rabies virus.

In an embodiment of the present invention, the light chain and heavy chain genes of the above-mentioned scFv antibody RV3A5 were respectively cloned into the VH/VK full antibody expression vector, and 293T cells were transiently transfected with the transfection reagent polyethyleneimine (PEI). The animal cell system realizes the secretory expression of the whole antibody, and obtains the whole antibody immunoglobulin IgG (that is, the whole human IgG antibody RV3A5).

The functional identification of the obtained whole antibody was carried out by ELISA, IFA, and SDS-PAGE. The results showed that the human IgG full antibody RV3A5 had specific binding to both aG strain and CTN strain rabies virus particles, and was expressed in a baculovirus/insect cell system. The glycoprotein of rabies virus aG strain has specific binding. The rapid immunofluorescence focus inhibition test was used to detect the neutralization reaction of the antibody with the international standard challenge strain CVS strain and the domestic vaccine strain aG strain in vitro, and the results showed that the CR4098-substituted antibody RV3A5 had higher activity against rabies virus aG strain and CVS strain The neutralizing titers were 834IU/ml and 779IU/ml, which were similar to the neutralizing activity of monoclonal antibody CR4098.

The present invention uses the phage antibody library technology to successfully obtain the human neutralizing antibody specific for rabies virus glycoprotein; the human neutralizing anti-rabies virus glycoprotein genetically engineered antibody variable region gene and Fab The antibody gene and the full antibody gene under the characteristics of each of the above antibody genes can realize the expression and production of this antibody in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system or the modified antibody gene based on this Any other gene can be used to obtain an antibody product that neutralizes rabies virus infection, and can be made into a specific antibody drug that is clinically used for the prevention and treatment of rabies.

Description of drawings

Fig. 1 is the ELISA detection result of the specific expression of human anti-rabies virus glycoprotein IgG antibody in Example 1 of the present invention.

Fig. 2 is the SDS-PAGE electrophoresis detection result of purified IgG in Example 1 of the present invention.

Fig. 3 is the result of immunofluorescence analysis of human IgG antibody and rabies virus glycoprotein protein expressed by sf9 cells in Example 1 of the present invention.

Fig. 4 is the result of the competition ELISA experiment of each antibody in Example 1 of the present invention.

Fig. 5 is the experimental results of non-competitive ELISA for measuring antibody affinity in Example 1 of the present invention.

Fig. 6 is the result of the determination of the neutralizing activity of the anti-rabies virus genetically engineered antibody in Example 1 of the present invention.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular cloning laboratory manual (Sambrook J & Russell DW, Molecular cloning: laboratory manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Materials and methods

1. Viruses, cells, and vectors: Rabies virus aG strain and CVS strain were provided by the Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention. 293T cells and insect cells Sf9 were from American Cell Culture Center (ATCC). The phage antibody library construction vector pHAL14 (HustM, ToleikisL, DubelS. Antibodyphagedisplay. Handbook of therapeutic antibodies. Weinheim: Wiley-VCH, 2007: 45-68.) was provided by Professor Stefan Dübel of the Technical University of Braunschweig, Germany. The whole antibody expression vector VH/VK (HustM, ToleikisL, DubelS. Antibodyphagedisplay. Handbook of therapeutic antibodies. Weinheim: Wiley-VCH, 2007: 45-68.) was provided by Professor Stefan Dübel, Braunschweig University of Technology, Germany. Rabies virus strain PM for human immunization is an imported human vaccine strain of the Weibo brand produced by Sanofi Pasteur in France, and the cultured cells used for rabies virus neutralization experiments in vitro are BHK-21. RNA extraction kits, RT-PCR kits, and PCR kits were all purchased from Roche. Trypsin, polyethylene glycol (PEG), and transfection reagent polyethyleneimine (PEI) were purchased from Sigma, USA.

2. Construction of pHAL14-VH _CR4098 clone: Firstly, clone the heavy chain gene VH of monoclonal antibody CR4098 into the pHAL14 vector as the vector for the construction of the light chain replacement library. The heavy chain PCR products of the vectors pHAL14 and CR4098 were digested with NcoI and HindIII respectively. After digesting at 37°C for 2 hours, the digested products were directly purified and recovered, ligated with T4 DNA ligase, transformed into XLI-BlueMRF competent cells, and positive cells were picked Clone pHAL4-VH _CR4098 assay identification.

3. Construction of light chain replacement antibody library: Lymphocytes were isolated from the anticoagulated blood of rabies virus vaccine injectors, total cellular RNA was extracted, reverse transcribed into cDNA, and a group of human antibody IgG1 heavy chain Fv and light chain were amplified Primers for Kappa and Lambda variable regions for PCR amplification. The PCR conditions are: 94°C for 1min, 55°C for 1min, 72°C for 10min, 30 cycles. Using the constructed pHAL4-VH _CR4098 as the library construction vector, after double digestion with MluI and NotI, the light chain diversity gene VL _Pool was connected to pHAL4-VH _CR4098 via T4DNA ligase, and electroporated XLI-BlueMRF competent to construct the light chain Replacement library, detailed library construction methods and primer sequence references (HustM, ToleikisL, DubelS.Antibodyphagedisplay.Handbookoftherapeuticantibodies.Weinheim:Wiley-VCH,2007:45-68, and PelatT,HustM,LafflyE,etal.Ahighaffinity,humanlikeantibodyfragment(scFv) neutralizing lethal factor (LF) of Bacillus anthracis by inhibiting the PA-LF complex formation. AntimicroAgents Chem, 2007, 51: 2758-2764).

4. Construction of the heavy chain replacement antibody library: the above-mentioned CR4098 light chain replacement library was screened to obtain positive clones as the backbone, and the light chain variable region gene VL _X was fixed, and VH _{CR4098 was} replaced. Firstly, the obtained light chain gene was cloned into the pHAL14 vector, the pHAL14-VL _X clone was constructed by MluI and NotI double digestion as the vector of the heavy chain replacement library, and the heavy chain gene was amplified using cDNA obtained from rabies vaccine injectors as a template. After being digested with NcoI and HindIII, ligated by T4 DNA ligase, and electroporated to XLI-BlueMRF competent, the heavy chain replacement library was constructed.

5. Enrichment screening of phage antibody library and induced expression of scFv single-chain antibody: Coat rabies virus at 4°C overnight with 0.1mol/L NaHCO ₃ (pH8.6) solution, add 1% skimmed milk and 0.05 % Tween-20) to block the coated wells and incubate at room temperature for 2 h, and then wash 3 times with PBST. Add 10 ¹¹ phage antibody library to it, and incubate at room temperature for 2 hours. Remove the supernatant and add 100 μl of 10 μg/ml trypsin to elute. The eluted phages were infected with fresh XLI-Blue bacteria solution (OD ₆₀₀ =0.5), packaged by helper phages and then subjected to the next round of screening. After repeating this for 3 times, a single clone was randomly selected to induce expression of ScFv by IPTG. For specific enrichment methods and induced expression of ScFv antibodies, please refer to PelatT, HustM, LafflyE, et al.

6. ELISA detection of scFv antibody: add induced expression single-chain antibody to the ELISA plate coated with rabies virus particles, and incubate at 37°C for 1h; add anti-myc mouse IgG antibody, and incubate at 37°C for 1h; add anti-mouse Enzyme-labeled IgG whole antibody, incubated at 37°C for 1 hour, developed color with chromogenic solution, terminated reaction with 2M H ₂ SO ₄ , detected absorbance A ₄₅₀ value with microplate reader, and determined anti-rabies virus scFv positive clones.

7. Gene sequence analysis: Use scFv sequencing primers to carry out sequence determination of antibody light and heavy chains on positive clones respectively. The determination work is completed by Beijing Qingke Biotechnology Co., Ltd., and the obtained recombinant scFv antibody nucleotide sequence is carried out by DNASTAR sequence analysis software Analytical processing is performed to obtain an amino acid sequence analysis deduced therefrom. Compare IgG family sequences in the InternetV-Base gene library to determine antibody light and heavy chain types.

8. Expression and purification of IgG antibodies: After sequencing, the positive clones of scFv antibodies were compared and analyzed with reference to the V-Base database. The obtained scFv antibody light and heavy chain genes were respectively cloned into VH/VK full antibody expression vectors, and the transfection reagent polyethyleneimine was used. (PEI) 293T cells were transiently transfected, cultured at 37° C. and 5% CO ₂ for 48 hours, then the supernatant was collected, and the IgG expression supernatant was purified by Protein-A affinity chromatography.

9. ELISA detection of IgG antibody: collect IgG expression supernatant, and perform ELISA detection of anti-human Fab and anti-rabies virus particles. Use 0.1mol/L NaHCO ₃ (pH9.6) solution to coat anti-human Fab antibody (US Sigma, used at 1:2000 dilution) and rabies virus particles (RV) on the microtiter plate, overnight at 4°C; 4% skimmed milk Block, 37°C for 1h, add IgG expression supernatant, 37°C for 1h; add enzyme-labeled anti-human Fc secondary antibody (US Sigma, used at 1:2000 dilution), 37°C for 1h; develop color with chromogenic solution, stop with 2M H ₂ SO ₄ Reaction, the microplate reader detects the absorbance value.

10. Indirect immunofluorescence (IFA) detection: use pAc-UW51 vector (BDPharmingen, USA) to transfect insect cells to prepare recombinant baculovirus for expression of rabies virus aG strain glycoprotein, prepare and express rabies virus glycoprotein antigen fragment, and add IgG antibody , incubated at 37°C for 30min, washed, added FITC-labeled anti-human IgG antibody (Sigma, USA), incubated at 37°C for 30min, washed, dried, and observed under a fluorescent microscope.

11. Competitive ELISA: use rabies virus aG strain virus as the coating antigen, use horseradish peroxidase-labeled antibody CR4098 against rabies virus glycoprotein III epitope as the detection antibody, and titrate the use of the enzyme-labeled antibody CR4098-HRP The dilution is 1:200. The competing antibody was purified whole antibody RV3A5, and the control competing antibody included CR4098, CR57 targeting epitope Ⅰ and human anti-interferon monoclonal antibody IFN-IgG. Add 100 μl of 5% skim milk-PBS solution to the 96-well plate, take 100 μl of each competing antibody, add them to 100 μl of 5% skim milk-PBS, mix and dilute at a volume ratio of 1:1, and then take 100 μl of the mixture from each well and add it to the next The wells were serially diluted, and then 1:200 diluted CR4098-HRP was added to incubate at 37°C for 1 h, the plate was washed, the color development solution was developed, the reaction was terminated with 2M H ₂ SO ₄ , and the absorbance A ₄₅₀ value was detected by a microplate reader.

12. Affinity determination by non-competitive ELISA method: Coat the purified rabies virus aG strain particles with 0.1M NaHCO ₃ (pH9.6) solution, the concentrations are 5μg/mL, 2.5μg/mL, 1.25μg/mL, 0.625μg/mL mL, after blocking with 5% skimmed milk, use 5% skimmed milk to dilute 100 μL of purified IgG antibody 2 times from 1.8mg/L, and the following dilutions are 1.8mg/L, 0.9mg/L, 0.45mg/L, 0.225mg/L By analogy, after incubating at 37°C for 1 hour, add the enzyme-labeled anti-human Fc antibody, incubate at 37°C for 1 hour, and detect the absorbance A ₄₅₀ value with a microplate reader after color development.

Judgment of results: Substituting the formula K=(n-1)/2(nAb'-Ab) to calculate the affinity constant, where Ab and Ab' represent the antibody concentration that produces half the absorbance value when the antigen concentration is Ag and Ag' respectively (mol/L), n=Ag'/Ag, the average K value is the antibody affinity.

13. Rapid immunofluorescence focus inhibition test (RFFIT) of human anti-rabies virus antibody: take 50 μL each of the antibody to be tested and antibody standard at different dilutions in a 96-well culture plate, and add 50 μL of 3×10 ⁶ pfu/mL to each well. And use virus CVS strain or aG strain, set blank control wells and neutralization virus control wells at the same time, mix well and place at 37°C for 1 hour, add 50 μL of 1×10 ⁶ /mL BHK-21 cell suspension to each well, place at 37°C Cultivate for 24 hours in a 5% CO ₂ incubator. After the culture was finished, blot the culture medium, wash with PBS and blot dry, add 50 μL of 80% acetone pre-cooled to 4°C to each well, fix at -30°C for 10 minutes, discard the acetone, and add the working concentration of fluorescently labeled antibody after it evaporated and dried. Rabies virus nucleoprotein antibody, 50 μL/well, incubate at 37°C for 30 minutes, shake off the liquid, wash the plate 2 to 3 times with PBS, dry the liquid, add 50 μL of 80% glycerol to each well, and observe with a fluorescence microscope. In the experimental group, the highest dilution factor of the antibody that can inhibit the fluorescence focus ≥ 50% is the neutralizing antibody titer of the tested antibody. According to the Reed&Muench formula, calculate the ED ₅₀ value of each antibody sample and standard substance, so as to obtain the titer of each antibody to be tested.

14. Antibody resistance to rabies virus after non-hypervariable region mutation

Based on the amino acid sequence of the variable region of the light chain of RV3A5, Val at position 6 of the amino acid sequence shown in SEQ ID No. 1 was replaced with Ala, and Ala at position 2 of the variable region of the heavy chain of RV3A5 (SEQ ID No. 2) was replaced with Gly. The heavy chain coding nucleic acid sequence of RV3A5 (the codon gtg was replaced by gcc at the corresponding position) and the light chain coding nucleic acid sequence (the codon gcc was replaced by ggg at the corresponding position) were synthesized respectively. According to the methods in 8-11 above, the light chain and heavy chain genes were respectively cloned into VH/VK full antibody expression vectors, transiently transfected into 293T cells, and the secretory expression of the full antibody was realized by using the mammalian cell system, and the mutation body for immunological testing.

result

1. Anti-rabies virus glycoprotein III epitope light chain replacement antibody library screening

The light chain PCR product and the vector pHAL4-VH _{CR4098 were} digested and ligated, and the ligated product was ethanol precipitated, then electrotransformed to construct a light chain replacement library, packaged with high-efficiency phage, enriched and screened with rabies virus aG strain, and enriched for 3 rounds Afterwards, 600 clones were randomly selected, and the scFv-induced expression supernatant was detected by rabies virus particle aG strain antigen ELISA. The results showed that the phage antibody library showed a strong enrichment trend during the three rounds of enrichment, especially the third round of enrichment. , the elution storage capacity has been improved by 2 orders of magnitude. ELISA detection showed that among the 600 screened clones, the ScFv-induced expression products of 34 clones had binding activity to the rabies virus particle aG strain. The yields of the three rounds of enrichment screening are shown in Table 2.

Table 2 Enrichment screening of human anti-rabies virus phage antibody library

2. Sequence analysis of human anti-rabies virus ScFv antibody

34 positive clones were sequenced, and the heavy chain and light chain genes of the antibody were compared with the IgG sequences in the InternetV-Base gene library, and 14 antibodies with sequence differences were found, of which 2 belonged to VK, 1 belonged to VL1 family, and 1 They belonged to the VL3 family, and 8 strains belonged to the VL6 family. The amino acid sequences of the light chain variable region CDRs are shown in Table 3.

Table 3 Replacement method screening anti-rabies virus scFv antibody light chain CDR amino acid sequence

3. CR4098 Antibody Heavy Chain Replacement Screening

Insert the above-screened light chain gene into the pHAL14 vector, and mix equal amounts of each positive clone as the vector pHAL14-VL _X of the heavy chain replacement library of the CR4098 antibody. The heavy chain PCR product and the vector pHAL14-VL _X are subjected to NocI and HindIII double enzymes Cut and recover the digested fragments and connect them. After ethanol precipitation, the ligation product was electrotransformed to a competent state, packaged with high-efficiency phage, and enriched and screened with rabies virus aG strain. There was no increasing trend of elution storage capacity during the enrichment screening, and 600 cells were randomly selected after the third round of enrichment. After IPTG-induced expression, rabies virus particles were used for ELISA detection. The results showed that no positive clones were detected in the ELISA detection of rabies virus particle binding activity, suggesting that the heavy chain of the CR4098 antibody played a major role in the antibody function. Once replaced, the antibody Binding activity was lost, so the heavy chain sequence of CR4098 was kept unchanged, and the screened light chain replacement antibodies were evaluated.

4. Human anti-rabies virus glycoprotein IgG antibody expression and ELISA detection

The positive clones screened by the rabies virus glycoprotein III episite replacement library were transiently transfected into 293T cells using the dual-plasmid system VH/VK, and the expression supernatant was collected for ELISA detection of anti-human Fab and anti-rabies virus particles. The results are shown in Figure 1 It shows that compared with CR4098, RV1C8 and RV4B8 (corresponding to 1C8 and 4B8 in Table 3) have higher anti-human Fab titers, indicating that the antibody expression level is higher, but the rabies virus detection titer is much lower than CR4098, indicating that the antibody Binding activity to rabies virus glycoprotein is low. Although the anti-human Fab titer of RV3A5 (corresponding to 3A5 in Table 3) is lower than that of CR4098, its antiviral activity is similar to it, indicating that at similar antibody expression levels, RV3A5 has good anti-rabies virus glycoprotein binding activity.

According to the above experimental results, RV3A5 with higher antiviral binding activity was selected for further research, and RV4B8 was used as a backup reference strain. RV3A5, RV4B8 and control CR4098 were purified by affinity chromatography using ProteinA affinity chromatography column, and the purified Ig antibodies were detected by SDS-PAGE electrophoresis. The results confirmed that all purified IgG proteins were obtained, which can be clearly seen at about 28KD and 55KD. strips (Figure 2).

5. Binding specificity (IFA) of human anti-rabies virus IgG antibody

In order to confirm the specificity of the obtained IgG antibody binding to rabies virus, the function of the antibody was further identified by indirect immunofluorescence test, as shown in Figure 3, the replacement library screening antibody RV3A5, RV4B8 strain monoclonal antibody and rabies virus aG strain glycoprotein Immunofluorescence was positive.

6. Competitive ELISA to analyze the epitope of antibody action

Rabies virus aG strain virus was used as the coating antigen, and the antibody CR4098 against the rabies virus glycoprotein III epitope was labeled with horseradish peroxidase as the detection antibody to detect whether there was mutual competition with RV35A. The control competing antibodies included CR4098, CR57 targeting epitope Ⅰ and human anti-interferon monoclonal antibody IFN-IgG. The results are shown in Figure 4. The monoclonal antibody CR57 targeting the No. I epitope of rabies virus glycoprotein and the human anti-interferon monoclonal antibody IFN-IgG did not compete with CR4098. There is obvious competition between human monoclonal antibody RV3A5 and CR4098, indicating that the two monoclonal antibodies may target the same epitope.

7. Determination of antibody affinity

The affinity of CR4098 antibody determined by non-competition ELISA was 2.8×10 ^-9 M, while that of RV3A5 was 2.1× ^10-9 M, which was slightly higher than that of CR4098. The affinity of RV4B8 is low, about 4.1×10 ^-8 , which is difficult to meet the practical application. The results of affinity determination of CR4098 and RV3A5 are shown in Fig. 5 .

8. Neutralization test of human anti-rabies virus antibody

In order to further study and compare the neutralizing activity of the rabies virus glycoprotein III episite displacement antibody RV3A5, a rapid immunofluorescence focus inhibition test was used to detect the neutralizing reaction of the antibody with the international standard challenge strain CVS strain and the domestic vaccine strain aG strain in vitro , the results are shown in Figure 6, CR4098-substituted antibody RV3A5 has high neutralizing titers of 834IU/ml and 779IU/ml for rabies virus aG strain and CVS strain respectively, which are similar to CR4098 neutralizing activity. Due to the low binding activity of RV4B8 to rabies virus glycoprotein, the neutralizing activity titer is low, about 200IU/ml. According to the provisions of the National Drug Control Agency, the anti-rabies serum titer greater than 200IU/mL means that the product is a qualified product. Therefore, the antibody RV3A5 obtained by the present invention has a neutralization effect equivalent to that of the monoclonal antibody CR4098. active.

9. Effect of non-hypervariable region mutation antibody on rabies virus resistance

According to the method of 14 in Materials and Methods (Study on the resistance of antibodies to rabies virus after non-hypervariable region mutation), the light chain gene and heavy chain gene based on RV3A5 changes were cloned into the VH/VK full antibody expression vector, and transiently The 293T cells were transfected, and the secretory expression of the whole antibody was realized by using the mammalian cell system, and the mutant RV3A5' was obtained. The mutant was tested immunologically. The indirect immunofluorescence experiment showed that RV3A5' can specifically target rabies virus glycoprotein G. The rapid immunofluorescence focus inhibition experiment was used to detect the neutralization reaction of the antibody with the international standard challenge strain CVS strain in vitro. , the results showed that its properties were basically the same as RV3A5.

discuss

The present invention uses ScFv phage antibody library technology to separate lymphocytes from the peripheral blood of vaccinators with high-titer anti-rabies virus antibodies, and combines with a high-efficiency phage packaging system to construct a neutralizing antibody against rabies virus glycoprotein III epitope CR4098. The library was replaced, and the purified rabies virus particle aG strain was selected for enrichment screening of the antibody library. Finally, a high-affinity human neutralizing antibody RV3A5 targeting glycoprotein III epitope was obtained. The light chain of this antibody was completely replaced with a new light chain gene, and the original VK1 family of CR4098 was replaced by the VL6 family. It is worth noting that in the present invention The heavy chain gene was not replaced successfully, suggesting that the heavy chain of the CR4098 antibody plays a major role in antibody function, and once replaced, the antibody binding activity is lost. The same antibody heavy chain and different light chain genes can be combined into a functional antibody with specific binding activity, indicating that the antibody heavy chain plays a key role in maintaining the structure and function of the antibody.

In the in vitro neutralization test of the present invention, the No. III episite substitution antibody RV3A5 has relatively high neutralizing titers of 779IU/ml and 834IU/ml for rabies virus CVS strain and aG strain, which are similar to CR4098 in neutralizing activity. Due to the low binding activity of RV4B8 to rabies virus glycoprotein, the neutralizing activity titer is low. The neutralization effect of antibodies depends on the joint action of various mechanisms. The differences in the neutralization efficiency of the above-mentioned antibodies against rabies virus may be closely related to the uniqueness of the antigenic epitopes they target and the affinity of the antibodies themselves for the antigens. Since there are many genotypes of rabies virus and the sequence of glycoprotein is not very conservative, the use of monoclonal antibodies against a single neutralizing antigenic site cannot achieve broad-spectrum and safe curative effect. Combining monoclonal antibodies against different neutralizing antigenic sites to make a monoclonal antibody cocktail has become the best choice to replace RIG as a passive immunization agent for post-exposure prophylaxis of rabies. Studies have reported that CL184, a monoclonal antibody cocktail composed of anti-rabies virus glycoprotein antibodies CR57 and CR4098, has completed phase I clinical trials in the United States and India. The clinical results show that CL184 is safe and effective, and can replace RIG for post-exposure prophylaxis. The inventor has successfully obtained 11 strains of human Fab antibodies against rabies virus glycoprotein through the Fab phage antibody library, combined with the new neutralizing antibody RV3A5 successfully transformed against the No. III epitope of glycoprotein in the present invention, all can be used as candidate antibodies. It will lay the foundation for the research and development of rabies monoclonal antibody cocktail formulations with independent intellectual property rights in my country.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

references:

[1] Yu Yongxin, editor-in-chief. Rabies and Rabies Vaccine (M). Beijing: China Medical Science and Technology Press, 2001

[2] KnobelDL, CleavelandS, ColemanPG, et al.

[3] World Health Organization. Strategies for the control and elimination of rabies in Asia [M]. Geneva, Switzerland, 2001, 2

[4] Suwansrinon, K., Jaijareonsup, W., Wilde, H., Benjavongkulchai, M., Sriaroon, C., and Sitprija, V. (2007). Sex-andage-related differences in rabies immunoglobulin hypersensitivity. .

[5] Suwansrinon, K., Jaijaroensup, W., Daviratanasilpa, S., Sriaroon, C., Wilde, H., and Sitprija, V. (2005).

[6] Schumacher CL, Dietzschold B, Ertl HC, et al. Use of mouse anti-rabies monoclonal antibodies in post exposure treatment of rabies. JClin Invest, 1989, 84 (3): 971-975

[7] Bakker, A.B., Marissen, W.E., Kramer, R.A., Rice, A.B., Weldon, W.C., Niezgoda, M., Hanlon, C.A., Thijsse, S., Backus, H.H., deKruif, J., Dietzschold, B. , Rupprecht, C.E., and Goudsmit, J. (2005).

[8] BakkerAB, PythonC, KisslingCJ, et al.Firstadministrationtohumansofamonoclonalantibodycocktailagainstrabiesvirus:safety,tolerability,andneutralizingactivity.Vaccine,2008,26(47):5922-5927

[9] Sun LN, Chen Z, YuL, WeiJS, LiC, JinJ, ShenXX, LvXJ, TangQ, LiDX, LiangMF.

[10] HustM, ToleikisL, DubelS.Antibodyphagedisplay.Handbookoftherapeuticantibodies.Weinheim:Wiley-VCH,2007:45-68.

[11] PelatT, HustM, LafflyE, etal.Ahighaffinity,humanlikeantibodyfragment(scFv)neutralisinglethalfactor(LF)ofBacillusanthracisbyinhibitingthePA-LFcomplexformation.AntimicroAgentsChem,2007,51:2758-2764.

[12] Susanne Rondot, Joachim Koch, Frank Breitling, and Stefan Dübel. A helper phage to improve single-chain antibody presentation in phage display. Nature Biotechnology, 2001, 75-78.

[13] Beatty JD, Barbara G. Beatty, William G. Vlahos. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods, 1987, 173-179.

Claims (10)

1. Human anti-rabies virus glycoprotein neutralizing antibody RV3A5, characterized in that the amino acid sequences of its light chain CDR1, CDR2 and CDR3 and heavy chain CDR1, CDR2 and CDR3 are: CDR1 CDR2 CDR3 VH GFTFSSYG ILYDGSDK AKVAVAGTHFDY VL TRSSGSIASNYVQ EDNQRPS QSHDGDSTSNPWV. 2. The neutralizing antibody RV3A5 according to claim 1, wherein the amino acid sequence of the light chain variable region and the heavy chain variable region amino acid sequence are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively. 3. The neutralizing antibody RV3A5 according to claim 2, which is a single-chain antibody ScFv or a whole antibody immunoglobulin IgG. 4. The gene encoding the neutralizing antibody RV3A5 according to any one of claims 1-3. 5. The gene according to claim 4, wherein the nucleotide sequence encoding the variable region of the light chain and the nucleotide sequence encoding the variable region of the heavy chain are shown in SEQ ID No: 3 and SEQ ID No: 4, respectively. 6. An expression vector comprising the gene of claim 4 or 5. 7. the engineered bacterium that contains the gene described in claim 4 or 5. 8. A transgenic cell line containing the gene of claim 4 or 5. 9. The use of the neutralizing antibody RV3A5 according to any one of claims 1-3 in the preparation of medicaments or diagnostic reagents for preventing or treating rabies. 10. A medicine or a diagnostic reagent containing the neutralizing antibody RV3A5 according to any one of claims 1-3.