CN116621975A - Humanized monoclonal antibody aiming at Nipah virus G protein and application thereof - Google Patents

Abstract

The antibody provided by the application can specifically and high-affinity bind with the nipah virus envelope protein, has effective neutralization activity on the nipah virus taking VSV as a framework, can effectively neutralize the nipah virus on a cellular level, and can completely protect hamsters infected with the nipah virus. Which are directed against epitopes different from the neutralizing epitopes reported. Therefore, the series of antibodies are hopefully used as novel specific antibody medicines for preventing and treating the Nipah virus, and provide more choices for preventing and controlling the Nipah virus.

Description

Humanized monoclonal antibody aiming at Nipah virus G protein and application thereof

Technical Field

The application relates to the technical field of biomedical engineering, in particular to a humanized monoclonal antibody aiming at Nipag virus G protein and application thereof.

Background

Nipah virus (NiV) is a negative strand RNA virus belonging to Paramyxoviridae, henry. Nipah virus has a wide host range and can infect multiple species of mammals, and in the natural ecosystem, the virus can infect humans through natural host bats in two ways, firstly, can be transmitted to humans through intermediate host poultry, and the other way is that the direct contact of humans and bats causes infection. Once infected, can cause acute, highly fatal infectious diseases with central nervous system, respiratory system as the primary pathology. Since the first infection of nipah virus in malaysia in 1988, the number of nipah virus infections published by the world health organization has reached 336, the mortality has reached 260, and the mortality has reached 77%. Due to the high virulence, broad host range of nipah viruses, and the current lack of specific effective prophylactic and therapeutic drugs, these are classified as Biosafety Level 4 pathogens.

The genome of nipah virus is an approximately 18kb single-stranded negative-strand RNA molecule encoding 6 proteins. Two of the protein adhesion proteins G and fusion protein F are major surface glycoproteins of pathogens. The G protein is a type II transmembrane glycoprotein, which is in tetrameric form on the viral surface. G-H loop of cell membrane surface protein erphrinB2/3 is inserted into the hydrophobic groove of G protein, and through hydrophobic interaction, the virus is attached to the cell surface to mediate the combination of the virus and the cell; the F protein promotes fusion of the virus with the cell membrane and fusion of the membrane of the infected cell with the membrane of the adjacent cell at the initial stage of the infection cycle to form a characteristic syncytium. The adhesion protein G binds to the cell surface receptor and acts with F to induce a conformational change in the F protein, which causes membrane fusion to occur, thereby releasing the viral ribonucleoprotein complex into the cytoplasm of the host cell.

Phage display technology is to fuse the displayed gene with phage self-protein gene (gIII or gVIII) and display it on the surface of phage. Can build a large capacity (10 ¹⁰ ～10 ¹¹ ) The display library of the system is screened, and the system is more and more valued by the unique advantages of high speed, short period and the like. The recombination of antibody molecule gene level can obtain various specific mouse-source, humanized and fully human antibody molecules and fragments. The technical field of phage display technology and antibody genetic engineering combined generation is currently in the fieldThe rapid development stage, the technology enables the development and research of the fully human monoclonal antibody to step into a substantial application stage and a development stage from a basic research stage, and brings new hopes for preventing and treating infectious diseases.

Disclosure of Invention

Aiming at the problems in the prior art, the application provides a humanized monoclonal antibody aiming at Nipagin G protein and application thereof.

The application uses human sourcesThe Fab phage display library uses Nipah virus G protein as antigen, carries out panning (panning) of anti-Nipah virus humanized antibody, uses a prokaryotic expression system to express and purify to obtain candidate clone protein, and carries out identification and evaluation on biological specificity, pseudovirus neutralization activity on cell level and protection effect on infected animals to obtain four fully human Nipah virus antibodies NiV41, 41-4, 41-6 and 41-9.

Specifically, the heavy chain variable region of the NiV41 antibody contains the amino acid sequence shown in Seq ID No.4 with amino acid sequences shown in SEQ ID No. 1-3 at positions 26-33, 51-58 and 97-120, respectively, while 41-4, 41-6 and 41-9 have identical heavy chains and light chains are different, the light chain variable region having three CDRs, LCDR1, LCDR2 and LCDR 3.

The light chain of NiV41 comprises the amino acid sequence shown as SEQ ID No.8, LCDR1 comprises the amino acid sequence shown as SEQ ID No.5, LCDR2 comprises the amino acid sequence shown as SEQ ID No.6, and LCDR3 comprises the amino acid sequence shown as SEQ ID No. 7.

41-4 comprising the amino acid sequence shown in SEQ ID No.12, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID No.9, LCDR2 comprises the amino acid sequence shown in SEQ ID No.10, and LCDR3 comprises the amino acid sequence shown in SEQ ID No. 11.

41-6, wherein LCDR1 comprises the amino acid sequence shown as SEQ ID No.16, LCDR2 comprises the amino acid sequence shown as SEQ ID No.14, and LCDR3 comprises the amino acid sequence shown as SEQ ID No. 15.

41-9 comprises the amino acid sequence shown as SEQ ID No.20, LCDR1 comprises the amino acid sequence shown as SEQ ID No.17, LCDR2 comprises the amino acid sequence shown as SEQ ID No.18, and LCDR3 comprises the amino acid sequence shown as SEQ ID No. 19.

The application also discloses an antigen binding fragment, which comprises the antibody heavy chain variable region aiming at the Nipah virus G protein, and a bispecific antibody, which comprises the antibody heavy chain variable region aiming at the Nipah virus G protein, or the Nipah virus antibody.

The application also discloses application of the antibody heavy chain variable region aiming at the Nipah virus G protein, the Nipah virus antibody and the antigen binding fragment in preparing preventive and therapeutic drugs aiming at the Nipah virus, detection probes, fusion polypeptides, fusion proteins, immunoconjugates and genetically engineered host cells.

The fully human monoclonal antibody against the Nipah virus provided by the application is obtained by the following method: first, constructing human sourceAnd (3) the Fab phage display library, and then 4 rounds of screening by taking the extracellular domain of the nipah virus envelope protein expressed by a mammalian cell expression system as an antigen, so as to obtain an enriched and high-affinity clone NiV41. The clone is expressed and purified, and is identified and evaluated, and the biological atopy, the pseudovirus neutralization activity on the cellular level and the infected animal protection effect of the clone are identified and evaluated, so that the antibody can effectively neutralize the Nipagin. Meanwhile, an affinity maturation library is constructed by a light chain substitution mode, and three clones 41-4, 41-6 and 41-9 which are enriched and have high affinity are obtained by taking the extracellular domain of the nipah virus envelope protein as an antigen and screening. The structure prediction of antigen-antibody complex based on Alpha Fold2 shows (see figure 6), that the series of antibodies mainly act through heavy chain, the heavy chain CDR3 acts on the central recess of antigen protein to compete for binding to envelope protein G protein and receptor binding site, thereby inhibiting virusInfection.

The beneficial effects of the application are as follows: the provided antibody can specifically bind with high affinity to the nipah virus envelope protein, has effective neutralization activity on the nipah pseudovirus taking VSV as a framework, can effectively neutralize the nipah virus at a cellular level, and can completely protect hamsters infected with the nipah virus. Which are directed against epitopes different from the neutralizing epitopes reported. Therefore, the series of antibodies are hopefully used as novel specific antibody medicines for preventing and treating the Nipah virus, and provide more choices for preventing and controlling the Nipah virus.

Drawings

Fig. 1: and (5) expressing and purifying the nipah virus envelope protein G protein. The target protein is detected by polyacrylamide gel electrophoresis (SDS-PAGE), lane M is a molecular weight standard, and lane G-Fc is the target protein.

Fig. 2: niV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG antibodies were purified and then detected by SDS-PAGE. Lanes NiV41, 41-4, 41-6, 41-9 are the proteins of interest.

Fig. 3: binding of NiV41 IgG, 41-4IgG, 41-6IgG to 41-9IgG and Nipah virus envelope protein as determined by ELISA. The binding concentration EC50 of NiV41 IgG and antigen protein was 1.8. Mu.g/ml, the binding concentration EC50 of 41-4IgG and antigen protein was 0.21. Mu.g/ml, the binding concentration EC50 of 41-6IgG and antigen protein was 0.1. Mu.g/ml, and the binding concentration EC50 of 41-9IgG and antigen protein was 0.06. Mu.g/ml.

Fig. 4: niV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG and Nipag pseudovirus experiments. The neutralizing activity IC50 of NiV41 IgG and Nipah pseudovirus is 0.01 mug/ml, the neutralizing activity IC50 of 41-4IgG and Nipah pseudovirus is 0.005 mug/ml, the neutralizing activity IC50 of 41-6IgG and Nipah pseudovirus is 0.002 mug/ml, and the neutralizing activity IC50 of 41-9IgG and Nipah pseudovirus is 0.002 mug/ml.

Fig. 5: niV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG and Nipag live virus experiments. The neutralizing activity IC50 of NiV41 IgG and Nipah live virus is 0.33 mug/ml, the neutralizing activity IC50 of 41-4IgG and Nipah live virus is 0.136 mug/ml, the neutralizing activity IC50 of 41-6IgG and Nipah live virus is 0.088 mug/ml, and the neutralizing activity IC50 of 41-9IgG and Nipah live virus is 0.059 mug/ml.

Fig. 6:41-6 interact with the antigenic protein NiV-G. Antigen-antibody complex structure prediction was performed by Alpha Fold2 prediction software, and antibody 41-6 was involved in the interaction with antigen primarily through the CDR3 region of the heavy chain.

Fig. 7: niV41 IgG, 41-6IgG animal protection experiments. NiV41 IgG provides complete therapeutic protection against infected animals, 41-6IgG provides complete prophylactic protection against infected animals, and the negative control is PBS.

Detailed Description

The present application will be described in detail with reference to the following examples and the accompanying drawings, wherein the following examples are given by way of illustration of the present application and specific embodiments and specific procedures thereof, but the scope of the present application is not limited to the following examples.

Example 1: expression and purification of nipah virus envelope proteins

According to the gene sequence of Nipag virus (GenBank NC-002728.1), the extracellular domain (amino acids 188-602) of the envelope protein is spliced with the Fc gene sequence of IgG1 type, and the spliced gene is connected with the vector pSecTag2A for transformation to construct the vector pSecTag2A-G-Fc. 293F cells (control cell density 5 to 10) were grown 1 day prior to transfection ⁵ 40 mL) was inoculated into 125mL suspension cell culture flasks. Mu.g of plasmid (pSecTag 2A-G-Fc) was diluted and gently homogenized in 4mL of DPBS buffer, and 120. Mu.L of PEI (polyethylenimine) was further diluted and gently homogenized in the culture medium. After incubation for 20 minutes at room temperature, they were added dropwise to the cells. The cells were placed in a suspension incubator at 125 rpm and incubated at 37 ℃.

After 144 hours, culture supernatants were collected and expression of antigen proteins was detected by Western blotting (Western Blot) using anti-Fc as primary antibody.

After the detection of G-Fc expression, the cell culture and transfection scale was enlarged and the G-Fc protein was expressed in large amounts. The culture supernatant was collected, the target protein was purified by protein A packing, and then the buffer was replaced by ultrafiltration with an ultrafiltration centrifuge tube having a molecular weight cut-off of 10kDa, and the purity was confirmed by SDS-PAGE, and the results are shown in FIG. 1.

Example 2: construction and screening of phage display libraries

Screening was performed with antigens expressed by mammalian cells using the constructed phage library. After the purified antigen is incubated in a 96-well plate at 4 ℃ overnight, panning is performed in a phage library, specific phage are captured by the antigen, PBS+0.05% Tween-20 is used for cleaning, and enrichment clone is obtained after 4 rounds of screening and named NiV41.

The gene encoding the antibody light chain fragment is obtained and amplified in large quantity by PCR on donor RNA which is derived from healthy and non-immune, a new affinity maturation library of light chain shuffling is obtained by enzyme digestion connection mode on the basis of the antibody gene of parent clone NiV41, and G-Fc protein is used for screening to obtain enrichment clones named 41-4, 41-6 and 41-9 respectively.

Example 3: expression purification of NiV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG

The heavy and light chains of NiV41 Fab, 41-4Fab, 41-6Fab and 41-9Fab were constructed into the IgG1 expression vector pivotr 2-neo-mcs (commercially available), expressed by PEI transfection of 293F cells, the expression supernatant was purified with protein A filler, and then the buffer was ultrafiltration-replaced with an ultrafiltration centrifuge tube of 30kDa cut-off, and the purity was confirmed by SDS-PAGE, as shown in FIG. 2.

Example 4: ELISA assay for binding of NiV41 IgG, 41-4IgG, 41-6IgG to 41-9IgG and envelope protein

Envelope proteins (4. Mu.g/mL) were coated on ELISA plates and incubated overnight at 4℃and blocked with PBS+3% mill for 1h at 37 ℃. Serial dilutions of antibodies were added, incubated for 2 hours at 37 ℃ and then washed four times with PBST (pbs+0.05% tween 20), and horseradish peroxidase (HRP) -labeled murine anti-human Fc monoclonal antibody was added, incubated for 1 hour at 37 ℃ and then washed four times with PBST and then ABTS was added for detection. Binding affinities of the individual antibodies were calculated by the data analysis software Prism fit. The results are shown in FIG. 3.

Example 5: niV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG antibodies neutralized Nipajamas in Vero cells.

mu.L of Vero cells (1X 10) ⁵ Each ml) was inoculated into a 96-well plate and cultured for 14 to 16 hours. Pressing the buttonAn equal volume of Nipah virus pseudovirus was mixed with the antibody dilution at an initial final antibody concentration of 100. Mu.g/ml, diluted in a three-fold gradient, and incubated for 1 hour at 37 ℃. The cell culture medium in the 96-well plate was discarded, and the cells were infected with a virus and antibody mixture, and were subjected to stationary culture at 37℃for 24 hours. The following day the cells were observed for fluorescence under a fluorescence microscope. By high content scanning, the fluorescence intensity of each well plate was analyzed, the weaker the fluorescence, indicating that fewer cells were infected with pseudoviruses, i.e., more pseudoviruses were neutralized by the antibody. Half maximal inhibitory activity (IC 50) of each antibody was calculated by data analysis software Prism. The results are shown in FIG. 4.

Example 6: antibodies NiV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG neutralize Nipah live virus.

0.5mL of Vero cells (1X 10) ⁵ Each ml) was inoculated with a 24-well plate and cultured for 14 to 16 hours. According to the constant concentration of 100ug/ml of antibody, three-fold gradient dilution is carried out, and the equal volume of Nipag live virus is mixed with the antibody diluent and incubated for 1 hour at 37 ℃. The cell culture medium in the 24-well plate was discarded, and the cells were infected with a virus and antibody mixture, and were subjected to stationary culture at 37℃for 1 hour. Removing virus-antibody mixed solution in cell plate, adding culture medium containing carboxymethyl cellulose, standing at 37 deg.C for 4-5 days. After the cell well plate was subjected to inactivation treatment, the formation and the number of plaques were observed and counted by crystal violet staining. Fewer plaques indicates fewer cells infected with the virus, i.e., more pseudoviruses neutralized by the antibody. Half maximal inhibitory activity (IC 50) of each antibody was calculated by data analysis software Prism. The results are shown in FIG. 5.

Example 7: interface prediction of interaction of antibody 41-6 with antigen

The sequences of the antibody Fab fragments and the antigen sequences are written into a text, the structure of an antigen antibody complex is predicted in a multimeric form through an Alpha Fold2 database, the structure generated by prediction is analyzed, and the structure with the highest score ranking is selected. Complex interaction interfaces were analyzed in the software PDBePISA. The results are shown in FIG. 6.

Example 8: protection of hamsters infected with Nipag virus by antibodies NiV41 IgG, 41-6IgG

Four-five week old female hamsters were inoculated with a dose of the Bengalenical strain (NiV by intraperitoneal injection _B ) After 6 hours of infection, 300 μg of IgG antibody NiV41 was injected into each experimental animal by intraperitoneal injection, PBS was selected as a negative control, hamster body condition was observed daily and survival was recorded;

four-five week old female hamsters were given 1mg of IgG antibody 41-6 per experimental animal by intraperitoneal injection, PBS as a negative control. One day after prophylactic administration, a dose of malaysia-series nipah virus (NiV _M ). Hamster physical status was observed daily and survival was recorded.

The above experiments were performed in a biosafety quaternary laboratory. The experimental results are shown in FIG. 7.

The present application may be better understood and appreciated by those skilled in the art by reference to the examples. However, the protection of the application and the scope of the claims are not limited to the cases provided. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present application, are within the scope of the present application.

Claims (10)

1. An antibody heavy chain variable region directed against nipah virus G protein, characterized by: the heavy chain variable region has three complementarity determining regions CDRs of HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence shown in SEQ ID No. 1; the HCDR2 comprises an amino acid sequence shown as SEQ ID No. 2; the HCDR3 comprises an amino acid sequence as shown in SEQ ID No. 3.

2. The antibody heavy chain variable region against nipah virus G protein as set forth in claim 1, wherein: the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID No. 4.

3. A nipah virus antibody comprising the antibody heavy chain variable region of claim 1 or 2 directed against a nipah virus G protein.

4. The nipah virus antibody of claim 3 having a light chain, wherein: the variable region of the light chain has three complementarity determining regions CDRs, LCDR1, LCDR2 and LCDR3, selected from the group consisting of:

the LCDR1 comprises an amino acid sequence shown as SEQ ID No. 5; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 6; the LCDR3 comprises an amino acid sequence shown as SEQ ID No. 7; or (b)

The LCDR1 comprises an amino acid sequence shown as SEQ ID No. 9; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 10; the LCDR3 comprises an amino acid sequence shown as SEQ ID No. 11; or (b)

The LCDR1 comprises an amino acid sequence shown as SEQ ID No. 13; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 14; the LCDR3 comprises an amino acid sequence shown as SEQ ID No. 15; or (b)

The LCDR1 comprises an amino acid sequence shown as SEQ ID No. 17; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 18; the LCDR3 comprises an amino acid sequence as shown in SEQ ID No. 19.

5. The nipah virus antibody of claim 4, wherein: the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID No.8, SEQ ID No.12, SEQ ID No.16, or SEQ ID No. 20.

6. An antigen binding fragment comprising the antibody heavy chain variable region of claim 1 or 2 against nipah virus G protein.

7. A bispecific antibody comprising the antibody heavy chain variable region of claim 1 or 2 against nipah virus G protein, or the nipah virus antibody of any one of claims 3-5.

8. Use of the heavy chain variable region of an antibody against nipah virus G protein according to claim 1 or 2 for the preparation of a prophylactic and therapeutic drug against nipah virus, a detection probe, a fusion polypeptide, a fusion protein, an immunoconjugate, a genetically engineered host cell.

9. Use of the nipah virus antibodies of claim 3 or 4 or 5 for the preparation of prophylactic and therapeutic drugs, detection probes, fusion polypeptides, fusion proteins, immunoconjugates, genetically engineered host cells against nipah virus.

10. Use of the antigen binding fragment of claim 6 for the preparation of a prophylactic and therapeutic drug against nipah virus, a detection probe, a fusion polypeptide, a fusion protein, an immunoconjugate, a genetically engineered host cell.