CN118878669A - An anti-chikungunya bispecific antibody 8D1-10D5 and its application - Google Patents

Abstract

The invention provides a camel-source nano-antibody 10D5 combined with chikungunya virus E2 protein, bispecific antibodies 8D1-10D5 based on the camel-source nano-antibody 10D5 and an anti-chikungunya virus humanized monoclonal antibody 8D1, and application of the camel-source nano-antibody 10D5 and the bispecific antibodies 8D1-10D5 in preparation of medicines for preventing or treating chikungunya virus diseases. Compared with the parent monoclonal antibody 8D1, the bispecific antibody provided by the invention has obviously improved neutralization activity, so that the bispecific antibody can be developed into a chikungunya heat specific drug.

Description

An anti-chikungunya bispecific antibody 8D1-10D5 and its application

Technical Field

The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to a bispecific antibody against Chikungunya virus and its use in preparing a drug.

Background Art

Chikungunya fever (CHIΚF) is a disease first discovered in Africa and caused by the Chikungunya virus (CHIΚV). Clinical manifestations include fever, rash, and body pain. It is one of the fastest-spreading mosquito-borne infectious diseases in the world after malaria and dengue fever. Since its discovery in 1953, CHIΚV has spread from Africa to more than 100 countries and regions in the world, infecting millions of people. In 2015, the World Health Organization identified the Chikungunya virus as one of the three sub-hazardous pathogens that may cause serious outbreaks. At present, there is no specific therapeutic drug developed for the Chikungunya virus, so the development of preventive vaccines and specific therapeutic drugs is crucial to the prevention and control of the disease.

At present, some studies have identified a variety of polyclonal or neutralizing monoclonal antibodies (mAbs) that are protective against CHIKV infection. Most of the identified neutralizing monoclonal antibodies against CHIKV target the surface-exposed domains of the E2 glycoprotein. The CHIKV virus E2 protein has three domains, two of which are the main targets of neutralizing antibodies, including the B domain and the adjacent A domain. In addition, protective monoclonal antibodies targeting the E1 protein have been isolated in animals, which function to block the fusion of the virus and host cell membranes and interact with innate immune cells to promote viral clearance. The inventors previously obtained a fully human monoclonal antibody 8D1 with high neutralizing activity against Chikungunya virus from plasma cells of peripheral blood of a Chikungunya fever infected person 15 days after recovery by flow sorting-single cell PCR technology, with an IC ₅₀ value of 0.12 μg/mL (see CN 110903386 A), but its binding activity and affinity to CHIKV E2 protein are relatively weak. There are currently no approved antibodies against CHIKV, so there is an urgent need to develop a new generation of neutralizing antibody drugs that can provide protective activity.

The concept of bispecific antibodies (bsAb) was first proposed in 1960. It is an antibody with two different antigen binding sites constructed by gene recombination, chemical coupling or quadruple hybridoma. This type of antibody can specifically bind to two antigens or two epitopes at the same time. Compared with traditional monoclonal antibodies, BsAb has higher sensitivity and specificity, and has the functions of recruiting immune cells and dual blocking signal pathways. In recent years, BsAb drugs have developed rapidly in the fields of tumor immunotherapy, anti-infection, autoimmune diseases, etc., thus achieving the therapeutic effect of "1+1>2".

The form of an effective recombinant bispecific antibody is crucial to maximizing the effectiveness of antibody therapy. At present, a variety of recombinant bsAb forms have been developed, among which the form of adding nanobody (VHH) structure to the multispecific antibody design is a new direction for drug development. VHH can be freely combined with various forms of antibodies to produce a variety of nano bispecific structures. The structure of VHH is relatively simple, and there is no problem of high production difficulty caused by excessively high proportions of VH and VL fusion errors, which makes the design of multi-antibody drugs more flexible, the production more efficient and stable, and can effectively respond to challenges in the drug development process. The purpose of the present invention is to provide a new bsAb form antibody that combines the advantages of IgG and nanoantibodies to supplement and strengthen existing CHIΚV antibody treatment methods.

Summary of the invention

Based on the above purpose, the present invention first immunizes camels with Chikungunya virus E2 protein (referred to as CHIKV E2 protein or antigen in the present invention) to construct an antibody library, and uses phage display technology to screen and provide a camel-derived nanoantibody against CHIKV E2 protein, wherein the camel-derived nanoantibody comprises a heavy chain variable region, and the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in the sequences at positions 26-33, 51-57 and 96-112 of SEQ ID NO: 1, respectively.

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-CHIKV E2 protein Nanobody is shown in SEQ ID NO: 1. The Nanobody with this specific technical solution in the present invention is named "10D5".

Secondly, the present invention provides a bispecific antibody against Chikungunya virus, wherein the bispecific antibody uses a monoclonal antibody against Chikungunya virus as a parent antibody, and the monoclonal antibody against Chikungunya virus is named "8D1" (monoclonal antibody 8D1 is disclosed in CN 110903386 A, and the disclosure thereof is incorporated into the specification of the present invention by reference). The bispecific antibody against Chikungunya virus is formed by fusing one end of the heavy chain or light chain of monoclonal antibody 8D1 with the camel-derived nanoantibody 10D5 that binds to CHIKV E2 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8D1 against Chikungunya virus is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

In a preferred embodiment, the amino acid sequence of the heavy chain constant region and the amino acid sequence of the light chain constant region of the anti-Chikungunya virus monoclonal antibody 8D1 are shown in SEQ ID NO: 7 and SEQ ID NO: 9, respectively.

In a more preferred embodiment, the fusion is fused in the form of (GGGGS)n connecting peptide, wherein n is a natural number of 1 to 4. Those skilled in the art can select other forms of connecting peptides to achieve the purpose of implementing the present invention.

In a preferred embodiment of the present invention, the bispecific antibody uses the anti-Chikungunya virus monoclonal antibody 8D1 as the parent antibody, and the N-terminus of its heavy chain is fused with the C-terminus of the camel-derived nanoantibody 10D5 that binds to the CHIKV E2 protein, that is, the "VHH(H)-IgG" form. In the present invention, the bispecific antibody is named 10D5-8D1-H; the amino acid sequences of the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 11 and SEQ ID NO: 13, respectively, or,

The N-terminus of the light chain of the anti-Chikungunya virus monoclonal antibody 8D1 is fused to the C-terminus of the camel-derived nanobody that binds to the CHIKV E2 protein, i.e., in the form of "VHH(L)-IgG", and the bispecific antibody in the present invention is named 10D5-8D1-k; the amino acid sequences of the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 15 and SEQ ID NO: 17, respectively, or,

The C-terminus of the heavy chain of the anti-Chikungunya virus monoclonal antibody 8D1 is fused with the N-terminus of the camel-derived nanoantibody that binds to the CHIKV E2 protein, i.e., in the form of "IgG(H)-VHH", the bispecific antibody in the present invention is named 8D1-H-10D5; the amino acid sequences of the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 19 and SEQ ID NO: 13, respectively, or,

The C-terminus of the light chain of the anti-Chikungunya virus monoclonal antibody 8D1 is fused with the N-terminus of the camel-derived nanoantibody that binds to the CHIKV E2 protein, that is, the "IgG (L) -VHH" format. In the present invention, the bispecific antibody is named 8D1-k-10D5; the amino acid sequences of the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 15 and SEQ ID NO: 21, respectively.

Third, the present invention provides a polynucleotide. In a preferred technical scheme, the polynucleotide is a polynucleotide encoding the above-mentioned camel-derived nanoantibody 10D5 that binds to CHIKV E2 protein, and the sequence of the polynucleotide encoding the camel-derived nanoantibody 10D5 that binds to CHIKV E2 protein is shown in SEQ ID NO: 2.

In another preferred technical solution, the polynucleotide is a polynucleotide encoding the above-mentioned anti-Chikungunya virus bispecific antibody, and the polynucleotide sequences encoding the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 12 and SEQ ID NO: 14, respectively. In the present invention, the bispecific antibody having the technical solution is named "10D5-8D1-H"; or,

The polynucleotide sequences encoding the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 16 and SEQ ID NO: 18, respectively. The bispecific antibody having the technical solution in the present invention is named "10D5-8D1-κ"; or,

The polynucleotide sequences encoding the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 20 and SEQ ID NO: 14, respectively. The bispecific antibody having the technical solution in the present invention is named "8D1-H-10D5"; or,

The polynucleotide sequences encoding the heavy chain and light chain of the anti-Chikungunya virus bispecific antibody are shown in SEQ ID NO: 16 and SEQ ID NO: 22, respectively. The bispecific antibody having the technical solution in the present invention is named "8D1-κ-10D5".

In a preferred embodiment of the present invention, the polynucleotide sequence encoding the heavy chain constant region and the polynucleotide sequence encoding the light chain constant region of the anti-Chikungunya virus monoclonal antibody 8D1 in the anti-Chikungunya virus bispecific antibody structure are shown in SEQ ID NO: 8 and SEQ ID NO: 10, respectively.

Fourth, the present invention provides a vector containing the above-mentioned polynucleotide. In a preferred technical solution, the vector is a vector containing the above-mentioned polynucleotide encoding the camel-derived nanoantibody 10D5 that binds to the CHIKV E2 protein. Preferably, the sequence of the polynucleotide is shown in SEQ ID NO. 2. In a specific embodiment of the present invention, the vector is a pcDNA3.4 vector. As known to those skilled in the art, other eukaryotic expression vectors can also be used to express the camel-derived nanoantibody 10D5 that binds to the CHIKV E2 protein described in the present invention.

In another preferred technical solution, the vector is a vector containing a polynucleotide encoding the above-mentioned anti-Chikungunya virus bispecific antibody. In a specific embodiment of the present invention, the vector is a pcDNA3.4 vector. As known to those skilled in the art, other eukaryotic expression vectors can also be used to express the anti-Chikungunya virus bispecific antibody vector of the present invention.

Fifth, the present invention provides a host cell containing the above-mentioned vector. In a preferred embodiment, the host cell is a host cell containing the above-mentioned vector containing the camel-derived nanobody 10D5 that binds to the CHIKV E2 protein. In a specific embodiment of the present invention, the host cell is an Expi 293F cell. As known to those skilled in the art, other eukaryotic expression host cells, such as CHO cells, can also be used to express the camel-derived nanobody 10D5 that binds to the CHIKV E2 protein described in the present invention.

In another preferred embodiment, the host cell is a host cell containing the vector of the anti-Chikungunya virus bispecific antibody described above. In a specific embodiment of the present invention, the host cell is an Expi 293F cell. As known to those skilled in the art, other eukaryotic expression host cells, such as CHO cells, can also be used to express the anti-Chikungunya virus bispecific antibody described in the present invention.

Sixth, the present invention provides the use of the camel-derived nanoantibody 10D5 that binds to CHIKV E2 protein in the preparation of a drug for preventing or treating chikungunya virus disease.

Finally, the present invention provides the use of the above-mentioned bispecific antibody against Chikungunya virus in the preparation of a drug for preventing or treating Chikungunya virus disease.

The present invention provides a camel-derived nanoantibody 10D5 that binds to CHIKV E2 protein. Due to the different binding activities of the human monoclonal antibody 8D1 and nanoantibody 10D5 against Chikungunya virus in the prior art with the E2 antigen, it is suggested that the two antibodies may have different binding targets with the CHIKV antigen. Based on this, the present invention uses human monoclonal antibody 8D1 and nanoantibody 10D5 as parent antibodies, designs bsAbs in the form of VHH(H)-IgG, VHH(L)-IgG, IgG(H)-VHH, and IgG(L)-VHH, and its specific antibody schemes are 10D5-8D1-H, 10D5-8D1-κ, 8D1-H-10D5, and 8D1-κ-10D5. The purpose is to utilize the advantages of bispecific antibodies, not only to better retain the specific functions of parent monoclonal antibody 8D1 and nanoantibody 10D5, but also to synergize the biological functions of the two parent antibodies, showing more obvious advantages in affinity, binding and blocking activity, and neutralizing activity against viruses, so as to provide candidate drugs for the prevention and treatment of CHIKF. The data of the embodiments of the present invention show that the bispecific antibodies have good affinity to the CHIKVE2 antigen. The data of the embodiments of the present invention show that the bispecific antibodies have good affinity to the CHIKVE2 protein, among which 10D5-8D1-H has the strongest affinity to the CHIKVE2 protein, reaching below 1.0E-12. In the protection effect and _IC50 evaluation on the cell model, compared with the antibodies in the prior art, the neutralizing activities of the bispecific antibodies 8D1-H-10D5, 10D5-8D1-κ, 8D1-κ-10D5 and the monoclonal antibody 8D1 are significantly improved; compared with the parent monoclonal antibody 8D1, The neutralizing activity of bispecific antibodies 8D1-H-10D5 and 10D5-8D1-κ was improved, among which 8D1-H-10D5 was more significantly improved, about 4.3 times that of monoclonal antibody 8D1, and 10D5-8D1-κ was 2.4 times that of monoclonal antibody 8D1, making it possible to develop it into a specific drug for Chikungunya fever.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: SDS-PAGE detection results of CHIΚV E2 protein (separation gel concentration 12%);

Figure 2: SDS-PAGE detection results of nanobody 10D5-Fc (separation gel concentration 12%);

FIG3 is a schematic diagram of the structure of a bispecific antibody, wherein FIG3A is 10D5-8D1-H, FIG3B is 8D1-H -10D5, FIG3C is 10D5-8D1-k, and FIG3D is 8D1- k -10D5;

Figure 4: SDS-PAGE test results of bispecific antibody and monoclonal antibody 8D1 (separation gel concentration 12%);

Figure 5: ELISA binding curves of bispecific antibody, monoclonal antibody 8D1, nanobody 10D5 and CHIKV E2 protein;

Figure 6: Binding kinetic curves of monoclonal antibody 8D1, bispecific antibodies 8D1-H-10D5 and 8D1-κ-10D5 to CHIKV E2 protein;

Figure 7: Binding kinetics of nanobody 10D5, bispecific antibodies 10D5-8D1-H and 10D5-8D1-κ to CHIKV E2 protein;

Figure 8: Neutralization activity of bispecific antibodies, parental antibodies and control antibodies against true CHIKV virus.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

Example 1. Screening and preparation of nanobodies against CHIKV E2 protein

1. Expression and purification of CHIKV E2 protein

The present invention adopts the insect cell-baculovirus system to express CHIKV E2 protein. First, the CHIKV E2 protein gene sequence (GenBank: AAU43881.1) is optimized according to the insect cell codon preference, and 6 histidine tags (N-terminal) are added, and then the gene fragment is synthesized by artificial methods and cloned into the pFastBac1 vector (insect cell baculovirus expression vector) by seamless cloning method. The recombinant plasmid was transformed into DH10Bac competent cells. After blue-white screening and PCR identification, the recombinant baculovirus bacmid (Bacmind) was obtained. The recombinant baculovirus bacmid was extracted by isopropanol precipitation method, and then Bacmind was transfected into Expi Sf21 insect cells using transfection reagent (ExpiFectamine ^TM Sf Transfection Reagent, A38915). After 96 hours of incubation at 27°C and 120 rpm in a shaking incubator, the culture supernatant was collected, centrifuged, and stored in aliquots at -80°C to obtain CHIΚV E2 protein P0 virus. Subsequently, the P0 virus was transferred to Expi Sf9 cells to amplify the virus. After further cultivation for 4 days, the supernatant was collected and purified by nickel ion affinity chromatography (HisTrap ^TM excel) to obtain CHIΚV E2 protein. Its expression and purity were identified by SDS-PAGE, as shown in Figure 1. The results showed that the molecular weight of the purified CHIΚV E2 protein was 50 kDa, which was in line with expectations and the purity also met the requirements.

2. Camel immunization and establishment of VHH nanobody library

1 mg of CHIKV E2 protein with 6 histidine tags prepared in the early stage was diluted to a final volume of 1 mL with PBS, emulsified with 1 mL of Freund's complete adjuvant, and mixed and injected subcutaneously at multiple points for the first immunization of camels. Immunization was then performed every two weeks. After the fifth immunization, the serum titer was detected by ELISA, and the serum titer was >10 ^5. Then 100 mL of blood was collected to separate peripheral blood mononuclear cells (PBMC). The total RNA was extracted from the separated PBMC according to the instructions of QIAGEN's Neasy® Plus MiniRNA Extraction Kit. Using the extracted total RNA as a template, cDNA inversion was performed according to the instructions of Invitrogen's Super Script®III First-Strand Synthesis System Extraction Kit. Gene template amplification was performed using the inverted cDNA library, and the first round of PCR reaction was performed using primers CALL001 and CALL002 (primer sequences are shown in Table 1), and the target band was recovered by running the gel. The recovered DNA was used as a template for secondary amplification, using VHH-FOR and VHH-REV primers (primer sequences are shown in Table 1), and the corresponding bands of VHH were recovered by running gel. Subsequently, the PCR product was double-digested with PstⅠ-HF enzyme and Not-Ⅰ enzyme, cloned into the pMECS phage display vector, and the ligation product was recovered using the PCR product recovery kit according to the operating steps in the instruction manual, transformed into competent cells TG1, and coated on a selective medium containing ampicillin. After overnight culture at 37°C, all colonies were collected in LB medium, centrifuged and the supernatant was discarded, and the cells were resuspended in LB, which was the antibody library.

Table 1. Reaction primers

3. Phage display technology to screen specific nanoantibodies

Take 100 μL of phage display vector library and inoculate it into 100 mL 2×YT/AMP GLU medium. Culture it at 37 ℃ and 200 r/min until the logarithmic phase (OD ₆₀₀ value is 0.6-0.8). Add 90 μL M13Κ07 helper phage (1.7×10 ¹³ PFU/mL), mix gently, and culture it at 37 ℃ overnight. Distribute the culture solution into 50 mL centrifuge tubes, centrifuge at 3800 g and 4 ℃ for 30 min, carefully collect the supernatant with a pipette (avoid inhaling the precipitate), add 1/5 volume of pre-cooled PEG/NaCl solution, mix it upside down, and let it stand on ice for 2 h; centrifuge it at 3800 g and 4 ℃ for 30 min, discard the supernatant, add 0.5 mL PBS to each tube, resuspend the phage precipitate, which is the collected phage particles, and determine the phage titer.

The CHIΚV E2 protein was diluted to 4 μg/mL with PBS buffer. Three duplicate wells of a 96-well ELISA plate were selected and 100 μL (400 ng/well) was added to each well. The plates were coated overnight at 4°C, and PBS was used as a negative control. The coating solution was discarded, and 150 μL 2% skim milk powder was added to each well. The plates were blocked for 1 h at room temperature. The plates were washed 4 times with PBST. The phage solution collected above was diluted to 5×10 ¹¹ PFU/mL with 2% skim milk powder, and added to the ELISA plate at 100 μL/well. The plates were incubated at room temperature for 2 h. The phage samples were discarded, washed 10 times with PBST, and then washed 5 times with PBS. 100 μL freshly prepared 0.1 M triethylamine was added to each well. The plates were allowed to stand at room temperature for 10 min. The eluate was aspirated and quickly neutralized with an equal volume of 1 M Tris-HCl (pH 7.4). 400 μL of the eluate was then taken and infected with 4 mL logarithmic phase TG1 cells, phage amplification. Prepare 96-well plates coated with CHIKV E2 protein again, and perform the second and third rounds of panning. Randomly pick 96 monoclones from the bacterial plate after the third round of screening, and after inducing VHH antibody expression with IPTG, prepare soluble recombinant VHH antibody crude extracts for ELISA detection, preliminarily identify the reactivity of VHH antibody clones with CHIKV-E2 protein, and judge as positive when OD ₄₅₀ value is more than 3 times of PBS control. Take the positive clone part of the bacterial solution for sequencing, and obtain the nucleic acid coding sequence of 10D5 clone as shown in SEQ ID NO:2, that is, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 amino acid sequences of the heavy chain variable region are shown in SEQ ID NO:1 amino acids 26-33, 51-57, 96-112, respectively.

4. Expression and purification of nanobody 10D5

The Fc sequence was added to the C-terminus of the nucleic acid coding sequence of 10D5, and it was constructed into the pcDNA3.4 vector through the restriction enzyme sites Eco RI and EcoRV, and transfected into Expi 293F cells. After 5 days of culture, the supernatant was collected, centrifuged at 3000 g for 30 minutes, filtered through a 0.22 μm filter membrane, and purified by Protein A column to obtain a relatively pure target protein. Its expression and purity were identified by SDS-PAGE. The results are shown in Figure 2, and the purified nanoantibody 10D5-Fc was obtained with a molecular weight of 40 kDa, which was in line with expectations.

Example 2. Construction of bispecific antibodies

1. Construction of bispecific antibodies

8D1 is a monoclonal antibody against Chikungunya virus, whose heavy chain variable region sequence is shown in SEQ ID NO:3, the nucleic acid coding sequence is shown in SEQ ID NO:4, the light chain variable region sequence is shown in SEQ ID NO:5 (note in the sequence list should be 8D1 VL), the nucleic acid coding sequence is shown in SEQ ID NO:6, the heavy chain constant region sequence is shown in SEQ ID NO:7, the nucleic acid coding sequence is shown in SEQ ID NO:8, the light chain constant region sequence is shown in SEQ ID NO:9, and the nucleic acid coding sequence is shown in SEQID NO:10 (see CN 110903386 A). The (G4S) ₂ linker was used to connect the VHH of 10D5 and 8D1 to synthesize the sequence, which was cloned into the eukaryotic expression vector pcDNA3.4 by enzyme digestion and homologous recombination. The construction forms included VHH(H)-IgG, IgG(H)-VHH, VHH(L)-IgG, and IgG(L)-VHH (175 kDa), as shown in Figure 3. The dark gray is the 8D1 heavy chain constant region, the dark blue is the 8D1 heavy chain variable region, the light gray is the 8D1 light chain constant region, the light blue is the 8D1 light chain variable region, and the red is the 10D5 heavy chain variable region. The key maps are as follows:

(1) VHH connected to the N-terminus of the IgG heavy chain (VHH(H)-IgG):

Eco RI restriction site -GCCGCCACC-start codon-signal peptide-10D5-VHH-(G4S)2-8D1-VH-CH-stop codon- Eco RV restriction site (Figure 3A); The newly synthesized plasmid was then co-transfected with the light chain expression plasmid of 8D1 into Expi 293F cells to express the bispecific antibody, referred to in the present invention as: 10D5-8D1-H (the heavy chain sequence is shown in SEQ ID NO:11, the nucleic acid coding sequence is shown in SEQ ID NO:12, the light chain sequence is shown in SEQ ID NO:13, and the nucleic acid coding sequence is shown in SEQ ID NO:14).

(2) The C-terminus of the IgG heavy chain is connected to VHH (IgG(H)-VHH):

Eco RI restriction site -GCCGCCACC-start codon-signal peptide- 8D1-VH-CH -(G4S)2- 10D5-VHH -stop codon- Eco RV restriction site (Figure 3B); the newly synthesized plasmid was then co-transfected with the light chain expression plasmid of 8D1 into Expi 293F cells to express the bispecific antibody, referred to in the present invention as: 8D1-H-10D5 (the heavy chain sequence is shown in SEQ ID NO: 19, the nucleic acid coding sequence is shown in SEQ ID NO: 20, the light chain sequence is shown in SEQ ID NO13, and the nucleic acid coding sequence is shown in SEQ ID NO: 14).

(3) VHH connected to the N-terminus of IgG light chain (VHH(L)-IgG):

Eco RI restriction site -GCCGCCACC-start codon-signal peptide-10D5-VHH-(G4S)2-8D1-VL-CL-stop codon- Eco RV restriction site (Figure 3C); the newly synthesized plasmid was then co-transfected with the heavy chain expression plasmid of 8D1 into Expi 293F cells to express the bispecific antibody, referred to in the present invention as: 10D5-8D1-κ (the heavy chain sequence is shown in SEQ ID NO: 15, the nucleic acid coding sequence is shown in SEQ ID NO: 16, the light chain sequence is shown in SEQ ID NO: 17, and the nucleic acid coding sequence is shown in SEQ ID NO: 18).

(4) VHH connected to the C-terminus of IgG light chain (IgG(L)-VHH):

Eco RI restriction site -GCCGCCACC-start codon-signal peptide- 8D1-VL-CL -(G4S)2- 10D5-VHH -stop codon- Eco RV restriction site (D in Figure 3); the newly synthesized plasmid was then co-transfected with the heavy chain expression plasmid of 8D1 into Expi 293F cells to express the bispecific antibody, referred to in the present invention as: 8D1-κ-10D5 (the heavy chain sequence is shown in SEQ ID NO: 15, the nucleic acid coding sequence is shown in SEQ ID NO: 16, the light chain sequence is shown in SEQ ID NO: 21, and the nucleic acid coding sequence is shown in SEQ ID NO: 22).

2. Transient Expression and Affinity Chromatography Purification of Bispecific Antibodies

Using the Expi 293 expression system, 15 μg of heavy chain and 15 μg of light chain were mixed and transfected into 30 mL of Expi 293F cells. Transient expression was performed according to the instructions (ThermoFisher Scientific, A14635). The supernatant was collected after 4 days. A 5 mL pre-packed Protein A affinity column was used. Before loading, it was balanced with 20 mM PBS. After the conductivity reached the baseline, the sample was injected. After the loading was completed, the column was washed with 20 mM PBS until the baseline was stable. The target protein was eluted with 0.1 M pH 2.7 glycine buffer. After UV280 was close to the baseline, the collection was stopped. Subsequently, ultrafiltration was performed through an ultrafiltration concentration tube, and the buffer was replaced with PBS to obtain the pure recombinant protein of the bispecific antibody, and its expression and purity were identified by SDS-PAGE. As shown in Figure 4: the heavy chain of antibodies 10D5-8D1-H and 8D1-H-10D5 is about 65 kDa, the light chain is about 25 kDa, the heavy chain of antibodies 10D5-8D1-κ and 8D1-κ-10D5 is about 50 kDa, and the light chain is about 40 kDa, which is in line with expectations, indicating that the light and heavy chains are fully expressed and a complete antibody structure is formed. Subsequently, the protein concentration was measured and statistical analysis was performed. The bispecific antibody disclosed in the present invention has a higher expression level than the parent monoclonal antibody. Nearly 30 ml of culture supernatant was collected, and the amount of purified antibody was about 15 mg. The transient transfection expression reached 0.5 g/L, which can meet the needs of subsequent production.

Example 3. Identification of bispecific antibody binding activity

The ELISA method was used to analyze the binding activity of the bispecific antibody and monoclonal antibody to the CHIKV E2 protein. The experimental steps are described as follows:

1. Coating: One day before the experiment, dilute CHIKV E2 antigen to a concentration of 2 μg/mL with coating solution in a 96-well ELISA plate. Coat the plate with 100 μL per well at 4°C overnight.

2. Blocking: Wash three times using a plate washer (BIO-TEΚ, 405_LS), add 100 µL of blocking solution to each well, and incubate at 37°C for 1 h.

3. Sample incubation: Wash the plate 3 times. Add 100 μL of diluent to each well except the first well. Dilute the antibody to 1 μg/mL in the first well. Perform 3-fold gradient dilution at 100 μL/well. Set up three replicate wells for each antibody and incubate at 37°C for 1 h.

4. Secondary antibody incubation: Wash the plate three times, dilute the HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1:10000 with diluent, add 100 µL per well to the corresponding wells of the ELISA plate, and incubate at 37°C for 1 h.

5. Color development: Wash the plate three times, add 100 µL of TMB single-component color development solution to each well, develop for 6 min at room temperature in the dark, then add 50 µL of stop solution to each well to terminate the reaction.

6. Detect the OD value at 450-630 nm on a microplate reader, draw the curve using the Logistic four-parameter fitting method, and calculate the EC ₅₀ value of the antibody.

ELISA test results are shown in Figure 5, bispecific antibodies are combined with CHIKV E2 protein, and have a dose-effect relationship. Under different antibody concentrations, compared with monoclonal antibody 8D1, EC ₅₀ (half maximum effect concentration) is significantly improved, compared with nano antibody 10D5, EC ₅₀ has no significant difference, as shown in Table 2.

Table 2 ELISA binding EC ₅₀ of bispecific antibodies and monoclonal antibodies to CHIKV E2 protein

From the EC ₅₀ results in Table 2, it can be seen that 10D5-8D1-H has the best binding activity. “-” indicates EC ₅₀ >1000 ng/mL).

Example 4: Affinity identification of bispecific antibodies

The BLI protein interaction system was used to measure the affinity of bispecific antibodies and monoclonal antibodies to the CHIKV E2 protein antigen and compare the affinity changes of different antibodies. The main steps are as follows:

1. Prepare buffer: Running Buffer (PBS + 0.02% Tween 20 + 0.2% BSA), Regeneration buffer (10 mM Gly, pH 1.75).

2. Sample preparation: dilute the antibody to 50 nM (7.5 μg/mL) with Running Buffer, dilute the antigen to 100, 50, 25, 12.5, 6.25, and 3.125 nM, and pipette 250 μL into the corresponding positions of the 96-well plate. The plate where the sample is located is plate A. Add the corresponding samples according to the design layout and place them on the tilted plate rack.

3. Turn on the Gator instrument, open the data acquisition software, and select the program module after the instrument completes the self-check. Use tweezers to carefully remove the SA probe from the box and soak it in a 96-well plate to which 200 μL buffer has been added. Place the 96-well plate on a horizontal plate rack, with the plate where the probe is located being plate B.

4. Select the K Assay program for sample detection: Select K Assay in the main interface Assay Setup, set Time to 600 s, Shaker A Speed to 400 rpm, and Shaker B Speed to 1000 rpm in Basic Parameters; select the sample type according to the experimental scheme of Baseline – Loading – Baseline– Association – Dissociation in Plate Set Up, and enter the sample information in the 96-well plate, including the sample name and concentration; define the Position, Time, Speed and Step Type of each step in Assay Steps, Step 1 is Baseline, Time is 60 s, Speed is 1000 rpm, Step 2 is Loading, Time is 100 s, Speed is 400 rpm, Step 3 is Baseline, Time is 60 s, Speed is 1000 rpm, Step 4 is Association, Time is 300 s, Speed is 1000 rpm, Step 5 is Dissociation, Time is 300 s, Speed is 1000 rpm; click Start in Preview to start running the program.

5. Result analysis: In the Results & Analysis module on the main interface, click New ΚAnalysis, and select the experiment to be analyzed in Experiment Selection; set Ref. Probe in Reference to define the control probe in the experiment, select the non-control well, click Edit Formula, select the multiple-minus-multiple operation mode, subtract the reference, and click Processed to display the processed data; in Binding Fitting, select Global in Fitting in Parameters, and click Binding Curve Fit to calculate the fitting curve; in Κinetic Analysis, select Binding Fitting Graph, and click Calculate Κinetics to calculate and display the binding kinetic data Κoff, Κon, and ΚD of the bispecific antibody and monoclonal antibody to the CHIKV E2 protein.

The BLI test results are shown in Figures 6 and 7. The affinity constants KD values of 8D1, 10D5, 8D1-H-10D5, 10D5-8D1-H, 8D1-κ-10D5, and 10D5-8D1-κ are 13.1, 2.08, 0.229, <0.001, 2.42, and 0.627 nM, respectively. The results show that the bispecific antibody has a good affinity with the CHIKV E2 antigen, making it possible to develop it into a specific drug for chikungunya fever. The detailed kinetic parameters are shown in Table 3 below.

Table 3. Binding kinetic data of bispecific antibodies and parental monoclonal antibodies to CHIKV E2 protein

Example 5: Analysis of Bispecific Antibody Neutralizing Activity against Viruses

1. One day before the experiment, dilute Vero cells with culture medium (DMEM + 10% FBS) to a concentration of 1.5×10 ⁵ cells/mL, inoculate into 96-well cell culture plates with an inoculation volume of 200 μL/well, and culture in a 37°C, 5% CO2 cell culture incubator;

2. On the day of the experiment, dilute the bispecific antibody and monoclonal antibody to the initial concentration (100 μg/ml) with culture medium (DMEM+2%FBS), dilute 4 times, and add to a 96-well culture plate with a volume of 100 μL/well; then add 100 μL of Chikungunya virus suspension (diluted 1:1000 with DMEM+2%FBS) to each well, mix well, and incubate in a cell culture incubator for 1 h;

3. Discard the cell culture supernatant in the 96-well plate, add 200 μL of the virus-antibody mixed suspension after co-incubation to each well; set up a survival control (no virus and antibody) and a death control (only virus), and continue to culture in a 37℃ 5% _CO2 cell culture incubator for 72 h;

4. After 72 h, discard the cell culture supernatant and add 50 μL/well crystal violet staining solution (50 mg crystal violet, 20 mL anhydrous ethanol, add water to 100 mL). Let stand at room temperature for 30 min, discard the staining solution, add 200 μL/well pure water, repeat washing 5 times, pat dry the 96-well plate, and take photos for record.

5. Discard the washing solution, add 100 μL/well decolorizing solution (50 mL of anhydrous ethanol, 0.1 mL of acetic acid, add water to 100 mL) to fully dissolve, use OD ₆₃₀ as a reference, and measure the OD ₅₇₀ value with a microplate reader; use (OD sample well-OD death control)/(OD survival control-OD death control) to calculate the cell viability, and use GraphPad Prism 8 to fit the cell viability and antibody concentration to calculate the antibody IC ₅₀ value;

6. The protective effect and _IC50 results of the dual antibody in the cell model are shown in Figure 8 and Table 4.

Results: Figure 8 is a curve diagram showing the neutralization activity of 10D5-8D1-H, 8D1-H-10D5, 10D5-8D1-κ, 8D1-κ-10D5, 8D1, and AB2 (control antibody, see CN 110903386 A) against CHIΚV true virus as a function of concentration. According to the _IC50 values in Table 4, the _IC50 values of bispecific antibodies 10D5-8D1-H, 8D1-H-10D5, 10D5-8D1-κ, and 8D1-κ-10D5 against CHIΚV are 0.14, 0.04, 0.07, and 0.37 μg/mL, respectively. The _IC50 value of monoclonal antibody 8D1 against CHIΚV is 0.17 μg/mL. The _{IC50 value} of control antibody AB2 against CHIΚV is 2.90 μg/mL. The results show that relative to control antibody AB2, The neutralizing activity of bispecific antibodies 8D1-H-10D5, 10D5-8D1-κ, 8D1-κ-10D5 and monoclonal antibody 8D1 was significantly improved; compared with the parent monoclonal antibody 8D1, the neutralizing activity of bispecific antibodies 8D1-H-10D5 and 10D5-8D1-κ was improved, among which 8D1-H-10D5 was more significantly improved, about 4.3 times that of monoclonal antibody 8D1, and 10D5-8D1-κ was 2.4 times that of monoclonal antibody 8D1. The neutralizing activity of bispecific antibodies, parent antibodies and control antibodies against CHIKV true virus is shown in Figure 8.

Table 4. IC ₅₀ of bispecific antibodies, parental monoclonal antibodies, control antibody AB2 and CHIΚV true virus

.

Claims (12)

1. The camel-source nanobody combined with the chikungunya virus E2 protein is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the camel-source nanobody are respectively shown in positions 26-33, 51-57 and 96-112 of SEQ ID NO. 1.

2. The camelid nanobody bound to the E2 protein of chikungunya virus according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the camelid nanobody is shown in SEQ ID No. 1.

3. The bispecific antibody against chikungunya virus is characterized in that the bispecific antibody takes a monoclonal antibody against chikungunya virus as a parent antibody, and is fused with a camel-derived nanobody combined with the E2 protein of the chikungunya virus at one end of a heavy chain or a light chain of the monoclonal antibody against chikungunya virus, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody against chikungunya virus is shown as SEQ ID NO:3, and the amino acid sequence of a light chain variable region of the monoclonal antibody against chikungunya virus is shown as SEQ ID NO: 5.

4. The bispecific antibody against chikungunya virus according to claim 3, characterized in that the amino acid sequence of the heavy chain constant region and the amino acid sequence of the light chain constant region of the monoclonal antibody against chikungunya virus are shown in SEQ ID No. 7 and SEQ ID No.9, respectively.

5. The bispecific antibody against chikungunya virus according to claim 4, wherein the fusion is in the form of a (GGGGS) n-linked peptide linkage, wherein n is a natural number from 1 to 4.

6. The bispecific antibody against chikungunya virus according to claim 5, wherein the bispecific antibody is a monoclonal antibody against chikungunya virus as a female parent fused at the N-terminus of its heavy chain to the C-terminus of a camelid nanobody to which the E2 protein of chikungunya virus binds, the heavy and light chain amino acid sequences of the bispecific antibody against chikungunya virus being shown in SEQ ID NO. 11 and SEQ ID NO. 13, respectively, or

The N end of the light chain is fused with the C end of the camelid nanobody combined with the E2 protein of the chikungunya virus, the amino acid sequences of the heavy chain and the light chain of the bispecific antibody of the chikungunya virus are respectively shown as SEQ ID NO. 15 and SEQ ID NO. 17, or

The C end of the heavy chain is fused with the N end of the camelid nanobody combined with the E2 protein of the chikungunya virus, the amino acid sequences of the heavy chain and the light chain of the bispecific antibody of the chikungunya virus are respectively shown as SEQ ID NO. 19 and SEQ ID NO. 13, or

And the C end of the light chain is fused with the N end of the chikungunya virus E2 protein-combined camel source nano antibody, and the amino acid sequences of the heavy chain and the light chain of the bispecific antibody against the chikungunya virus are respectively shown as SEQ ID NO. 15 and SEQ ID NO. 21.

7. A polynucleotide encoding the camelid nanobody bound to chikungunya virus E2 protein of claim 2, wherein the polynucleotide has a sequence shown in SEQ ID No. 2.

8. A polynucleotide encoding the bispecific antibody against chikungunya virus according to claim 6, wherein the polynucleotide sequences are shown in SEQ ID NO. 12 and SEQ ID NO. 14, respectively, or

Polynucleotide sequences encoding the heavy and light chains of the bispecific antibody against chikungunya virus are shown in SEQ ID NO. 16 and SEQ ID NO.18, respectively, or

The polynucleotide sequences of the heavy chain and the light chain of the bispecific antibody for resisting the chikungunya virus are respectively shown as SEQ ID NO. 20 and SEQ ID NO. 14, or

The polynucleotide sequences encoding the heavy and light chains of the bispecific antibody against chikungunya virus are shown in SEQ ID NO. 16 and SEQ ID NO. 22, respectively.

9. A vector comprising the polynucleotide of claim 7 or 8.

10. A host cell comprising the vector of claim 9.

11. Use of a camelid nanobody bound to chikungunya virus E2 protein according to claim 1 or2 in the manufacture of a medicament for the prophylaxis or treatment of chikungunya virus diseases.

12. Use of a bispecific antibody against chikungunya virus according to any one of claims 3-6 for the preparation of a medicament for the prophylaxis or treatment of chikungunya virus disease.