WO2011137570A1

WO2011137570A1 - Human-derived neutralizing antibody rvfab8 against rabies virus glycoprotein

Info

Publication number: WO2011137570A1
Application number: PCT/CN2010/001575
Authority: WO
Inventors: 梁米芳; 孙丽娜; 陈哲; 李川; 李德新
Original assignee: 中国疾病预防控制中心病毒病预防控制所
Priority date: 2010-05-06
Filing date: 2010-10-09
Publication date: 2011-11-10
Also published as: CN101812131A; CN101812131B

Abstract

The present invention discloses a human-derived neutralizing antibody RVFab8 against rabies virus glycoprotein. The present invention also discloses the use of the antibody for the manufacturing of a medicament for the prevention or treatment of rabies.

Description

Human anti-rabies virus glycoprotein neutralizing antibody RVFAB8 TECHNICAL FIELD

The present invention relates to genetically engineered antibody technology, and more particularly to a human anti-rabies virus glycoprotein neutralizing antibody; the present invention also relates to the use of the antibody for the preparation of a medicament for preventing or treating rabies. Background technique

Rabies is a worldwide zoonosis caused by rabies virus, which kills 100% of the disease. There are currently rabies reports in 87 countries around the world, and more than 50,000 people die each year from rabies (Knobel DL, et al. 2005). Post-exposure prophylaxis for rabies is a major measure to prevent rabies. For people who are severely exposed, the World Health Organization (WHO) recommends the use of rabies vaccine in combination with anti-rabies immune globulin (RIG). The two types of RIG currently used are human rabies immune globulin (HRIG) and Equine rabies immune globulin (ERI). Due to the serious side effects of ERIG and the inhibition of antibody responses to certain vaccines, HRIG is expensive, limited in supply and potentially pathogenic. Therefore, it is our goal to prepare a passive immunological preparation with high efficiency, low cost and low side reaction.

Human or animal serum immunoglobulins containing specific antibodies have a long history of preventing and treating infectious diseases. The in vitro antiviral neutralizing activity of monoclonal antibodies and the protection of the body against viral challenge in vivo have been proved by many experiments, such as murine anti-hepatitis A virus, hantavirus, measles virus, RSV virus, CMV virus and other neutralizing monoclonal antibodies can be 100% protect animals from viral attack. The use of antigens to immunize animals to obtain multiple antiserum pathways has been a classic method of obtaining antibodies, but lacks specificity and homogeneity. The B-lymphocyte hybridoma technology established by the company has enabled many scientists to prepare various monoclonal antibodies (MCAbs) in vitro through cell engineering, which are highly specific, uniform in nature, and easy to mass-produce. However, McAb is mostly murine, and the heterologous response of murine McAb greatly limits the use of McAb as a therapeutic preparation in humans. Immunoglobulin The protein (Voccinia immune globulin, VIG) is mainly used as the antibody component from the donor (recovery patient) immune serum. It takes a lot of manpower and financial resources to obtain positive serum and pass the safety test, which limits its large-scale preparation. At the same time, since it is derived from serum, it is prone to infection of blood-borne diseases. Therefore, the use of human genetic engineering products instead of blood products can overcome these shortcomings, and the deepening of human genetic engineering antibody research has brought new hope and broad prospects to the development of biological products in this field. A variety of specific murine and human antibodies can be obtained by recombination of antibody molecules at the gene level, which has made breakthroughs in the research of monoclonal antibodies and has increasingly shown its significance and practical application prospects. The development of monoclonal antibodies against human anti-rabies virus and the production of phage antibody library technology have provided new ideas for solving the problem of passive immunologic preparations. The monoclonal antibody cocktails made from rabies monoclonal antibodies CR57 and CR4098 neutralized 26 typical street strains. Protective experiments on animals have shown that the use of monoclonal antibody cocktails for treatment is feasible and superior (Goudsmit J, et al. 2006).

The rise of phage antibody gene library technology in the late 1980s and early 1990s and the development of the whole field of genetic engineering antibody technology have made great progress in the development of human or genetically engineered antibodies in the world and have gone from the basic research stage to the essence. Sexual application research and development phase. The successful research of human antiviral genetic engineering antibodies, especially human whole antibodies, has brought new hopes for the specific prevention and treatment of various viral infectious diseases, and gradually formed a class in the field of antiviral infection biopharmaceuticals. A new antiviral drug, the so-called Antibody Drug. As with the transition from a blood-borne vaccine to a genetically engineered vaccine, there is now an urgent need to replace blood-derived VIG with genetically engineered antibodies, such as by chimeric antibody technology (Boulianne, G丄. et al., 1984; Morrison, SL et al. , 1984), Humanized Antibody Technology (Jones, PT et al., 1986), Transgenic Mouse Technology with Human Monoclonal Antibody (Green, LL et al., 1994), Allogeneic Hybridoma Technology (James, K.et Al., 1987), phage display technology (Barbas, CF et al., 1991) and other humanized antibodies have become a major research direction at home and abroad, and are gradually becoming successful. Summary of the invention

A first object of the present invention is to provide a human anti-rabies virus glycoprotein neutralizing antibody and an active fragment thereof.

A second object of the present invention is to provide a gene encoding the above antibody or an active fragment thereof.

A third object of the present invention is to provide the use of the above antibody and its active fragment for the preparation of a medicament for the prevention or treatment of rabies or a diagnostic reagent.

The invention adopts the phage surface presentation technology to collect a plurality of peripheral blood lymphocytes of vaccine injections with high titer rabies virus antibodies, and constructs a human anti-rabies virus genetic engineering antibody library by genetic engineering means, and screens for specific anti-madness. Large virus genetically engineered antibody Fab segment. The obtained Fab segment antibody was named RVFab8.

These three recombinant antibodies are determined by the high-variable region (CDRs)-specific gene sequences present in the variable regions of the antibody light and heavy chain genes, and are specifically expressed in prokaryotic cells to specifically bind to the function of rabies virus. Sexual antibodies. They specifically recognize rabies virus granule antigens, and both target rabies virus glycoprotein G, and have obvious immunofluorescence reaction (IFA) and enzyme-linked immunosorbent assay (ELISA) reactions with rabies virus, and have neutralizing activity against rabies virus infection.

The RVFabS-specific light and heavy chain variable region genes are derived from the specific enrichment screening of the human anti-rabies antibody gene pool, which is derived from the peripheral blood lymphocyte gene of the Chinese rabies virus vaccine. . The corresponding three CDR region sequence combinations of the light and heavy chain variable regions and the framework region sequences between the CDR regions constitute the sequence characteristics of each antibody variable region, and RVFab8 belongs to the antibody light chain family VL2. The function of the antibody protein is determined by the specific nucleotide sequence and its complement in the CDR1, CDR2 and CDR3 of the determinant complementary region of the light chain and heavy chain variable regions of the antibody gene, and the corresponding amino acid sequences of the corresponding CDR regions constitute the antibody. The specific antigen binding region determines the antigen binding characteristics and anti-rabies virus function characteristics of each antibody in the invention. The detailed amino acid sequence of the light chain and heavy chain variable regions of the antibody which determines the function of each neutralizing antibody and its comparison results are shown in Table 1: Table 1

The amino acid sequence of the RVFab8 light chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 2.

The gene sequence encoding the light chain variable region of RVFab8 is shown in SEQ ID No. 3, and the gene sequence of the heavy chain variable region is shown in SEQ ID No. 4.

It will be understood that those skilled in the art can perform various substitutions, additions and/or deletions of one or several amino acids on the amino acid sequence shown in SEQ ID No. 1-2 to obtain equivalent functions without affecting the activity of the Fab antibody. The amino acid sequence, for example, replaces an amino acid having similar properties in a non-hypervariable region, such as replacing Val at position 8 of the heavy chain VH sequence of RVFab8 with Ala.

Further, in consideration of the degeneracy of the codon, for example, the gene sequence encoding the above Fab 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 engineer the gene according to the preference of the expression of the antibody host to improve the expression efficiency of the antibody.

Further, the present invention recombines the light chain variable region and the heavy chain variable region of the above Fab antibody to obtain a single-chain antibody (ScFv) having a smaller molecular weight, which antibody can also specifically recognize a rabies virus surface antigen, and has intracellular The role of immunity. Single-chain antibodies have strong penetrating power and are easy to enter local tissues.

The gene encoding the Fab antibody and the ScFv gene can be cloned into an expression vector, and then transformed into a host, and a Fab antibody and a single-chain antibody can be obtained by inducing expression.

Furthermore, the light chain coding gene and the heavy Fd fragment gene of the above Fab antibody can be cloned into a total anti-expression vector and introduced into a host cell to obtain a total anti-immunoglobulin expressing anti-rabies virus.

In an embodiment of the present invention, the light chain and heavy Fd fragment genes of the above Fab antibody RVFab8 are separately cloned into the whole antibody expression vector pAC-L-Fc and transfected into insect Sf cells, Secreted expression of whole antibodies was achieved using a baculovirus/insect cell system to obtain a full antibody RV IgG8.

Functional identification of the obtained whole antibodies by ELISA, IFA and SDS-PAGE showed that the human IgG whole antibody RVIgG8 specifically binds to the aG strain and the CTN strain rabies virus particles, and expresses ERA with the baculovirus/insect cell system. The four rabies virus glycoproteins of CVS, CTN and aG strains all specifically bind. The rabies virus rapid immunofluorescence inhibition assay (RFFIT) was used to identify the whole antibody. The results showed that: RVIgG8 has good neutralizing activity, can reach 876.6 IU/mg, and fully possesses the neutralizing international standard attack strain CVS- The capacity of 11 strains.

The present invention successfully obtains a human neutralizing antibody specific for a rabies virus glycoprotein by using a phage antibody library technology; using the human neutralizing anti-rabies glycoprotein genetically engineered antibody variable region gene obtained above, The Fab antibody gene and the whole antibody gene under the characteristics of each of the above antibody genes can express and produce the antibody in prokaryotic cells, yeast cells, eukaryotic cells and any recombinant system or the modified antibody gene based thereon Any other gene that obtains an antibody product that neutralizes rabies virus infection, and is made into a specific antibody drug for clinical use in the prevention and treatment of rabies. DRAWINGS

Figure 1 is an immunofluorescence analysis of human rabies virus Fab antibody and rabies virus glycoproteins of ERA, CVS, CTN and aG strains;

Figure 2 is a SDS-PAGE electropherogram of purified IgG;

Figure 3 shows an anti-rabies virus human IgG antibody against CVS-11 rabies virus rapid immunofluorescence inhibition assay. detailed description

The specific embodiments of the present invention are further described in detail below with reference to the drawings and embodiments. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise specified. Materials and Method

1. Virus, cell, vector: Rabies virus aG strain, CTN strain, CVS strain and ERA strain are provided by the Chinese Center for Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. The human immunization strain PM strain is an imported human vaccine strain, and the cell used for the in vitro neutralization experiment of rabies virus is BHK-21 (ATCC). The strain was XLI-Blue (Stratagene, USA) and the vector was _P Comb3 (40 kb) supplied by the Scripps Research Institute of the United States. The baculovirus expression vector is pAC-L-Fc (PROGEN PR3003, Germany) (Liang, MF, Stefan, D., Li, DX, Queitsch, I., Li, W., and Bautz, EF Baculovirus expression cassette vectors for rapid production Of complete human IgG from phage displayselected antibody fragments. Journal of Immunological Methods. 247: 119-130. Insect cell Sf from the American Cell Culture Center

(ATCC).

2. Preparation of antigen

2.1 Eukaryotic expression of rabies virus glycoprotein (ERA, CVS, CTN, aG strain) PCR amplification of rabies virus aG strain, CTN strain, CVS strain and ERA strain provided by the Chinese Center for Disease Control and Prevention, respectively. The glycoprotein (G) gene was designed to introduce a Bamffl restriction site at both ends of the primer, and a 6-His tag was added to the 3' segment. The purified amplified product was digested with BamHI, and the digested fragment was recovered and directly cloned into the baculovirus expression vector pAcUW51 which was also digested with BamHI, and the HA gene was identified by PCR under the control of the polyhedrin promoter. After the target gene was inserted into the direction, the recombinant plasmid was obtained as: pAc-HA. Recombinant baculovirus was prepared by transfecting the recombinant plasmid into insect cells for protein expression, and the expression was carried out using the BaculoGold co-transfection kit of Pharmogen, USA. The method of operation is as follows: After mixing 5Mg of recombinant plasmid DNA with 0.5Mg of BaculoGold linear DNA, the Sf cells with a growth density of 50% were transfected with the transfection reagent in the kit, and cultured at 27 ° C for 4 days, collected. The supernatant was titrated and amplified as a virus of the recombinant virus. See the Baculovirus expression vector system manual for specific instructions. Obtained expression of rabies virus glycoprotein (ERA, CVS, CTN, aG strain) recombinant baculovirus infected Sf cells, and infected cells were harvested 4 to 5 days later to prepare recombinant rabies virus glycoprotein (ERA, CVS, CTN, aG strain) antigen tablets.

2.2 rabies virus purification

The rabies virus aG strain and the CTN strain were respectively infected with BHK-21 cells, and the supernatant was cultured. After formaldehyde inactivation and safety examination, the virus particles were purified by centrifugation for 3 h at 20 °C with a continuous sucrose density gradient of 35000 g (Beckman). SW28).

3. Construction of phage antibody library: Lymphocytes were isolated from anticoagulant of vaccination with high titer rabies virus antibody using lymphocyte separation solution (Sigma, USA), and total cellular RNA was extracted with RNeasy Mini Kit (QIAGEN, Germany). The extracted RNA was reverse-transcribed into cDNA using the Invitrogen First-Strand Synthesis System for RT-PCR. Cat No. 18080-051 using Oligo-dT primers. Human antibody IgG1 heavy chain Fd and light chain Kappa and Lambda primers were used for PCR amplification of human light and heavy chain Fab genes. The PCR conditions were: 94 ° C lmin, 54 ° C 1 min, 72 ° C 2 min, 35 cycles. The method of building the library is basically carried out according to the literature (Barbas, C. Fill., Kang, AS, and lamer, RA Assembly of combinatorial antibody libraries on phage surface: the genelll site. Proc.Natl.Acad.Sci.USA.1991;88( 18): 7978-7982).

4. Enrichment screening of phage antibody library and induction expression of Fab fragment antibody: The screening antigen was an inactivated virus particle aG and CTN strain purified by ultracentrifugation. Dilute with O.lm/L NaHC0 ₃ (pH 8.6) solution, coat the immunotube, and block with 4% skim milk-PBS, 37 V for 2 h, then add the above phage antibody library, 1 ml per tube, 37 Incubate for 2 h at ° C, and wash repeatedly with 5% Tween-20-TBS for 20 times. Finally, elute with 1 ml of a pH 2. 2 glycine-hydrochloric acid eluate, and neutralize the pH 9.6 Tris solution. The eluted phage continued to infect 2 ml of fresh XLl-Blu bacteria with OD _{6 (X)} = 1. 0, and the next round of screening was performed after infection with helper phage VCSM13 (Stmtagene, USA). This is repeated 4 to 5 times. Specific enrichment screening methods and induced expression of Fab fragments were performed according to the literature (Barbas, C. Fill., Kang, A. S" and lamer, RA Assembly of combinatorial antibody libraries on phage Surface: the genelll site. Proc. Natl. Acad. Sci. USA. 1991; 88(18): 7978-7982).

5. Human anti-rabies virus Fab antibody 'ELISA test

5.1 Detection of Fab expression The antibody was coated with anti-human Fab antibody (Sigma, 1:2000 dilution) in a solution of 0.1 m/L NaHC0 ₃ (pH 9.6) on the plate, overnight at 4 ° C; 4% skim milk was blocked , 37 °C lh, added Fab antibody, 37 ° C lh; added enzyme-labeled anti-human Fab secondary antibody (US Sigma, 1: 2000 dilution), 37 ° C lh; color development liquid, 2M H ₂ The reaction was terminated by S0 ₄ , and the absorbance A value was measured by a microplate reader.

5.2 Indirect enzyme-linked immunosorbent assay for Fab binding to rabies virus Purified inactivated rabies virus particles were used as coating antigens, followed by the same procedure.

6. Indirect immunofluorescence (IFA) assay: Sf9 cells were infected with recombinant baculovirus expressing rabies virus glycoprotein, and infected cells were harvested 4 to 5 days later to prepare recombinant rabies virus glycoprotein antigen tablets. The expressed Fab was added, incubated at 37 ° C for 30 min, washed, added with FITC-labeled anti-Fab antibody (Sigma, USA), incubated at 37 ° C for 30 min, washed, air-dried, and observed under a microscope.

7. Nucleic acid sequence analysis of human Fab antibody variable region gene: Plasmid DNA was prepared using Qiagen Miniprep Kit (QIAGEN, Germany) for nucleic acid sequence analysis. The primers for the light and heavy chains are 5'-AAACTAGCTAGTCGCCAAGGA-3' (as shown in SEQ ID No. 5) and 5'-CCGCGGTGGCGGCCGCAAAT-3' (as shown in SEQ ID No. 6). The sequencing results were compared with the IgG gene sequence in the Internet V-Base gene pool.

8. Construction of the whole antibody recombinant expression plasmid: The light chain of the obtained Fab antibody was first digested with Xba1, and then incubated at 37 ° C for 3 h, then filled with Klenow enzyme, and the target fragment was recovered at 37 ° C for 2 h; The target fragment was digested at 37 ° C for 3 h. At this time, a fragment of about 700 bp, which is a light chain fragment, was detected in agarose gel electrophoresis, and the light chain fragment was recovered and cloned into SacI/EcoRV at 37 ° C for 3 h double digestion. pAC-L-Fc vector (PROGEN PR3003, Germany) (Liang, MF, Stefan, D., Li, DX, Queitsch, I., Li, W., and Bautz, EF Baculovirus expression cassette vectors for rapid production of complete human IgG from Phage displayselected antibody fragments. Journal of Immunological Methods.247: 119-130.), and then clone the heavy chain Fd fragment into a full antibody expression vector using the ^Tzo/ZS^/site.

9. Transfection and recombinant virus infection and proliferation: The BaculoGold co-transfection kit from Pharmogen, USA. The method of operation outlined as follows: The recombinant plasmid was linearized DNA mixed with DNA 5 g of the BaculoGold _0.5μ β, using transfection reagent kit transfection growth density of 50% of the Sf9 cells, the culture 4d 27 ° C, The supernatant was collected for titration and amplification as a virus of the recombinant virus. See the Baculovirus expression vector system manual (BD Biosciences Pharmingen, USA) for specific procedures.

10. Whole-body IgG secretion expression and purification: Recombinant virus infected Sf cells with a growth density of about 70%, adsorbed at 27 °C for 1 h. Switch to SF-900 II SFM serum-free medium, and culture at 27 ° C for 3 ~ 5 days. clear. The expression supernatant was directly purified using Amersham's Protein-A affinity chromatography column (Harlow E, Lane D. Antibodies: A Laboratory Manual [M]. New York: Cold Spring Harbor Laboratory Press, 1988.). The functional properties of the purified IgG antibodies were identified by ELISA and IFA. See the material method 5, 6 for specific operations.

11. Human anti-rabies virus antibody rapid immunofluorescence inhibition assay (RFFIT): Take antibody and antibody standard dilutions of 50μ1 in 96-well culture plates, add neutralizing virus CVS-11, 50μ1/well, and set The blank well control, and the neutralizing virus control wells, were mixed and neutralized at 37 ° C for 1 hour, and each well was added with lxl0 ⁶ /ml BHK-21 cell suspension 50 μl, placed in a 37 ° C 5% C0 ₂ incubator. Cultivate for 24 hours. After the culture is finished, the dried culture solution is sucked, and each well is added with ΙΟΟμΙ/PBS and dried. After each well, add 80 μl of acetone 50 μl pre-cooled to 4 ° C, fix at -30 ° C for 10 minutes, discard acetone, and wait for evaporation. After drying, add the working concentration of fluorescently labeled anti-rabies virus nucleoprotein antibody, 50μl / well, incubate at 37 °C for 30 minutes, remove the liquid, wash the plate with PBS 2 ~ 3 times, dry the liquid, add 80% glycerol 50μ1 per well , observed by fluorescence microscopy. In the experimental group, the fluorescent antibody can inhibit the highest dilution factor of ≥50% of the antibody, which is the neutralizing antibody titer of the antibody to be tested. According to the Reed & Muench formula, the ED _{5Q of} each antibody sample and the labeled product was calculated to obtain the titer of each antibody to be tested.

12. Study on the resistance of rabies virus to antibodies in non-hypervariable region Based on the RV IgG8 heavy chain variable region amino acid sequence, the Val at position 8 of the amino acid sequence shown in SEQ ID No. 2 was replaced with Ala, and the fifth position of the RV IgG8 light chain variable region (shown by SEQ ID No. 1) Replace Ala with Gly. The heavy chain encoding nucleic acid sequence of RV IgGS (replacement of the codon gtg to gcc at the corresponding position) and the light chain encoding nucleic acid sequence (replacement of the codon gcc to ggg at the corresponding position) were separately synthesized. The light chain gene and the heavy chain Fd fragment were cloned into pAC-L-Fc according to the above methods 8 to 11, and transfected into insect Sf cells, and the secreted expression of the whole antibody was achieved by the baculovirus/insect cell system, and The mutant was subjected to immunological detection. result

1. Screening of human anti-rabies virus antibody library

The phage antibody library was enriched and screened with purified rabies virus particle aG strain, and 6000 clones were randomly picked after 2 rounds of screening. The anti-human Fab antibody (sigma, 1 : 2 000 diluted use), rabies virus particle aG strain antigen coated 96-well plate, added to the test sample supernatant, with enzyme-labeled anti-human Fab secondary antibody (Sigma, 1: 2 000 dilutions used) detection. The results showed that a total of 2984 human Fab-positive clones were obtained, as shown in Table 2. Of the 2984 human-derived Fab-positive clones, 181 clones specifically bind to the rabies virus granule aG strain, of which 36 Fab clones were identified against rabies virus glycoproteins.

The phage antibody library was enriched and screened by the purified rabies virus particle CTN strain, and 2400 clones were randomly picked after 2 rounds of screening. Anti-human Fab antibody (sigma, 1:2 000 diluted use), rabies virus particle aG strain antigen coated 96-well plate, added to the supernatant of the test sample, and detected with enzyme-labeled anti-human Fab secondary antibody (Sigma, 1:2 000 diluted use). The results showed that a total of 1833 human Fab-positive clones were obtained, as shown in Table 3. Of the 1833 human-derived Fab-positive clones, 79 clones specifically bind to the rabies virus particle CTN strain, and 34 of the Fab clones were identified as directed against rabies virus glycoproteins.

Screening of phage antibody library by CTN strain

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

The DNASTAR sequence analysis software was used for analysis and comparison of the IgG sequences in the Internet V-Base gene pool. Among the 70 human Fab monoclonal antibodies that specifically bind to the rabies virus glycoprotein, 11 of them were different in sequence. Therefore, this study successfully screened and cloned 11 antibodies with different antibody light and heavy chain variable region sequences and their combinations. The heavy chain variable regions were mainly classified into IgG VH4 and VH3 families, and their light chain variable regions were mainly classified. For the IgG VL1, VL2, VL3 and VK1 families, 11 human Fab monoclonal antibodies that specifically bind to rabies virus glycoprotein were named RVFabl-l. The sequences of RVFabl, RVab3, RVFab5, RVFab8 and RVFab9 were selected as follows: Table 4 Comparison of amino acid sequences of human anti-rabies virus glycoprotein Fab antibody variable region gene

RVFab5 VH SVNFYWS YIYYSGSTNYNPSLKS ERLTTGAMDV

VL SGSSSNIGSNYVS DN KRPS GTWDSSLSAWV

RVFab8 VH SVNSYWG NFYYSGNTHYNPSL S QSTIGGFFDY

VL TGTSSDIGNYNLVS EVT RPS SSYTAT NYWI

RVFab9 VH SVNSYWG NFYYSGNTHYNPSLKS QSTIGGFFDY

VL TGTSSDVGSYNLVS EGS RPS CSYAGSSTWV

VR, variable region; VH, heavy chain in VR (heavy chain variable region); VL, light chain in VR

(light chain variable region).

CDR, complementarity determining region

3. Specific binding of human anti-rabies virus Fab antibody to rabies virus glycoprotein In order to confirm the binding specificity of the obtained recombinant Fab antibody to different rabies virus glycoproteins, we further identified prokaryotic by indirect immunofluorescence assay (IFA). Express the functional activity of the Fab antibody. As shown in Figure 1, RVFabl was positive for glycoprotein immunofluorescence of four rabies virus strains of ERA, CVS, CTN and aG strains, negative for normal sf9 cells, and the other 10 human Fab antibodies with different rabies The glycoprotein immunofluorescence results were identical to those of RVFabl. The results of immunofluorescence showed that 11 Fab-positive antibodies obtained from antibody library screening were directed against rabies virus glycoprotein, and the response spectrum was broad.

4. Whole antibody IgG expression and purification

The light chain and heavy chain Fd genes of five Fab antibodies (RVFab 1, RVFab3, RVFab5, RVFab8, RVFab9) that have been functionally identified were cloned into the whole antibody expression vector pAC-L-Fc and transfected into insect Sf cells. The secreted expression of whole antibodies was achieved using a baculovirus/insect cell system, designated as RVIgG1, RVIgG3, RVIgG5, RVIgG8 and RVIgG9, respectively. The expression supernatant was directly purified by Amersham's Protein-A affinity chromatography column. The expression and purification of the whole antibody IgG were confirmed by SDS-PAGE. The results showed that the pure protein was obtained, and the antibody after melting was clearly observed. , heavy chain, located at about 28kD, 55kD. as shown in picture 2.

5. Human anti-rabies virus antibody rapid immunofluorescence inhibition assay (RFFIT) To further study 5 full-antibody IgGs (RVIgGl, RVIgG3, RVIgG5, The neutralizing activity of RVIgG8, RVIgG9), we used a rapid immunofluorescence inhibition assay to detect the neutralization reaction of the antibody in vitro with the international standard challenge strain CVS-11, and the results showed that 5 people were resistant to rabies virus. The neutralizing titers of the antibodies RVIgGl, RVIgG3, RVIgG5, RVIgG and RVIgG9 were 866.6 IU/mg, 813.3 IU/mg, 689.8 IU/mg, 876.6 IU/mg, 428.5 IU/mg, respectively, according to the WHO Rabies Expert Committee. When the antibody level is equal to or higher than 0.5 IU/ml, the antibody has neutralizing activity, and thus the five human monoclonal antibodies obtained in the present study have neutralizing activity against rabies virus. Among them, the neutralizing activity of RVIgG9 was weak, and the other 4 strains had good neutralizing activity, and fully possessed the ability to neutralize the international standard attack strain CVS-11, as shown in Fig. 3.

6. Effect of antibody pairs after mutation in non-hypervariable region on rabies virus resistance

According to the above methods 8~11, the light chain gene and heavy chain gene modified based on RVIgG8 were cloned into pAC-L-Fc, and transfected into insect Sf cells, and full antibody secretion was achieved by baculovirus/insect cell system. Expression of the type, the mutant RVIgG8' was obtained. Immunological detection of the mutant, indirect immunofluorescence experiments showed that RVFab8' can specifically target rabies virus glycoprotein G, and use rapid immunofluorescence inhibition assay to detect antibody in vitro and international standard challenge strain CVS-11 strain. The neutralization reaction showed that the properties were substantially the same as those of RVIgG8. Industrial applicability

The human neutralizing anti-rabies virus glycoprotein genetically engineered antibody variable region gene, the Fab antibody gene and the whole antibody gene under the characteristics of each of the above antibody genes obtained by the present invention can be used in prokaryotic cells, yeast cells, eukaryotic cells and The expression and production of this antibody in any recombinant system, or any other gene containing this antibody gene based on this, obtains an antibody product having a neutralizing rabies virus infection, and is clinically used for the prevention and treatment of rabies. Specific antibody drugs.

Claims

Claim

A human anti-rabies virus glycoprotein neutralizing antibody, characterized in that the amino acid sequences of the light chain CDR1, CDR2 and CDR3 and the heavy chain CDR1, CDR2 and CDR3 are shown in the following table:

The antibody according to claim 1, wherein the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence are as shown in SEQ ID No. 1 and SEQ ID No. 2.

The antibody according to claim 2, which is a single-chain antibody ScFv or a full-antibody immunoglobulin IgG.

4. A derivative antibody engineered according to any one of claims 1 to 3, which comprises deletion, substitution and/or insertion of an amino acid without altering the activity of the antibody.

5. A gene encoding the antibody of any one of claims 1-4.

6. The gene according to claim 5, wherein the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region are SEQ ID No. 3 and SEQ ID No. 4. Shown.

7. An expression vector comprising the gene of claim 5 or 6.

8. A host comprising the expression vector of claim 7.

Use of the antibody according to any one of claims 1 to 4 for the preparation of a medicament for preventing or treating rabies.

10. A medicament or detection reagent comprising the antibody of any one of claims 1-4.